4. CHAPTER-I

CONSTRUCTION OF cDNA LIBRARY USING E. GRANULOSUS

GERMINAL MEMBRANE mRNA AND IMMUNOSCREENING

Cystic echinococcos~s (CE), caused by Echinococcus grunulosus, is a disease of high

prevalence in developing countries lncludlng Indla (Parija, 199 1). The accurate assessment

of the prevalence of CE is therefore a major component to understand the magnitude of the

~roblem and evaluate the success of the control strategy. Therefore, early diagnosis and

treatment of CE is most important (Ammann et al., 1990). The applicat~on of modem

immunodiagnostic or molecular diagnostic techmques has enhanced diagnost~c effic~ency

in the diagnosis of many infectious d~seases including the CE. One of such promising

approaches mcludes development of recombinant antigens and their appl~cation in the

diagnosis of CE.

Several immunological methods have been developed and evaluated us~ng crude and

purified antigen fractions obtalned from both metacestode and adult worm components of

E, granulosus. In recent years, many studies have focused mainly on the ldent~ficatlon and

purification of species-specific echinococcal antlgens (Gottstein, 1985; Al-Yaman and

Knobloch 1989). Various antigens such as antigen B (A@) and antlgen 5 (Ag5) have

been found to be the most relevant antigens for the diagnosis of CE. The smallest 8 kDa

subunit of AgB is believed to be Echrnococcus-specific and to have potentla1 d~agnost~c

value (Gonstein, 1985). The laminated layer antigens. 25-29 kDa, of hydatid cyst are found

to be important markers of disease status (Taherkhani et al., 2007).

To date, there is no standard, hlghly sensitive and specific test available for

~mmunodiagnosls of CE in humans (LI el al., 2003). Desplte the development of sensltlve

and spec~fic techniques, such as ~mmunoblottlng, the ~mmunodiagnosis of CE In clinical

practice still continues to remain a diagnostic challenge. The differentral d~agnos~s of CE

cannot be made specifically by use of natlve antlgen extracts of E, grunulosus cysts. The

sera from patients infected with other helmlnthic parasites have been found to exh~b~i

cross-reactions with crude ech~nococcal antlgens (Chemta~ et al., 1981; Wattal el al., 1986;

Hira et al.. 1987; Gonstein. 1992). The crude hydatid antigens obtalned from different

t~aturally infected animals also showed vanable ant~genic composition (Peuella et al.,

1984; Gonstein et al., 1987: Shepherd et al., 1991) The frequent cross-reactions and varied

antigenic composition from hatch to batch preparation made the evaluation of native

antigens in different endemic areas as well as the standard~zation of immunodiagnostic

assays using native antigens are inherent problems associated with diagnosis of CE

(Shepherd et al., 1991). Moreover, the extensive intra- and inter- strain vanation among E.

gronulosus isolates causing CE in a particular endemlc m a as well as in different areas ,

enhances further the difficulty in identification and evaluation of sensltlve and specific

diagnostic antigens for diagnosis of CE (Gotstem, 1992).

In this context, immunodiagnosls of CE may be ~mproved by combin~ng several defined

antigens and the desrgn of new E. granulosus-specific peptides (Zhang et al., 2003).

Thearetically, a mixture of recomblnant protelns with ~mmunodiagnostic potentral can help

to solve these drawbach of uslng native antigens. Unlimited amounts of antigen can be

under controlled conditions, and it may even be possible to identify and remove

the cross-reactive epitopes without loosing the diagnostic efficiency.

Hence, the current studies in serod~amnostics of CE are focused on investigation and

production of recombinant antlgens and synthetic pephdes derived from sequences of AgB

and antigen 5, for their use as hagnostic antigens (Ersfeld and Craig, 1995; Colebmok and

Lightowlem. 1997). Recently, several recombinant protems, synthets peptides or

combinations of welldefined antigens have been evaluated with enhanced diagnostic

specificity (Zhang et al., 2003) with low diagnostic sensitivrties in many cases (LI et al.,

2003). The vaned sens~tivities of the recombinant antlgrns suggest the need lo look for

new recombmant antigenic peptides with high d~agnostic sensltlvlty and specific~ty for

diagnosis of CE.

Moreover, till date efforts are lacking in India lo develop recomblnant antigen based

diagnostic assays for diagnosis of CE .The present study, therefore, was undertaken lo

~dentify and produce sensitive and spec~fic E. grunvlosus recombinant antigens for use in

simple, rapid and reliable unmunoassays for serodiagnosis of CE.

The objectives of this pbase of the study are:

I. To construct the ZAP Express cDNA libmy using mRNA extracted from E.

granulosw metacestode germinal membrane.

2. To identify and isolate the ~mmunodominant diagnostically relevant recomb~nant

antigens by immunoscreemg the E.vcherichru coli expression cDNA I l b r q .

3. To clone, express and punfy diagnostlcally relevant E, grunulosus recombinant

GST-fus~on protelns for rvaluat~on In rnzyme l~nked ~mmuno-transfer blotting

(EITB) and enzyme linked immunosorbent assay (ELISA).

4. To identify the native antigen fractions corresponding to the diagnostlcally

relevant E. granulosus recomb~nant antigens in hydatid cyst wall and hydatid cyst

fluid antigens.

Patients

The present study was conducted in the Department of Microbiology, Jawaharlal lnst~tute

of Postgraduate Medical Education and Research (JIPMER), P u d u c h q . The study was

conducted after obtaining the institutional ethical committee approval. An informed

consent was also obtained from all human adult participants involved in this study. A detail

history of all participants involved in thls study was taken, a thorough cllnical examination

was done in each case and the clinlcal presentation was recorded. Subsequently, semm

samples were collected from cases w~th CE, other parasltlc d~seases and healthy

individuals.

Study groups

Group-I: Surgically confirmed CE cases (n=lO): This group lncluded 10 cases of

surgically confirmed CE cases. The cysts removed dunng surgery were confirmed to bc of

hydatid cyst etiology by lilstopathologlcal evldence of germinal layer and by

demonstration of scolices and hooklets in the aspirated HCF.

Croup-11: Controls with other parasitic diseases (n=ZS): This group included patients

with other parasitic dlseases such as 10 cases w~th neurocysticercosls, 3 cases each with

filariasis, schistosorniasls, malana, amoebiasls and toxoplasmosis.

Croup-III: Healthy controls (n=lO): This goup included healthy adults (blood donors

and students) with no chnical history of CE or any other disease m the recent past

Specimens

Serum

Five milliliters of venous blood was collected from all surg~cally confirmed CE cases

(n=lO) and controls (n=35) under aseptic precautions and was allowed to clot. The serum

was separated and stored in aliquots at - 2 0 ' ~ till use.

The surgically removed hydatld cysts were dissected free from surounding tissue into

sterile phosphate buffered saline (PBS) (pH 7.2) at room temperature. The cysts were

washed in several changes with sterile PBS @H 7.2). The hydatid cyst fluld (HCF) was

aspirated with a sterile disposable synnge and stored in aliquots at - 2 0 ' ~ till use. The cyst

membrane was dissected open and washed thoroughly in several changes of PBS (pH 7.2)

to remove the protoscoleces. The germmil membrane was separated out from the

laminated layer and ~mmed~ately placed in liqu~d mtrogen and stored at - 2 0 " ~ until use.

Preparation of E. granulosus natlve antigens

Preparation of hydatid cystfluid (HCF) antigen

The hydatrd cyst fluld antlgen was prepared as per the method descr~hed by Kanwar et al.

(1992) with minor modifications. The HCF was aspirated asept~cally from the fertile

hydatid cyst and was collected m sterile centrifuge tube. The flu~d was centrifuged at 5000

x g for 30 min at 4°C; supernatant was collected and dialyzed extensively agalnst distilled

water. To the dialisate. I mM of phenylmethylsulfonyl fluoride (PMSF) was added and

stored at -20°C. The HCF was dialyzed against PBS (pH 7.2) for 48 h at 4'C, with 8

changes of PBS (pH 7.2). Fmally, the HCF was concentrated ten-fold using the centrlcon

concentmor tubes (Millipore Indza Ltd.. Indra) and stored at -70°C unt~l used.

Preparation of hydatid cysf wall (HCW) antigen

The HCW antlgen was prepared as per the method described by Rafiei and Craig (2002)

AAer aspiration of cyst fluid. the collapsed cyst membranes were washed three times in

PBS @H 7.2). The cyst membrane was homogen~zed using monar and pestle and in glass

tissue homogemzer wlth PBS (pH 7.2) containlng PMSF (I mM) under cooling conditions.

The homogenized tissue was collected m a sterile contaner and kept at -20°C overnight.

The homogenate was thawed and sonicated 8 tlmes at 12 kH w~th 90 sec coohng interval

using the sonicator (Lobmore, Indzn.). Each cycle of sonication was for 1 mln. The

sonication was done under cooling condition in an ice bath. The sonicated material was

kept a1 4°C overnight and then centrifuged at 4 ' ~ for 30 mm at 14,000 x g. The supematant

was used as HCW antigen.

The protein content of each antlgen preparation was estimated by standard method of

Lowry (1951) and readings were taken at 280 nm in an W-visible spectrophotometer

(Cintra 5. Australia). After the estimation of protein concentration, all antigens were

aliquoted to lml clyoprotected vials and stored at - 2 0 ' ~ till use.

Preparatlon of hyper immune rabblt serum agalnst HCF and HCW somatic antigens

Antibodies were raised in rabbits agalnst both E. granulosus native antigens (HCF antigen

and HCW antigen). Two rabbits were immunized for each antigen preparatlon. The rabb~ts

were bled one day before to stan ~mmunization. This serum was used as the normal rabbrt

serum control.

Immunization of the rabbns agalnst each E grunuiosus native antigen was done as per thc

method described by Shariff and Parija (1993). Briefly, each of tbe antigen preparatlon

(400 hg) either HCF or HCW antigen was emulsified w~th equal volume of Freund's

complete adjuvant (Dgco Lnhoratoies. Detrott, MO. Adult rabbits (3-5 kg) were injected

wlth 0.5 ml of tbls emulslon in all 4 limbs ~ntramuscularly. After 6 weeks they were

injected with 0.5 ml of the same antigen preparation with Freund's incomplete adjuvant

(Difco Laboratoies, Detroit, MI). Test bleed was done from the ear veln of rabbit 10 days

after the booster. Approximately, 0.5 ml of blood was collected from each rabbit.

The level of antibody raised m rabbit was tested by ELISA using goat anti-rabha-lgG

conjugate (Bangalore Genei, India) as per the procedure of Crowther (1995). Up to 4 ml of

blood was collected from rabb~ts with high-level ant~body as seen in the test bleedrng. The

serum was separated and stored In 1 ml al~quots at -20°C after being pre-adsorbed against

Ercherichio coh lysate following the standard protocol (Samhrook and Russell. 2001).

Purlneation of mRNA from hydatid cyst wall germinal membrane

The total RNA was extracted from freshly collected E granulosuii g e n n d l membrane

uslng RNA extraction !at following the manufacturers' instructions (Lifetechnologies,

Rockviiie. MD).

The procedure of mRNA purification consisted of the following steps:

I. The germinal membrane was dissected out from the l?eshly collected human hydatid

cyst and placed immediately in liqu~d mtrogen.

2. The germinal membrane was ground to a fine powder in liquid nitrogen using mortar

and pestle.

3. The total RNA was then extracted from powdered germinal layer usmg Tnzol reagent.

4. The Messenger &'A (mRNA) was extracted *om 2 mg of total RNA uslng Ol~gotex

mRNA Mrni Kit (Qtagen. Hilden, Germany) accordrng to the manufacturers'

instructions.

5. The mRNA was precipitated using 1/10 vol of 3 M sodium acetate (pH 5.2). and 0.8

vol of isopropanol. The tube was kept at -20'42 overnrght and centr~fuged at 12,000 x

g for 30 min at 4-42.

6. The mRNA pellet was washed with 70% (vtv) ethanol, alr-dried at room temperature

(RT) for 10-15 min, and dissolved in 20 p1 of dlethyl pyrocarbooate (DEPC) treated

water.

The resulted mRNA was used for constructron of E, granulosus cDNA library.

Construction of ZAP Express cDNA library from E. granulosus germinal membrane

mRNA

The ZAP Express cDNA library was const~ucted from E. granu1osu.s metacestode germinal

membrane cells usmg ZAP Express cDNA synthesrs k ~ t (Straragenr, CA. USA) and ZAP

Express cDNA glgapack 111 gold clon~ng kit (Strotagene, CA. USA) accord~ng to the

manufacturers instructions. Const~ction of E granulosus cDNA library ~ncluded the

following steps (Appendix-I: Prrpurutzon of media, rrugenrs and buffers used In cDNA

lrbrary cowtrctron):

Synthesis offlrst-strand cDNA from mRNA

Synthesis of first-strand cDNA from E. granulosus metacestode mRNA consisted of the

following steps:

I. The first strand cDNA was synthesized in a final volume of 50 p1 of the fmt-strand

reaction mix.

2. In an RNase-free m~crocentnfuge tube, the following reagents were added in order; 5

pl of 10X first-strand buffer, 3 p1 of first-strand methyl nucleot~de mlxture, 2 p1 of

linker-primer (1.4 pdpl), 12.5 p1 of DEPC-treated water and I pl of RNase block

ribonuclease inhibitor (40 UIpI).

3. The components were mixed and approximately 5 vg of mRNA (25 p1) was added.

4. The primer was allowed to anneal to the template (mRNA) for 10 minutes at RT.

5. Then 1.5 pl of Strata Scnpr RT (50 Ulpl) was added to the fmt-strand synthesis

reaction mix.

6. The sample was mixed gently and spun down the contents briefly in a

microcen~fuge.

7. Subsequently, a control reaction was also set for first-strand synthesis using 5 pl of the

fmt-strand synthes~s reaction mix (from the previous tube) and 0.5 pl of [a - 32 PI

dNTP (800 CVmmol) in a separate tube.

8. Both fmt-strand synthesis reaction mlx and the control reactlon were incubated for 1 h

at 42'C In a water bath. After ~ncubation, both reaction tubes were removed from

water bath.

9. The non-radioactive first-strand synthesis reaction tube was immediately placed on

ice.

10. The radioactive first-strand synthesis control reaction mix was resolved by

electrophoresis on an alkal~ne agarose gel to observe the efficiency of the first strand

synthesis.

Synthesis of Second-Strand cDNA

Synthesis of second-strand cDNA from E grirnulosus metacestode mRNA conslsted of the

following steps:

I The second strand cDNA reaction components such as 20 pl of IOX second-strand

buffer, 6 p1 of second-strand dNTP mixture, 114 p1 of stenle DEPC-treated water. 2 p1

of [a - 32 PI dNTP (800 CVmmol), 2 p1 of RNase H (1.5 Ulpl) and 1 l pI of DNA

polymerase 1 (9.0 Ulpl) were added to the 45 p1 of non-radioact~ve, first-strand cDNA

synthesis reaction mlx on ice.

2. The contents of the tube were vortexed gently.

3. The reaction contents were spun in a microcentrifuge, and incubated for 2.5 h at 16°C.

Proper care was taken to ensure that the temperature does not rise above 16%

4. Afler second-strand synthesis, the reaction tube was immediately transferred on to ice.

Blunting of cDNA Termini

Blunting of cDNA tennlnl conslsted of the following steps:

1. To the second-strand synthesis reaction mix, 23 p1 of blunting dNTP mix and 2 pI of

cloned Pfu DNA polymerase (2.5 Ulpl) were added.

2. The reaction mix was quickly vortexed and spun down the components in a

microcentrifuge.

3. The reaction nux was incubated at 72°C for 30 min.

4. To the reaction tube, 200 pl of phenol (pH 7 ~ h l o m f o m [I : ] (vlv)] was added,

briefly voltexed the tube and spun in a microcentrifuge at 10000 x g for 2 min at RT.

5. The upper aqueous layer, containing the cDNA, was transferred carefully to a new

tube without disturbing the interface.

6 . An equal volume of chloroform was added to the reaction mix, vonexed and spun at

10000 x g for 2 min at RT.

7. The upper aqueous layer, contalnlng the cDNA, was transferred to a new tube

8. To the blunting reaction mix, 20 pl of 3 M sodium acetate and 400 p1 of 100% (vlv)

ethanol were added, vortexed and mcubated overmght at -20DC.

9. After incubation, the cDNA prec~pitate was spun down ~n a m~crocentrifuge at 10000

x g for 60 min at 4'C.

10. The radioactive supernatant was carefully removed and d~scarded In a radioact~ve

waste container without d~sturbing the cDNA pellet.

1 I . The pellet was washed gently by adding 500 p1 of 70% (vlv) ethanol to the s~de of the

tube away from the preclpltate. The components were spun ~n a m~crocentrifuge at

10000 x g for 2 min at RT.

12. The ethanol was asp~rated carefully and air dried the pellet.

13. The pellet was =-suspended in 9 p1 of EcoR I adapters and mcubated at 4'C for 2

days.

14. At this point, 1 p1 each of the samples of second strand cDNA synthesis reactions was

mn on an alkaline agarose gel to determ~ne the slze of the cDNA and to check the

presence of any secondaly structure.

Ligation of EcoR 1 adapters to the blunted eDNA

The EcoR I adapters were l~gated by adding I p1 of 10X ligase buffer, 1 pl of 10 mM rATP

and 1 pl of T4 DNA ligase (4 Ulpl) to the tube containing the blunted cDNA and the EcoR

1 adapters. The components were spun down m a micmcenuifuge and incubated for 2 days

at 4°C. Afier incubation, the T4 DNA ligase enzyme was heat mact~vated by placing the

tubes in a 70'C water bath for 30 min.

Phosphorylation of EcoR I Ends

Afler the T4 DNA ligase was heat inactivated, the reaction mix was spun in a

microcentrifuge for 15 sec and cooled the reaction for 5 mln at RT. The adapter ends of the

cDNA were phosphorylated uslng 1 pl of LOX ligase buffer, 2 p1 of 10 mM rATP, 5 pl of

sterile water and 2 pl of T4 polynucleot~de kinase (5 Uipl). The reaction mix was

incubated for 30 min at 37°C. Afler phosphorylat~on, the T4 polynucleotide lunase was

heat inactivated for 30 min at 70°C. The components and condensation were spun down

for 15 sm and allowed the reaction mix to equilibrate to RT for 5 min.

Digestion of phosphorylated cDNA using Xho 1

The phosphorylated cDNA fragments were digested using Xho 1. To the phospborjlated

cDNA fragments, 28 p1 ofXho I buffer supplement and 3 pl ofXho I(40 U!pl) were added

and incubated for 90 mm at 37'C. The cDNA was precipitated by adding 5 pl of 10X STE

buffer (Appendix-I) and 125 p1 of 100% (v!v) ethanol to the reaction mix and incubating

the tube overnight at -20°C. The reaction mix was spun in a mlcrocentnfuge at I0000 x g

for 60 min at 4'C. The supernatant was carefully discarded, dried the cDNA pellet

completely and resuspended in 14 p1 of IX STE buffer.

Sue fractionation of Xho I digested cDNA fragments

TheXho I digested cDNA fragments were size fractionated usmg drip column according to

the manufacturer's instructions (Stratagene, CA. USA). About 3.5 pl of the column loading

dye was added to the cDNA fragments and run through a drip column contalmng

Sepharose CL-2B gel filtrat~on medium. The whole process of size fractionation includ~ng

the preparation of drip column and elution of cDNA from drip column was completed in 1

day.

For standard cDNA size fractionation (>400 base pairs), approximately 12 fractions were

collected. The progression ofthe leading edge of the dye through the column was used as a

guideline for monitonng the collection of cDNA fractions. All the fractions were assessed

for the presence of cDNA on a 5% nondenaturing acrylam~de gel. The eluted samples

which did not contain cDNA were hscarded.

The suitable cDNA fractions were determined by electrophoresis using 8 pi of each

fraction on 5% nondenaturing acrylamide gel. The size-selected cDNA fragments were

recovered using phenol-chloroform []:I (vlv)] and 80% (vlv) ethanol. The pellet was

dissolved in 5 pl of sterile water and the cDNA was quant~fied using spectrophotometer

(Cintra 5. Australia).

Llgntlon of E. granulosus cDNA fragments into ZAP Express Vector

Approximately, 100 ng of the size fractionated cDNA fragments were llgated into 1 pg of

ZAP Express vector. In a fresh tube. 1.5 PI(-100 ng) of resuspended cDNA was taken and

0.5 p1 of 10X ligase buffer, 0.5 pl of 10 mM rATP @H 7.5). 1.0 p1 of the ZAP Express

vector (1 pdpl). I pl of stenle DEPC water and 0.5 p1 of T4 DNA ligase (4 UIpI) were

added in the order.

A control ligation reaction was also set by ligating the test insert (pBR322 test lnseR

digested with Sol 1 and EcoR I) into the ZAP Express vector using 1.0 pl of the ZAP

Express vector (I pg), 1.6 p1 of test insert (0.4 pg) (provided with the kt). 0.5 pl of 10X

ligase buffer, 0.5 pl of 10 mM rATP (pH 7.5), 0.9 pl of water and 0.5 pl of T4 DNA llgase

(4 Ulpl) m the order ment~oned. The test and control tubes were incubated for 2 days at

4OC.

Two mcroliters of each ligation m x , mcluhng the control ligatlon, was packaged uslng

Gigapack In Gold packagmng extracl according to the packaging instmctions outlmed by

the manufacturer. The packaging of ligatlon mix involved the following steps:

Preparation of host bacteria

The XLI-Blue MRF' and VCS257 cells were streaked onto LB agar plates (Appendix-I)

containing antibiotic and incubated overnight at 37OC. A separate 50-ml cultures of XLI-

Blue MRF' and VCS257 cells were prepared in LB broth (Appendix-I) with supplements

and without antibiotic and ~ncubated with shaking at 37OC for 6 h (up to an ODw of

cultures 1.0).

AAer incubation, the bacteria were pelletted at 1000 x g for 10 min and gently

resuspended each cell pellet in 25 ml of sterile LO mM MgSO,. Fmally, the cells were

diluted to an ODm of 0.5 using sterile 10 mM MgS0, and used immediately.

Packagingprotocol for the Gigapack III Goldpackaging extract

The Gigapack IIl Gold packaging extract was thawed quickly until the contents of the tube

just begin to thaw. Two m~crollters (approximately 1.0 pg) of l~gated DNA was added

immediately to 25 pl of Gigapack 111 gold packaging extract. The reaction contents were

mixed gently and spun quickly for 5 sec. The tube was incubated at RT for 2 h.

Then 500 p1 of SM buffer (Appendix-I) and 20 11 of chloroform were added to the tube

and spun briefly to sediment the debris. The supernatant containing the phage was then

t~tered.

Similarly, 2 pl of ligated test insert was also packaged using 25 p1 of Gigapack 111 gold

packaging extract as described for sample DNA.

Testing the effleiency of Gigapack III Gold packaging extract using wild-lype Lambda

control DNA

Testing the efficiency of G~gapack 111 Gold packaging extract consisted of the follow~ng

steps:

I . Approximately 0.2 pg of ?c1857 Sam7 wild-type lambda control DNA (I pl) was

added to 25 p1 of Gigapack I11 gold packaging extract.

2. The reaction contents were m~xed gently and spun quickly for 5 sec. The tube was

incubated for 2 h at RT.

3. To the pacbged control DNA, I pl of SM buffer (Appendix-I) and 20 p1 of

chlorofom were added and spun briefly to sediment the debris.

4. Two consecutive 10-' dilutions (fmal dilution was lo4) of the packaging reaction were

prepared from the supernatant containing the wild-type Lambda control DNA.

5 . 10 pl of the lo4 diluted wild-type lambda control DNA was added to 200 p1 of the

VCS257 host strain at an ODm of 0.5 and incubated at 37°C for 15 mm.

6. After incubation, 3 ml of NZY top agar (Appendix-I) was melted and cooled to -48°C

and was added to the host cells and control DNA mlx.

7. The NZY top agar mix was quickly poured onto dry, pre-warmed NZY agar plates and

incubated for 12 bat 37°C.

8. The plaques obtained on NZY agar were counted. (Approximately 350 plaques were

obtained with Gigapack 111 Gold packaging extract on the lo4 dilut~on plate.)

CHAPTER-I

The efficiency of the Gigapack III Gold packaging extract was calculated using the

following equation:

(Number of plaques) X (Dilution factor) X (Total packaging volume)

(Total number of micrograms packaged) X (Number of microliters plated)

plating and tinering ofrecombinant phage vector

The amplified phage stock was diluted to 1: 10,000, 1: 100,000, 1 : 1,000,000 and 1 pl of

each dilution were added to 200 pI of host cells (XLI-Blue MRF' host cells diluted in 10

mM MgSO, to ODm of 0.5).

The phage and bacteria were ~ncubated for 15 min at 37°C to allow the phage to anach to

the cells. AAer incubation, 3 ml of NZY top agar (melted and cooled to -4E°C), I5 pl of

0.5M IPTG (in water) and 50 p1 of X-gal [250 mgiml (in dimethyl formam~de)] were

added to phage-bacteria mix.

Finally, the whole mix was immediately plated onto dry, pre-warmed NZY agar plates and

allowed the plates to set for 10 min. The plates were incubated for up to 18 h at 37°C in

inverted position.

AmpUflentlon of ZAP Express cDNA library

Amplification of ZAP Express cDNA library consisted of the following steps:

I . The XLI-Blue MRF' cells were grown in 50 ml of in LB broth with supplements

(Appendix-I) for overnight at 30°C with shaking.

2. Gently spun down the XLI-Blue MRF' cells (1000 x g), discarded the supernatant.

3. The cell pellet was resuspended in 25 ml of 10 mM MgS04.

4. The cells were diluted to an ODm of 0.5 in 10 mM MgSO..

5. 300 p1 of packaged mixture or library suspension (containing -5 10' plaque forming

units @fu) of bacteriophage) was added with 600 pl of XLI-Blue MRF' cells at an

ODm of 0.5 in polypropylene tubes.

6 . The tubes were incubated for I5 min at 37°C to allow the phage to attach to the cells.

To amplify 1 x lo6 plaques, a total of 20 aliquots were used (each aliquot contained 5

lo4 plaques1150 mm plate).

7. The NZY top agar (melted and cooled to -48°C) was nuxed with each allquot of

infected bacteria and spread evenly onto a freshly poured NZY agar plate (150 mm)

and allowed the plates to set for 10 min.

8. The plates were incubated at 37% for 8 h in inverted position.

9. After incubation, the plates were overlaid with 10 ml of SM buffer and stored the

plates overnight at 4°C to allow the phage to d~ffuse into the SM buffer.

10. The bacteriophage suspension was recovered from each plate and pooled Into a sterile

polypropylene contamer. The plates were nnsed with an additional 2 ml of SM buffer

and pooled.

I I. To the bacteriophage suspension, cblorofonn (5% (viv) final concentration) was

added.

12. The components were mixed well and incubated for 15 min at RT.

13. The cell debris was removed by centrifuging the contents for 10 min at 500 x g

14. The supenlatant was transfemed to a fresh sterile polypropylene container containing

chlorofom~(0 3% (vlv) final concentration) and stored at 4°C.

Performing plaque lifts

The plaque lifting protocol consisted of the following steps:

1. Approximately 200 p1 (1.5 x 10' pfu 1100 mm plate) of titered llhrary suspension or

amplified mixture was added to 600 p1 of freshly prepared XLI-Blue MRF' cells at an

ODmoof 0.5.

2. The bacteria and phage mixture were incubated at 37 OC for 15 nun to allow the phage

to attach to the cells.

3. The bacteria and phage mixture were added to 3 ml of LB top agar (-48'C) and

quickly poured onto a dry, pre-warmed 2 days old 100 mm LB agar plate.

4. The cells were evenly distnbuted by carefully swirling the plate and allowed the plates

to set for 10 min.

5. The plates were incubated at 37°C for 8 h in an inverted position followed by at 4'C

for 2 h. Sufficient number of plates was seeded to screen I x 10' pfu.

6. A nitrocellulose membrane (NCM) was placed onto each LB agar plate for 2 min to

allow the transfer of the phage particles to the membrane.

7. The membrane and agar were pricked using a needle for orientation.

8. AAer two min, NCM was removed with forceps and a second NCM was placed onto

each LB agar plate for 4 min.

9. The nitrocellulose-bound DNA was denatured by submerging the membrane in a 1.5

M NaCl and 0.5 M NaOH denatwation solution for 2 min.

10. The NCM were neutralized for 5 min by submerging the membrane in a 1.5 M NaCl

and 0.5 M Tris-HCI (pH 8.0) neutralization solution.

11. Following neutralization, membranes were rinsed for 30 sec by submerging the

membranes in a 0.2 M Tris-HCI (pH 7.5) and 2X SSC buffer solut~on (Appendx-I)

and blotted briefly on a Whatman (3MM) filter paper.

12. The nitrocellulose-bound DNA was cross-linked to the membranes by incubating the

membranes at 80°C for 2 b in an oven.

13. The plates and NCM were stored at 4 ' ~ and 2 0 " ~ respectively, for further analysis.

Preparation of insert DNA by single-clone excision

The extraction of insert DNA from the recombinant clone involved the following steps:

I. A single XLI-blue colony was added to NZY broth (Appendix-I) containing 0.2%

(w/v) maltose and incubated overnight at 30°C.

2. Similarly, a single XLOLR (strain) E, colt (Siraragene. CA, USA) colony was also

incubated overnight at 30°C in NZY broth.

3. The cells were pelleted (1,000 x g for 10 min at room temperature) and resuspended at

an ODm of 1.0 in 10 mM MgS04.

4. To a sterile Falcon 2059 polypropylene tube containing 250 p1 (>1 x 10' phage

particles) of purified phage stock and I pl (>I x lob pfdpl) of ExAss~st helper phage,

200 pl of XL1-blue MRF cells were transferred.

5. The tube was incubated at 37°C for 15 mln and 3 ml of NZY broth was added.

6. The tube was incubated for a further 3 h at 37°C w~th shaking (100 rpm).

7. The cells were lysed by heat~ng at 68'C for 20 min.

8. The cell debris was pelleted by centrifuging at 1,000 x g for 15 min.

9. The supernatant (10 pl) was incubated with 200 @ of washed XLOLR cells at 37'C

for 15 min and 300 @ of NZY broth was added.

10. The whole mix was incubated at 37°C for 45min.

I I . The infected XLOLR cells (I00 pl) were plated onto LB agar plates contaming 50

pglml of Kanamycin and incubated at 37°C for ovenught.

12. Single colonles were plcked from LB agar plates and inoculated Into 10 ml of LB

broth containing Kanamycin (50 pgiml).

13. The tubes were incubated agaln at 37OC for overnight and plasmid DNA was extracted

from the cells the following day.

Immunoscreenlug of cDNA Ubrary

The E. coli expression E. granulosus cDNA library was immunoscreened using a pool of

sera collected from patients with surg~cally confirmed CE and rabbit plyclonal

hyperimmune serum raised agamst HCW and HCF using picoBlue immunoscreening lut

(Stratagene, CA. USA) according to the manufacturer's instmctions.

This was done to ldentify and isolate immunodominant diagnostic relevant recombinant

antigen producing clones. The cDNA libraries were always screened in duplicate sets of

NCM to eliminate most false positive clones. Immunoscreening of the cDNA library

consisted of the following steps (The reagents and media composrrions used in

immunoscreening were depicted in Appendix-10:

Testing of antibodies and other reagenLr used in immunoscreening by Dot BIol assay

The antibodies and other reagents were tested for theu efficiency by dot blot assay before

employing in immnnoscreening of cDNA. The dot blot assay consisted of the following

steps:

1. SIX serial dilutions (-1 pg-10 pg) of the HCW antigen and E. coli phage lysate were

prepared.

2. Six NCM strips (each size -5 2 10 cm) were cut out and marked a grid pattern on the

strips using a soR pencil.

3. The E. coli denved cross-reactive ant~bod~es were pre-absorbed from the first ant~body

(a pool of sera collected from surgically confirmed CE cases and rabb~t polyclonal

hyperimmune serum) uslng E coli phage lysate provided in the same kit.

4. One microliter of each dilution of antigen and E coli phage lysate were spotted In

separate a d s on the NCM stnps and alr dned for 5 min.

5. The test strips were immersed In blocking solution for 1 h at RT to block nonspecific

protein binding sites.

6. The strips were washed three times for 5 mln each using 50 ml of TBST (Tns-

buffered saline (TBS) (20 mM Tris-HCI (pH 7.5) and 150 mM NaCI) containing

Tween 20 (0.05% (vlv)).

7. Five serial dilutions (1:10&1:1,000) of the first antibody (a pool of sera collected

from surgically c o n f i e d CE cases and rabbit plyclonal hyperimmune s e m ) were

prepared in blocking solution.

8. Five NCM strips were ind~vidually incubated in the various fist antibody dilutions for

1-2 hat RT agitating gently and washed three times for 5 min each m 50 ml ofTBST.

9. The sixth NCM test strip was used as control for conjugate.

10. Entire SIX test stnps were incubated with the second antibody conjugate (alkalme

phosphatase (AP)-conjugated goat anti-rabbit 1gG; 1:lOOO dilutions in blocking

solution) provided in the k ~ t for 1 hat RT, agitating gently.

11. The strips were washed three times for 5 min each m 50 ml of TBST.

12. The test strips were incubated in color development solution (0.3 mglml of nitroblue

tetrazolium (NBT) and 0.15 mg/ml of 5-bromo-4-chloro-3-indolyl phosphate (BCIP))

for 15-20 min in the dark.

13. The residual precipitated dye was rinsed off from the test strips uslng TBS.

14. The test strips were immersed in stop solution (20 mM Tris-HC1 (pH 2.9) and I mM

EDTA).

Absorption ofprimary antibody with Escherichia coliphage lysate

A !:I0 (vIv) dilution of E. coli phage lysate, provlded in the picoBlue ~mmunoscreening

kit (Stratugene, CA. USA), was prepared in TBST. Four anligen quoted NCM stnps (each

slze -5 x 10 cm) were immersed into the E coli phage lysate and incubated for 30 min at

RT with occasional agltatlon The strips were removed from E colr phage lysate and alr

dried on Whatman (3MM) filter paper. The membranes were washed three times for 5 mm

each with 50 ml of TBS.

The non-specific sites on the membranes were blocked by immersing the stnps in 50 ml of

blocking solution for 1 h with shaking at RT. The strips were rinsed three times w~th 50 ml

of TBST. The test strips were incubated, one at a time, in the fust antlhody (1:s dilutlon

(v/v) in TBST) for 10 min at 37'C wrth shalung and discarded the test membrane. Thls was

repeated 5 times using 5 more NCM test strips and collected the first antibody solution.

Colony immunoscreening assay

The colony immunoscreening assay protocol consisted of the follow~ng steps:

1. A NCM (82 mm diameter) was placed directly onto a 100 mm LB agar plate (LB agar

with 100 p g l d of ampicillin) containmg 10' colonies and grew the colonies to I mm

in diameter (- 16 h).

2. The NCM was removed from the master plate containing the library and placed the

membrane on a stack of three sheets of Whatman (3MM) filter paper with the colony

side facing upward.

3. A second NCM (replica membrane) that has been wetted on a fresh LB agar plate

containing Isopropyl-0-D-thiogaIactopyranoside (IPTG) (10 mM) was placed on top

of the h t NCM.

4. The orientation of the two NCM relative to one another was marked by piercing the

membranes with an 18-gauge needle in several places.

5. Both NCM strips were overlaid with three additional sheets of Whatman (3MM) filter

paper.

6. The two strips were separated and the repllca membrane was placed onto the fresh LB

agar plate contalnlng IPTG ( I0 mM).

7. The transfer process was repeated four times to make four more repl~ca plates in the

same orientation each time.

8. The master and replica plates were incubated at 37'C for 6 h until small colonies

appear and then stored the plates at 4OC.

9. After incubation, the NCM strips were removed from LB agar plates containing IPTG

and exposed to saturated chloroform atmosphere for 10 min under a fume hood by

placing 10 ml of chloroform in the bottom of a 4-liter beaker and by hanging the NCM

from a pipette placed across the top of the beaker

10. The strips were transferred onto Whatman (3MM) filter paper and allowed the residual

chlorofonn to evaporate.

I I . The membranes were immersed and incubated in lysozyme buffer (Append~x-11) for I

h at RT with gentle agitation.

12. The strips were transferred to a fresh solut~on of lysozyme buffer and Incubated for I

h with gentle agitat~on.

13. The strips were washed twice for 10 min each in TBST.

14. The nonspecific binding sites of the membranes were blocked by incubat~ng the strips

in blocking solution (I% (wlv) howne serum albumin (BSA) in TBS) for 1 h wlth

gentle agitation. These strips were used for antibody treatment and color development.

Plaque intrnunoscreening assay

The plaque immunoscreenmg assay protocol consisted of the following steps:

1. The titered library suspension or amplified library m~xture (200 pi) was added to 600

pl of freshly prepared XL1-Blue MRF' cells (at an ODw0f 0.5)

2. The bacteria and phage mixture were incubated at 37 'C for 15 minutes to allow the

phage to attach to the cells.

3. Then 3 ml of LB top agar (-48'C) supplemented with 10 m M MgS04 and 0.2% (vlv)

maltose was added to the bactena and phage mixture and quickly poured onto a dry,

pre-warmed 2 days old LB agar plate (LOO mm diameter).

4. The cells were evenly distributed by carefully swuling the plate and allowed the plates

to set for 10 m.

5. The plates were incubated at 37°C for 8 h in an inverted position followed by at 4-C

for 2 h. Sufficient number of plates was used to screen 1 x lo6 pfu.

6 . The NCM (82 mm for 100 mm plates) strips were processed by immersing the strips

in 10 mM IPTG (in sterile distilled water) solution for 30 mn.

7. The strips were air dried by placing the membranes on Whatman (3MM) filter paper.

8. The agar plates contaming plaques and processed NCM strips were labeled

appropriately using a soft pencd.

9. The numbered IPTG-treated strips werc placed on to the appropriately numbered agar

plates w~thout creating air bubbles.

10. The strips were allowed to be wet followed by Incubating the agar plates for 4 h at

37°C.

11. The orientation of the strips and agar plates were marked using a needle by pierclng

the membrane in several places.

12. The strips were carefully removed with forceps and washed thoroughly using TBST.

13. Plaque lifting procedure was repeated once again using another set of processed NCM

strips.

14. The strips were removed from LB agar plates and immersed in TBST.

15. The strips were washed three times for 15 min each with 10 mlimemhrane of TBST.

16. The remaining nonspecific protein bmd~ng sites on stnps were blocked by immersing

in blocking solution (1% (wiv) BSA in TBS) for I b at RT.

17. The strips were washed three times for 15 min each with 10 mllmembrane of TBST.

These strips were used for antibody treatment and color development

Antibody and conjugate incubations and color development

The antibody treatment and colour development of the NCM test stnps consisted of the

following steps:

1 . The NCM strips were transferred into 8 dmembrane of fint ant~body solutlon (]:SO0

and 1:100 dilutions in blocking solution).

2 The stnps were mcubated for 1 hat RT with gentle agitat~on

3 The stnps were washed three tlmes for 5 mm each ln 8 mumembrane of TEST

4 The membranes were ~ncuhated m 8 Mmemhrane of fresh bloclng solut~on

contatmg the AP-goat anti-rahblt I@ conjugate (1 1000 &lution) for 1 h at RT w~th

gentle agttatlon

5 The stnps were washed three tlmes for 5 mm each m 8 mumembrane of TBST

6 The residual Tween 20 was removed by wash~ng the membranes IU 8 rnlimemhrane of

TBS alone

7 The stnps were Immersed In a freshly prepared color development solut~on (0 3 mgiml

of Nitroblue Tetrazolium (NTB) and 0 15 mg/ml of 5-Bromo-4-chloro-3-lndolyl

Phosphate (BCIP) contalnrng 100 m M Tns-HCI (pH 9 5). 100 mM NaCl and 5 mM

MgCI2) and incubated in the dark untll the posltlve reactions were clearly vlslble (20

mnutes)

8 The stnps were nnsed wlth TBS and lmmersed the membranes m stop solutlon (20

mM Tns-HC1 (pH 2 9) and 1 mM EDTA)

9 The membranes were air dned and stored protected from llght

Synthesis and purification of glutathlone S-transferase (GST) fusion proteins

The glutath~one S-transferase (GST) fuslon proteins of recomb~nant-phage DNA obta~ned

from all positlve clones were synthes~zed and punfied as per the method described by

Helbig et al (1993) The recombinant-phage DNA fragments from all posltlve clones were

sub-cloned Into the Sma I and EcoR 1 sltes of the expresslon vector pGEX-3X (Pharmrrcla

b'rosyslem, USA) Fmally, the GST fuslon proteln was synthesized in E colr DH5a straln

by ~nductlon of gene expresslon using IPTG for 3 h

The synthesis and punficat~on of E gmulosua GST-fus~on pmtems consisted of the

followmg steps

I A colony of E colr DH5a stram was plcked from LB agar late using a tooth pick and

inoculated mto 30 ml LB hroth contauung Lanamycm antlhlotlc at a fiml

concenhatlon of 30pgImI and Incubated overmght at 37'C

2 The culture broth was diluted 10 tlmes to 300 ml and furlher grew the bacteria by

measuring the optlcal dcns~ty at 600nm (00603)

3 When the cell concentration reached to the ODm of 0 6, 0 2 mM IPTG (final

concentration) was added to the culture hroth

The culture hroth was incubated for 6 hat 37'C

5. The cells were harvested by centrifugation for 30 min at 6000 x g and discarded the

supernatant.

6. The cell pellet was resuspended in GST binding buffer (25 mM Tris pH 7.5, 150 mM

NaCI, 10 mM DlT, 1 mM EDTA and 1 mm PMSF) (10 ml of GST binding buffer per

1 g of wet cells).

7. The cells were lysed using lysozyme (IOpgIml) and Triton X-100 (final concentration,

1% [viv]).

8. The cells were lysed by ultrasonication (90% output and 50% duty cycle) for 3 min for

three times with a 3 mm cooling interval after each sonication. The son~cation was

performed in cool conditions by placing on ice.

9. The sonicate was centrifuged at 14000 x g for 30 mln at 4°C.

10. The resulted supernatant (10 pl) was subjected to SDS-PAGE to estimate the synthesis

of the fusion protein.

I I. The proteln concentration of the lysate was measured by Lowry's (195 I) method.

12. The lysate was diluted to I mdml of total protein in GST blnd~ng buffer and loaded

onto a gluthatluone sepharose column (Pharmacia Biosystems, USA) at 4OC (2 ml of

gluthatione sepharose was used for 10 mg of recombinant protein).

13. The column was washed by flowing through with GST bind~ng buffer until no protein

could be detected.

14. The fusion proteins were eluted by adding elution buffer (50 mM Tns-HC1 (pH 8.0)

with 10 mM reduced glutathione) at a flow rate of I mumin until no protein rema~ned

m the column.

15. The protein concentration of the eluted sample was measured by Lowry's (1951)

method and stored in ahquots at -70°C until use.

16. The eluted recomblnant GST-fusion proteins were treated with factor X (Phrrrmucra

Biosystems. USA) following the manufacturers' ~nstmct~ons to cleave the GST moiety

from the GST-fus~on protems.

Sodium dodecyl sulphate-polyacrylamlde gel electrophoresis (SDS-PAGE)

The somatic HCF and HCW antigens and the whole cell protein preparations of the

recombinant E. coli cells were subjected to SDS-PAGE in 12% acrylamide gel using a

submerged gel electrophoresis appamtus (Genei. India), as per the method described by

Laemmli (1970). m e concentration and preparation of the reagents and bufers used in

electrophoresis and sta~ning, destaining so1u:ions are described in Appendix-111.)

Briefly, 2Opg each of all antigen preparations along with prestained molecular welght

markers, (broad range protein of apparent Mr 175 kDa - 6.5 kDa) (New England Bioluhs)

was subjected to electrophores~s with 5% stacking gel and 12% separatrng gel under

reducing and denaturing condit~ons 10 an electrophoretic cell at IOOV for 1.30 hours.

The gel, after electrophoresis, was sta~ned In Coomassie blue R-250- 0.25% staining

solution and destained to remove excess stain by appropriate desta~ning solution.

Molecular weight of the antigen bands was determined by comparing with standard

molecular weight marker (NEW Englund Biolabs) run along side, using the sofiware

provided with the gel documentation system (Em-Rod, USA). Non-recombinant expression

cell whole cell protein profile was also run In parallel to the recombinant cell profiles.

Elution of recombinant and native antigens from acrylamide gels

After identifying the appropriate recombinant GST fusion proteins and native antigens, the

respective protein fractrons were eluted from the SDS-PAGE gels as per the method

described by Hager and Burgess (1980) with few rnod~fications. Eaclr antlgen preparatron

was processed and purified separately uslng the same protocol.

Briefly, 150-200 pg of antigen preparation was applied to slngle well of polyacrylam~de

gel and subjected to SDS-PAGE as described above under the same conditions. After the

electmphores~s, gel was removed into a clean tray and one antigen line front of the gel was

cut, while the remaining gel was sealed in air-tight cover and stored carefully at 4 ' ~ . The

cut gel was stained with 0.25% Coomassie brilliant blue R-250 for 1 hour and desta~ned

for 30min. The stained gel was kept along with unstaned gel on a transilluminator (Bio-

Rod. USA) and the band of interest was cut out and put into a sterile homogenizer tube.

The gel pieces were soaked in two changes of 1 ml double distilled water for I5 min.

Finally, the protern was eluted from the gel using electronic gel eluter (Millipore India

L~mifed, India) according to the manufacturer's instntctions.

The SDS-PAGE was performed using eluted recombinant and native hydatid antigens

along with pre stained molecular weight markers, (broad range proteln marker of apparent

Mr 175 kDa to 6.5 kDa) (New EnglondBiolubs), as described above, to check the pur~ty of

the desired antigens. The molecular weight of the eluted hydat~d antigens was detected

using the software provided with the gel documentation system (Bio-Rod. USA).

Demonrtntion of lmmunogenlcity and cross-reactivity of E. grcrnulosus recombinant

antigens by enzyme linked Immuno-transfer blotting (EITB)

The E. granulosur recombinant antigens (Eg-KWI2 and Eg-KW24). were subjected to

EITB separately using a panel of sera collected from cases with surgically confinned CE,

other parasitic diseases and healthy individuals. (Composition ondpreprrrarion ofreagenrr

and buffers for EITB were given in Appendix-/IIj

The procedure of ElTB consisted of the follow~ng steps:

I. SDS-PAGE: The respective antlgene mixtures were separated based on the difference

In molecular weight by SDS-PAGE as per standard method described earher.

2. Elecrroblorring The separated antlgens were blotted onto nltro cellulose membrane

(NCM) ( 0 . 2 ~ pore size) (Hybond ECL, Amersham bioscrence, Germany) by uslng the

blotting apparatus (Pharmucia Biosysrem, USA). The transfer was done at constant

voltage (50 V) for 90 min transfer time.

3. Blocking: The free reactive sltes on the NCM (cut to strips) were blocked by PBS (pH

7.2) containing 3% BSA by incubating for 3 h at 3 7 ' ~ under constant rocking.

4. Washing: Membrane strips were washed three times with PBST as mentioned before

5. Sample serum dilulion and incubrrlion: A 1:100 dilutlon of the patient sera was

prepared m PBST. Finally. 100 p1 of each diluted SeNm was added to the membrane

strip and ~ncubated for 1.5 h at 3 7 ' ~ undcr constant rocking.

6 . Washing. The membrane stnps were washed three tlrnes wlth PBSr as before to

remove the unbound antibodies.

7. Second anrihody conjugal^) incubrrlion: Goat anti-rabbit-IgG-HRP conjugated second

antibody (Genrr. India) was used as per the manufacturers instruction (1:2000) wlth

PBS (pH 7.2) conta~nlng Tween-20 (0.05%); 100 pl volume was dispensed to all the

strips and incubated for 0.5 h at 3 7 ' ~ in dark under constant rocking.

8. Washing. The membrane strips were washed three times with PBST as before to

remove the unbound ant~bodies.

9 Colour developmenl: Substrate solution was prepared freshly by adding 6 mg of DAB

(Sigma, USA) in 10 ml of PBS (pH 7.2) contalmng 0.05% Tween-20 and finally, 10 p1

of H20Z was added just before addlng to the wells. Five millilitres of the substrate

solution per strip was dispensed and incubated for 15-20 min at 37°C in dxk under

constant rocking.

10. Stop reaction: The reaction was stopped by washing the stnps w~th double distilled

water.

Cloning, expressi0II and purification of E. granulosus recombinant antigen B812 (Rec

Eg-AgBW

Purifwdon of DNA from hydatid cysr waN germinal membrane

The genomic DNA was extracted from freshly collected E gronulosus germinal

membrane as per the standard method described by Sambrook el al. (1989).

The procedure of DNA purification consisted of the following steps:

1. The germinal membrane was pound to a fine powder in lrquid nltrogen using mortar

and pestle.

2. Approximately 200 mg of ground tissue was drgested m 0.5 rnl of homogenisatiun

buffer (10 mM Tns-HC1 (pH 7.5). l0mM EDTA. 50 mM NaCI. 2% sodium dodecyl

sulfate, and 20 rnM d~th~othreitol and 900 vg of proteinase K) at 56°C for 8 hours

with occasional gentle inversion of the tube.

3. The Iysate was extracted twlce w ~ t h Phenol: Chloroform.

4. The extracted supernatant was ~ncubated In 2% CTAB (Stgma, USA) and 1. I M NaCl

for 10 min at RT and extracted wrth chloroform.

5 . The nucleic acids were precipitated with an equal volume of isopropanol and the

pellet was washed two times using 70% ethanol.

6 . Finally, the nucleic ac~ds were Incubated In 20 mg of ribonuclease-Nml for 1 h at

37Oc.

7. The DNA pellet was dissolved in 50 p1 of DEPC treated water and stored at - 2 0 ' ~ 1111

use.

8. The concentration and purity of DNA was estimated by measuring the optical density

at 2601280 nm usmg spectrophotorneter (Systronics, hdiu).

Preparation of genomic DNA insert of E. granulosus recombinant antigen B8/2 (Rec

Eg-AgBW2)

The antigen-B812 coding gene was produced from E. grundoslosu.~ genomlc DNA uslng Eg-

AgB812 specific primers (5'-ATITGTGGAGACAATCGC-' and 5'-AGGCAAATCAT

GTGTCCC-3') (Bio Corporals, India) as per the method described by Femandez el al.

(1996).

Briefly, PCR was performed in a final volume of 50 pl using 100 mM of each dNTF'. 100

ng of each primer and 1 U of Taq DNA polymerase (Invitrogen. USA). The amplification

reaction was carried out in a Palm Cycler (Corbel, Australia) under the following

conditions: 20 cycles of 1 min denaturation at 94OC, I min annealing at SOT and 2 mm

extension at 72°C. with a touch down of I'C at every cycle, followed by 20 more cycles at

an annealing temperature of 40PC and a final extension of 10 min.

Synthesis andpurification of Eg-AgBEYZ GST-fusion protein

The Eg-AgB812 GST-fus~on proteln was synthes~zed and purified as per the method

described by Helbig et al. (1993). The PCR ampl~fied fragment corresponding to Eg-

As812 encoding gene was cloned Into pGEXJX expression vector (Pharmacia

B~nsysystems, USA) and expressed in E. coli DH5a cell using the same method described

above.

Conshuctlon of cDNA library from germinal layer of E. granulosus metacestode

The ZAP Express cDNA library derived from mRNA of metacestode germrnal layer of E

p n u l o s u s was constructed in the E. coli. The E coli expression cDNA library was

I-unoscreened using a pool of sera collected from patients wrth surgcally confi rmed CE

and rabbit polyclonal hyperimmune serum for the identification of diagnostrc relevant

recombinant antigen productng clones for diagnosis of CE.

The total RNA was extracted from the E grunulosus metacestode germma1 membrane.

collected from human cases. The total RNA was Intact and formed two distinct bands on

agarose gel stained w~th sta~ned wrth eth~dium brom~de (Fig 4-1). The mKNA was

extracted from 2 mg of total RNA obtained from the E, grunulosus metacrstode germlnal

membrane.

The cDNA was synthesized from 5 pg of purified E. grunulosus metacestode germinal

membrane mRNA using oligo(dT) primer. About 5 pg of purified E, granu1usu.v

metacestode germinal membrane mRNA produced approximately 300 ng of double

stranded cDNA (Fig 4-2). The total cDNA obtained from E. grunulosus mRNA was size

fractionated using drip column. The cDNA fiactlons with srze greater than 0.4 kb

(Fractions 5, 6, 718) (Fig 4-2) were pooled and used for ltgatlon Into the ZAP Express

phage vector. The pooled size fractionated cDNA, resolved on agarose gel, showed an

average fragment size of 1.0 kb.

The packaged recombinant E. granulosus cDNA-phage insert produced v~sible plaques on

the XL1-Blue MRF' E, coli cells (McrA McrB strain). The tltre of the unampl~fied cyst

wall cDNA library was estimated as lo6 pfu per I ml of packaged extract. Approxtmately

95% of the phages in the cDNA library were found to be recombinants phages.

Immunoscreeoing of E. granulosus ZAP Express cDNA library

All three sera pools (pool of sera from CE cases, anti-HCW and anti-HCF) were confirmed

to be highly reactive against both HCW and HCF nattve antigens by EITB, before they

employed for immunoscreening of cDNA library.

I I Fig 4-1: Total RNA (resolved on 0.8% ag.roac gel s&d

with Ethidium bromide) exhactcd from =srmind membrane of hydatid cyst o ' w d from h u k pstienfs.

Lane M: standard DNA Ladder (A DNA-Hind 111 digest and 0 X174-Hae 111 digest mix); ~ a & s 1-3: Total RNA &&ind in

three subsequent extractions

m ~ g e r m ~ l m a n t m n c m R N ~ & d r m n d ~ ~ ~ Lnrr M .Pnd.rd DNA Lsdda (XDNA-Hcd In d 9x174-Hw IIl

Qsat mn); Lsna 1 4 -of dmbb4rm-d cDNAbsrm sue hXumbaa 1

The E m usmg HCW and HCF natlve antigens demonstrated 8 and 6 reactive bands

respectively, ranging from 8 kDa to 172 kDa on testing with the pool of sna collected

from 10 surgically confirmed cases of CE. The EKE using both HCW and HCF nat~ve

antigens demonstrated I2 reactlve bands ranging from 8 kDa to 200 kDa on testing w~th

rabbit plyclonal antl-HCW antibody. The EITB using both HCW and HCF native

antigens demonstrated 10 reactlve bands ranging from 8 kDa to 200 kDa on testing with

rabb~t plyclonal anti-HCW antibody. All the three serum pools showed strong signals

w~th 8-12 kDa, 16 kDa and 24 kDa sub-units of the HCW and HCF antrgens. The EITB

using both HCW and HCF native antigens demonstrated only 2-3 reactlve bands on testlng

w~th sera collected from patlents with ueurocysticercus, filarias~s, schistosom~as~s, malarla

and amoebiasis.

The ZAP Express cDNA library derived from 5 vg of purified E granulosus metacestode

germinal membrane mRNA consisted of approximately lo6 recombinant phages. The

vlsual screening of bluetwhite selection on LB agar plates revealed that the wild t p e to

recombinant phage ratio was approximately 1:20. The majority of cDNA inselts were

found to be more than 1 kb (ranging between 1-3.0 kb) in size (Fig 4-3).

lmmunoscreemng of lo6 pfu (1.5 x 10~pful10 cm LB agar plate) of the ZAP Express

cDNA library using a pool of sera from CE cases (HS), anti-HCW (CW) and anti-HCF

(CF) hyper immune sera separately identified 120, 90 and 50 putative pos~tive plaques by

plaque immunoassay. The 260 nnmunoreactlve plaques by subculture on to fresh LB agar

also produced visible plaques.

lm~uunoscreen~ng of these 260 reactive plaques uslng same sera (HS, CW and CF) showed

stronger signals against 27 definitive clones, including 12 clones w~th HS (designated as

HSP1-I2), 8 clones with CW (CWPl-8) and 7 clones w~th CF (CFPl-7). All the 27

defin~tive reactive clones were plated again on E coli cells (sub-library) for

lmmunoscreening with affmity purified antibodies.

The immunoscreening of the sub-cDNA library uslng affinity purified CW and CF

antibodies showed strong signals against only nine clones (HSP2. HSP4, HSP9, CWP4,

CWP5, CWP9, CFPZ. CFP3 and CFP7). The colony immunoassay using all nine clones

separately did not show reaction with the sera from 10 healthy individuals.

Fig 4-3 t-- The following

sentence was added to the legend the

legend of the Fig 4-3.

The cDNA inserts wrrr nroducrdh I'CK us in^ T3 and

T7 primers LStrofawene, C'A. 0.

Fig 4-3: cDNA insert sizes of the nine positive clones selected by inmunoscreening of the E. granulosus

germinal memtmne cDNA library. Lane M: standard DNA ladder (1 D N A - H i IIl digest and 0x174-Hee I11 digest mix); Lanes 1-9: cDNA insmts of

nine clones uxespodmg to the n k profiles of the lysstes of E colr expression cells in Fig 4-4.

The cDNA insens were produced by PCR using

T3 and T7 primers (Stratagene, CA, USA).

CHAPTER-I

-ka&Eg- CHAPTER-]

C W 9 , CFP2, CFP3 and

pGEXJX vector and expres

I ~ ~ 1 2 , ab& ~mmunologiuri analysis o f t ' C W X rd ati-Roc

k q e ~ m wen

8 0 , The cDNA inserts of the

~ndlvldual sub-clones conta I inserts on SDS-PAGE along %

The designation of the E. granulosus

recombinant antigen ~ roduced by the clone HSP. was P4, C W 5 ,

In EITB, four GST-fuslon

changed to EE doned Into rHS24 or Eo-

rCW24 as per the Of the nine

examiners osus cDNA suggestion.

ed stronger

ins (HSP9,

P4, CWP9,

ZE cases.

The E granu/osus recombin; . 1 espectlvely, showed 100% sensit~vity and spec~fic~ty by EITB. Th- b r o d u c e d by

other seven clones (HSP2, HSP9. CWP4, C W 9 , CFPZ. CFP3 and CFP7) showed cross-

reactions with sera from cases with other pdrasltlc dlseases (both speclfic~ty and sensitivity

< 90%).

Purification of E. granulosus GST-fusion protelns

The affiity purified HSP4-GST, CWS-GST and Eg-A@R-GST fus~on proteins,

resolved on polyacrylamide gels (12%) under reduclng conditions showed an apparent Mr

of 52 kDa. 38 kDa and 35 kDa respectively (Flg 4-6). After cleamng the GST moiety.

these recombinant antigens showed an apparent Mr of 24 kDa, 12 kDa and 8 kDa

respectively (Fig 4-7). The GST expression protein showed an apparent Mr of 25 kDa-27

kDa. The E granuiosus recomhlnant antlgens produced by the clones HSP4, CWPS and

Eg-AgBIZ were deslbmated a~Eg-rCW24. Eg-rCW 12 and Rec Eg-AgB812 respect~vely Eg-rHS24 or

The EITB using anti-Eg-rCW12, ant,-Eg-KW24 and ant,-Rec Eg-AgB812 monospecific

sera against both HCW and HCF native antigens showed that the Eg-rCWIZ corresponds

to a 10-12 kDa protein. Tbe epitopes coded for by Eg-rCW24 and Rec Eg-AgB812 were

observed on four bands including 60 kDa, 24 kDa, 16 kDa and 8-12 kDa.

I J

Fig 4-5: Colonies of -binanl E. coh cells on LB agar plates.

I I

Fin 4-6: Whole cell motein d l e s of recombinant E. col~ Cells (DHS~) -baining 'E. granu~osus antigens and

purified E. granulosus GST-fusion proluins.

1 I

Fig 4-7: Purified recanbinant antigens afbx cleaving GST moiety. Laa I. atEnuv olm6ed En-rCWZ4 0 4 LI*).

Page - 83 CHAPTER4

Pnra-1; Lane-2 . granulosus GST-fusion protelns by ELISA

c G o ~ ~ ~ ~ $ t h " 2usion proteins (Eg-rCW24, Eg-rCW1Z and Rec Eg-AgB8/2)

was changed as specificitywhen tested usingsera collected from cases with

"when tested using" : diseases and healthy individuals. The ELISA using GST

,tical dens~ty (OD4Y1) values ranglng from 0.07 to 0.19 on

i control sera. The ODlPl values in ELISA using sera from

:s showed in a range of 0.50 to 2.00. The values in

lisease controls and healthy individuals did not exceed 0.29.

using HSP2 and CFP7 fusion proteins did not exceed 0.30

,Is. The ELISA using HSP2 and CFP7 fusion proteins also

).40 < OD192 < 0.75) w~th five control sera from patlents with

4 Us~ng HSP9. CWP4, CWP9, CFP2 and CFP3 GST-fusion

discrimlnat~on between of control sera and sera from

CE is characterized by the triggering of an intense humoral response with a comspon&ng

rise in the titers of specific antibodies (Zhang et al., 2003), which forms the basis for the

development of serodiagnostic assays for diagnosis of CE. Till date, several E granulorus

native and recombinant antlgens have been evaluated and employed for the diabnosis of CE

hut still lack optimum sensitivity or specificity (Enfeld and Craig, 1995: Colebrook and

Llghtowlers, 1997; Zhang et al., 2003; LI et al., 2003). Hence, in the present study, efforts

have made to identify suitable E. granulosus recombinant antigens by cloning, expression

and purification of E. ~ u n f i l u s u r antigenic components to develop simple, rapid and cheap

immunodia~ostic assays for the dlabnosis of CE.

Construction of cDNA library from E. granulosus metseestode germinal lsyer mRNA

Complementary DNA libraries represent the information encoded m the mRNA of a

particular tissue or organism (Hemmings and McManus, 1989; Muller et al., 1989; Li et al..

2004). The RNA molecules are exceptionally labile and difficult to amplify in then natural

form (Li et al., 2004; Fernandez et al., 2002: Helbig et al., 1993; Frosch et al., 1991). Once

the genetic information was available in the form of a cDNA library, individual processed

segments of the ong~nal genetlc ~nformatron could be lsolated and examlned wlth relauve

ease in identification of immunodominant and specific diagnostic antigens (Frosch et al.,

1991; Helbig et al., 1993; Fernandez et al., 2002; LI et al., 2004). lience, m the prcsent

study the informat~on encoded by the RNA was first converted Into a stablr DNA duplex

(cDNA) and then was inserted Into a self-replicating lambda vector.

Sufficient quantity and high quality mRNA has been cruclal for construction of the E.

sranulosus cDNA library (Gasser et al., 1990; Frosch et al., 1991; Helblg et al., 1993;

Femandez et al., 2002; Li et al., 2004). Hence in the present study, appropriate precautions

were taken in collection and processing of hydatid cyst material and extraction of mRNA.

The RNA extraction was performed under cold condihons. All plastic ware and other

utensils used in mRNA extraction were also treated with DEPC to ensure that they are frec

from RNAses. To obtain good quality and intact mRNA, the E ffanulosus germinal

membrane was dissected out from hydatid cysts within 2 h after surgical rejection from

human cases. After the fmal wash with PBS, the germinal membrane was immediately

placed into liquid nitrogen directly and stored at - 7 0 ' ~ until use. The mortar and pestle for

grinding the tissue were pre-cooled with liquid nitrogen. l l e tissue was gound by

,p~craphical lrror - The .,t,,,n mark (7) .,I aas deleted.

luously adding liquid nitrogen to keep the mortar cool. Once the tlssue was powdered.

bder was added to the Trirol reagent

ZAP Express cDNA synthesis kit, employed in the present study, used a hybrld oligo

linker-primer that contained an Xho I restriction site. In first-strand synthes~s, the

A was primed with the linker-pnmer and reverse transcribed by StratlScript reverse

jriptase enzyme (StrataScript RT) usrng 5-methyl dCTP.

bhataScript RT 1s a novel maloney murine leukemla virus reverse tmscriptase

Fv-RT) without any detectable RNase H activity (Sambrook el al., 1989; Jerpseth et

692; Altmg-Mees et al., 1992; Kretz et al., 1994). The recombinant StrataScrlpt RT

(ynthesized and punfied by cloning and expression of a genetically englneered mutant

CV-RT gene in E coli expression cells A point mutatlon in the highly conserved

$e of the RNase H region resulted m the loss of undesired RNase H degmdative

ity without affecting the desired reverse transcriptase function (Sambrook et al.. 1989,

kth et al., 1992; Alting-Mees et al.. 1992; Kretz et a1 , 1994). The result was a nuclease-

free mutant of MMLV-RT (StrdtaScript RT) that could produce larger ylelds of full-length

cDNA transcripts than wild-type MMLV-RT. The StrataScnpt RT was also found as the

enzyme of choice for production of full length cDNA hgments from E gru~~ulosus mRNA

(LI et al., 2004).

In the present study, the StrataScript RT enzyme was employed for applications ~nvolv~ng

the preparation of full-length, complete cDNA transcripts, including fust-strand cDNA

synthesis and E. b~u~iulosus cDNA l~brary construction. The nuclease-free MMLV-RT

produced larger yields of full-lenbqh cDNA transcripts kom E. granulosus germlnal

membrane mRNA. About 5 l g of purified E granulosus metacestode germinal membrane

mRNA yielded approximately 300 ng of double stranded cDNA.

The use of 5-methyl dCTP during first-strand cDNA synthesla hem~merhyldtes the cDNA

fragments, wh~ch protects the cDNA from dlgestlon with Xho I restnctlon endonucleases

(Alting-Mees et al., 1992; Sambrook et al., 1989). In the absence of Xho I recognltlon sites

on E granulosus cDNA f r a ~ e n t s , a 50-base ol~gonucleotide pnmer used in first-strand

CDNA synthesis with the following sequence: 5'-GAGAGAGAGAGAGAGAGAGAACT

A G T C T C G A G m m - 3 ' provided an Xho I recognition site ( b e t 2 et

al., 1994; Jerpseth et a]., 1992). The GAGA" sequence present in oligonucleotide pnmer

Protects the Xho 1 recogition site from hemimethylation. Therefore, on Xho I dipstion of

the E. granulosus cDNA, only the unmethylated site within the linker-primer was cleaved

(Li et al., 2003; Li et al., 2004).

h the present study, the fimt-strand cDNA was synthesized from E. grunulri,~us metacestode

germinal membrane mRNA template using StrataScript RT, m the presence of nucleotides

(dATP, dGTP, dlTP and 5-methyl dCTP) and a 50-base oligonucleotide primer. The usage

of 5-methyl dCTP ensured the complete E. granulosus first strand cDNA to possess a

methyl group on each cytosine base, which protects the cDNA from restriction enzymes

used in subsequent cloning steps. Subsequently, the 50-base oligonucleotide primer

employed in the fmt-strand cDNA synthesis prov~ded the Xho I restnctlon enzyme

recognition site on each cDNA fragment.

In second-strand cDNA synthesis, the RNase H nicks the mRNA bound to the first-strand

cDNA to produce a multitude of mRNA fragments (Samhrook et al., 1989). The mRNA

fragments bound to first-strand cDNA serve as primers for DNA polymerase I enzyme to

synthesize second-strand cDNA (Samhrook et al., 1989). Hence, in the present study; the

second-strand cDNA was synthesized using DNA polymerase I by n~ck-translation of the E

grunulosus mRNA fragments. The second-strand nucleot~de mixture was also supplemented

w~th dCTP to reduce the probability of 5-methyl dCTP. The dCTP nucleotides

supplemented In the second-strand nucleotide mlxture competitively inhib~t the

incorporation of 5-methyl dCTP into the second-strand cDNA.

During the second strand synthesis, the temperatures above 16'C have been found to cause

the formation of hairpin structures in the double stranded cDNA which are unclonable In the

ZAP Express vector (Samhrook et al., 1989; Jerpseth et al., 1992; Altmg-Mees et a]., 1992;

Kretz et al., 1994). The ha~rp~n structures interfere w~tb the efficient insertion of correctly

synthesized cDNA into the prepared ZAP Express vector (Li et al.. 2004: Fernandez et al.,

2002). Therefore, appropriate care was laken to maintain the temperatures below 16-C

during the E. granulosus second-strand cDNA synthesis.

In the present study, the uneven termini of the double-stranded cDNA were nibbled back or

filled-in to make blunt ends using cloned PJb DNA polymerase. The blunt ends of the

CDNA fragments were ligated to the EcoR 1 adapten (5'-OH-AATTCGGCACGAGG-3'

and 3'-GCCGTGCTCCp-5'). AAer adapter ligation, the l~gase enzyme was heat mactivated.

The 14-mer oligonucleotide was phosphorylated to enable 11s ligation to the

dephosphorylated vector arms. Then the EcoR I adapter ]]gated cDNA fragnents were

digested with Xho I restriction enzyme to release the EcoR I adapter and resldual linker-

From the 3' end of the cDNA fragments. These two fragments were separated on a

)I- containing Sepharose CL-2B gel filtration medium.

nsuuction of hlgh quality and full length cDNA library also depends on the careful b n and punficatlon of the full length double stranded cDNA (Gasser et al.. 1990;

et a].. 1991; Helbig el a!.. 1993; Li et a].. 2004). The full length cDNA could be I. by careful slze fractionation of the double stranded cDNA fragments (Sambrook et

(89; Fernandez et al., 2002; LI el al., 2004). The dnp-column provided m the ZAP

cDNA library construction lut (Srrurag.fnr, CA, USA) was found to be very efticlent

k fractlonation and isolation of E gronulosur full length cDNA fragments (Sambrook

1989; Li et al., 2004).

( in the present study, in order to obtain good qual~ty cDNA, a drlp column was used

btionate the cDNA afler the addit~on of synthetic EcoRl adapters (5'-OH-

CGGCACGAGG-3' (14-mer), 3'-GCCGTGCTCCp-5' (10-mer)). Stringent protocol

.tons were followed dunng the slze fract~onation of the cDNA fragments. Thc cDNA

fragments w~th slzes greater than 400 bases (Fractions 5, 6 . 718) (Fig 4-2) were purified and

used for libmtion mto the ZAP Express phage vector. The average fragment slzr of the

pooled size fract~onated cDNA was found to be about 1.0 kb. The s~ze-fractionated E.

gronulosur cDNA was then precipitated and ligated to the ZAP Express vector.

Approximately 100 ng of size fractionated cDNA was ligated Into 1 pg of ZAP Express

phage vector.

Tlie ZAP Express vector, used In the present study, allowed both eukaryotic and prokaryohc

cxpresslon, whlle also increasing both cloning capaclty and the number of unlque lambda

clorung sites (Alting-Mees et al.. 1992). The ZAP Express vector conslsled of 12 unlque

cloning sites including Aprr 1. BumH I, EcoR I, Hind 111, Kpn I. Nut I, Suc I . Sol I. Smu I .

.(ye 1, Xba I, and Xho 1. The ZAP Express vector was found to accommodate the cDNA

Inserts up to 12 kb In length, indicat~ng that the resulted E grunulo.~u\ cDNA librdry

contained full length cDNA frabments (Short el al., 1988; Alting-Mees el al., 1992).

Moreover, the cDNA inserts cloned into the ZAP Express vector could be exclsed out of the

phage in the form of the kanamycin-resistant pBKCMV phagemld vector (Short el al..

19.38, Alting-Mees et al., 1992).

The E granulosur ZAP Express cDNA library was packaged m a highsfficlency system

Such as Gigapack 111 Gold packagmg extract (Kretz et al., 1994). The Gigapack IU Gold

Packaging extract with restrict~on minus (HsdR- McrA- McrBC McrF Mri) was also

confinned to package the recombinant lambda phages with high efficiency (Kretz et al..

1994). The high efficiency packaging of the recombinant lambda phages in turn Increases

the s l u of cDNA libranes (Kretz el al.. 1994) and produces full lenyh and good quality

cDNA libranes.

The packaged E. granulosus ZAP Express cDNA Ilbrary , in the present study .was plated

on the E colr cell line XLI-Blue MRF' s t m n (McrA McrB straln) (Stratagene, CA. LISA).

Smce. tbe most E. coli strains could dlgest the cDNA fragments contaimng 5'-methyl dCTP,

11 IS important to plate the packaged E &~unulosus ZAP Express cDNA library on McrA

McrB strain (Jerpseth el al., 1992; Altlny-Mees el al., 1992). In slm~lar studies reported

carher, the hemimethylated cDNA Introduced into a McrA' McrB' stratn was found lo be

drgested by mcrA and mcrB restriction systems. AAer passing the cDNA library through

XLI-Blue MRF' cells, the DNA was found to be no longer hemimethylated and could be

grown on McrA' McrB* strains (XL1-Blue straln) (Jerpseth et al., 1992; Altlny-Mees ct al..

1992).

The resulted unamplltied E granulosu.\ cDNA library was tllrated in the present study The

titre of the unampllfied E g-runulous cDNA library was estimated as lO\Iaquc form~ng

units (pfu) per 1 ml of packaged extract About 95% of the phages in the cDNA library were

found to be recombinilnts phages. The plaques were observed aRer 6-8 hours of ~nooulat~on.

although color detection rcqulred overnight rncubatlon. The backb~ouound plaques were

nppeared to be blue, wblle recomblnant plaques were whlte (clear). The recomblnant flakes

were found 50-60 folds above the background.

lmmunoscreening of E. granulosur Z A P Express cDNA library

The successful and efficient immunoscreening of the cDNA library is also very Important to

ldentify the most sensitive and specific recombinant antlgen produc~ng clones (Gasscr et al..

1990; Frosch et al.. 1991; Helbig et al., 1993; Li el al., 2004) The efficient

lnununoscrerning of the cDNA library malnly depend on the quality and antlbody tlter of

the primary anllhody or sera used to detect the praleln of interest (Gasser el a1 . 1990;

Frosch et al., 1991; Helblg et al.. 1993: LI et al.. 2004).

The immunoblotting or immunodot assay has been evaluated and found to be simple,

sensitive and to be most efficient method for lmmunoscreenlny of E gronulosus cDNA

libraries (Gasser n al., 1996; Frosch et al., 1991; Helbig el al., 1993; F e m u a and Zaha,

1994; F d e z h d., 2002; Li ei a)., 2004). Furthermore, immunoblotting directly shows

the reactivity of target protein with a positive serum sample (Siracusano el al.. 1991;

loppolo et al.. 1996; Onona et al.. 2000). Hence, in the present study, both plaque

mun no assay for immunoscnol~ng of the E. coli expression E. granulosur cDNA library

and colony immunoassay for immunological analysis of E coli expresslon GST-fislon

proteins were performed by immunoblotting or ~mmunodot assay due to its s~mpl~c~ ty and

sensitivity.

In the present study, approximately 10b pfi (1.5 x 104pfd10 cm LB agar plate) of the ZAP

Express cDNA library were screened with each of the three sera pwls (HS. CW and CF).

The eff~ciency and appropriate pnmary antibody dilutlon of all three sera pools (HS. CW

and CF) used In the lmmunoscreenlng of E grunu1o.w cDNA library was detemlned by

dot blot assay The avidity of the second antibody conjugate for the pnmary antibody was

also checked by dot blot assay. The three sera pools (HS. CW and CF) used In the

immunoscreemng of E grunulusus cDNA library were tested to determtne the opt~mum

serum for screenlny the E. 6(ronulosus cDNA libnuy by ElTB uslng HCW and HCF

antlgens. All the three sen pools were ~nd~vtdually probed against HCW and HCF antlgens

~n EITB analysis.

In EITB analysis, all the three sera pools showed strong signals agatnst both HCW and HCF

antigens producing 8-12 reactive bands The EITB analysis also showed that almost all

HCW and HCF antlgens could be recognized by the three sera pools used In

~mmunoscreening. These results were also found to be sunllar to the prevlous studles (LI et

al., 2004).

The polyclonal antibody preparations ofien contaln antibodies that are reactlve with E ioli

and phage proleins (Hrmmmgs and McManus, 1989: Muller et al.. 1989; Gasser et ai..

1940: Frosch et al., 1991; Facon et a]., 1991; Helb~g et al., 1993). These contaminat~ng

antibodies were found to increase background and create false poslttve reactions thereby

affecting the sens~tiv~ty and rel~ab~lity of the immunoscreemng assay (Hemmmgs and

McManus, 1989; Muller et al., 1989; Gasser et ai., 1990; Frosch et al., 1991; Facon el al.,

1991; Helbig et al., 1993). In contrast, m the present study. the immunoscreening of cDNA

library using three sera pools gave relatively strong posit~ve signals, and low background

rC.<\:tlvity of the ant~bodies. Subsequently all the sera pools were also treated wlth E cull-

phage lysate to remove the cross-reacting antibodies with E. coli phage antigens.

In the present study, immunoscreenlng of lo6 pfu of the ZAP Express cDNA l i b w

Produced nine pufative positive clones (designated as HSP2. HSP4, HSP9. CWP4. CWPJ,

cWP9, CFP2. CFP3, CFP7 respectively). The cDNA lnseRs of the nlne positive plaques

were subcloned into the pGEX-3X vector and expressed as GST fusion proteins to ident~fy

the potential serodiagnostlc value of thew expression products. All nine clones produced

GST-fusion protems in E coli host cells.

~n.lysls of the E. grannlosus GST-fusloo protelns by EITB and ELISA

The GST-fusion proteins, produced by mne clones, were tested for their ~mmunoreacnvity

by ElTB using sera collected from surgically confirmed cases of CE. The four GST-fusion

protens (HSP2, HSP4. CWP5 and CFP7) were found to be expressed In larger quantities

than the other five (HSP9. CWP4. CWP9, CFP2 and CFP3). The four GST-fusion proteins

(HSP2, HSP4, CWP5 and CFP7) that were found to be expressed m larger quantities gave

mong positive signals wrth all 10 sera collected from surglcally confirmed cases uf CE The

other GST-fusion protelns showed weak signals wlth the surglcally confirmed patient sera

by EITB. Thus the clones HSP2. HSP4. CWPS and CFP7 that produced recomblnant E

yranulosus anllgens were selected to test thelr speclficlty to use as dlabwostlc anttgens for

the d~agnosis of CE.

The GST used to produce E granulusu.~ fuslon proteins was found lo be derived fion~

Schrsrosomajaponlcunt. However, the recombinant GST fuslon proteins d ~ d not show any

cross reaction \nth the three sera from patients with sch~stosom~asis (Frosch el al.. 1993; LI

el al., 2003). The reasons for t h s lack of react~vity might be due to the fact that the E colt

expressed GST did not contain conformatlonal epltopes required for recognlt~on by the

rch~stosomiasis infect~on sera or the antigemclty of GST was too weak to be reco&nlzed

(Frosch ef al., 1993; Li et al., 2003; LI el al., 2004). However, in the present study, all serum

~pecimens were also treated wlth recomblnant E, o l ~ cell lysate containing GST alone.

These sera also did not show sign~ficant difference m the background nose.

In the present study, the GST moiety from three E granulosus recomblnant protelns (Eg-

CW24, Eg-CW12 and Rec Eg-A$8/2) was cleaved using factor X (Phurnwcro

Biosysrems, USA) to eliminate the posslble back~ound reactlvlly with tllc ant]-GST

ant~body, whlch may be present In human sera. The three E grunulo.susur recomblnant

pr~.:eins, Eg-rCW24, Eg-rCWl2 and Rec Eg-AgE48I2, showed a Mr of about 24 kDa, 12

kDa and 8 kDa respectively, after cleavmg the GST moiety (Fig 4-7 and 4-8). The E. coli

expression GST protein showed a Mr of about 25-27 m a . The predicted Mr of the GST

expression protein (25-27 kDa) was ach~eved afler cleaing from the E. flanulosur

recombinad proteins. The Mr of GST expression protein was also found to be about 27 kDa

~revious studies (Gasser et al., 1990; Facon et al.. 1991; Fmsch et al., 1993; Li et al.,

2003: LI el al., 2004). The E grnnulosur recombinant pmteins did not show any additional

break do^ during the treatment with factor X. These results indicate that the E. granuloslcr

recombinant proteins obtained in the present study were stable. These results were s~milar to

the previous studies of unmunoscreenlng of cDNA libraries wtth lnfectlon SeNm and

h ~ n m m u n e serum (Gasser el al.. 1990; Facon et al.. 1991; Frosch el al.. 1993: LI et a1,

2003, Li et a].. 2004).

In the present study. the three E. grunulosus recomblnant antlgens (Eg-rCW24. Eg-tiW12

and Kec Eg-AgB812) were evaluated as diagnostic mt~gens by BLlSA and EITB uslng a

panel of sera collected from surgically confirmed cases of CE, cases w~th other parasit~c

dtseases (neurocysticercosis, filariasls, schistosomias~s, malaria, amoebiasis). and healthy

~ndivlduals. In the ~mmunological assay, three recombinant antigens showed 100%

sensltlwty and specificity by both ELlSA and EITB. The affinity punfied E. granubruv

recombinant protelns w~thout GST molety also did not show any cross reactions wlth sera

troln patients w~th schistosom~as~s. Moreover, the three E granulosus recombman1

antlgens produced hyperimmune serum when rabblts were Injected ~ntramuscularly w~tli

those of purified recomblnant antigens.

Subsequently, in the present study, the anti-Eg-tiWI2, ant,-Eg-tiW24 and Rec Eg-AgBXR

n~otioapec~fjc antibodlcs were purlfied by affin~ty chromatography The ElTB using ant,-Eg-

C W l 2 nionospec~fic SeNm showed strong reactlona with 8-12kDa and a weak reactlon

ivlth 60 kDa and 24 kDa protein fract~ons of the HCW and HCF natlve antlgens

rcspect~vely. The ElTB us~ng anti-Eg-tiW24 and ant,-Kec Eg-AgB812 monospecific acrum

illowed strong react~ons with fbur natrve antigen bands rnclud~ng 60 kDs. 24 m a . 16 kDa

and 8-12 kDa. These results indlcate that the epltopes coded for by Eg-tiW24 and Kec Eg-

hgB812 are present on all proposed sub-units of the antlgen B Hence, the both E

gmnulosus recomb~nant antigens ~dentlfied In the present study are also appeared to be

antlgen B fractions. Them results were similar to the previous stud~es of ~mmunoscreenlng

of cDNA libraries w~th infection serum and hyperimmune serum (Gasser el al . I99Q Facon

et al., 1991; Frosch et al., 1993; Li el al., 2003: LI ct al., 2004).

Tl.c most frequent cross-react~ons In immunod~aposis of CE were round to be due to E

mulrrbcular~s and T. solrum (Schantz et al., 1980). The E grunulu~us AgS showed frequent

cross-reactions with sera fmm pat~mts infected wlth E mulriluculun~. E vrjgeb, T ro1zum

and other parasites (Leggatt et al., 1992). Where as, the antigen B was found to be more

specific (lm et &., 1999; Mamuti el al.. 2004). although cross reactlon occurs wlth

antibodies in sera Iium patients infected with E. mulfilocularis and E vogeli (Shepherd and

McManus. 1987). and T solium (kggatt el al.. 1992). The ELISA using purified E

F~nulosw antigen showed cross reactions with the sera collected from T soginoru,

Enrerobiw vermicuhris and Fusciolo heporrco (Guisantes ct al., 1981). The seFodiagnostlc

tests uslng E. pnu losur antigens also showed cross tractions with sera from patlents with

AE, schistosomiasis and vlclUn0~iS (Sjolander et al.. 1989; Force el a].. 1992). sera from

unchocerciasis, schistosomiasis, and ascariasrs (Sjolander et al.. 1989) and sera from

~~s t~ce rcos i s (Mad&son et al.. 1989; Wen and Craig, 1994).

In the present study, the affinity purified E gronulosus recombinant protelns were tested for

the~r specific~ly using a panel of sera from cases with neumcysticercons, filanasls.

\chistosom~asis, malaria, amoebias~s and sera from healthy ~ndrv~duals. Both EITB and

tLISA using E grunulosus recombinant antlgens. Eg-rCW12. Eg-rCW24 and Rec Eg-

AgB812 did not show any cross reactions wlth sera from other disease controls and llealtlly

lndlv~duals In the preliminary evaluat~on. Therefore, these recomb~nant antigens were

selected as the diagnostm anllgens of choice for the further evaluat~on and to ~dn i t~ fy the~r

dlagnosts efficacy In developing serodiagnostic assays for CE. Though these antlgens were

not tested for the cross-reactivity with that of sera from patients w~th AE and PE, either of

these antlgens alone could be employed as diagnost~c antigen at leas1 in countries where CE

alone is prevalent.

Cystic eohinococcosis WE), caused by E. gmnulosvr. is a disease of high prevalence in

developing countries including India. Though the actual country wide prevalence and

~ncideiux of h u m CE in India is not yet known, the increasing rrpons and information,

obtainad mostly from hospital-based studies, indicates the disease is still very active or is

r e d and probable endemicity of CE in India, which h the subject of concern.

Therefore, the dmlopment of immunodiagnosis tosts that dsteots species-specific

antibodies or antigonr in senun or 0th- body nuids is a nocepsity.

Hence, in the first phase of the present study, ZAP Expross oDNA library was consmcted

from E. granulosus metacestode germinal mombra00 mRNA to identify and evaluate

sensitive and specific antigen for the diagnosis of CE.

The E. coli expmssion cDNA library was immwoscmned using a pool of sera collected

from patients with surgically confirmed CE and rabbit polyclonal hyperimmune serum to

~dentify and isolate inmunodominant diagnostic relevant recombinant antigen producing

clones. lmmunoscreening of lo6 ( I S x 10'110 cm plate) pfu of the ZAP Express cDNA

l~brary produced nine posltive clones (HSPZ. HSP4, HSP9. CWP4, C W 5 . CWP9, CFPZ.

CFP3, CFP7) with high intensity.

Having shown the potential scxodiagnostic value of thcii uprsssioe prodyts, the cDNA

mzrts of HSP2. HSP4, HSPP, CWP4, C W 5 , CWP9, CFPZ, CFP3 and CFP7 plaques

were sub-cloned into the pGEX-3X vector and oxpmsaod ar GST firsion proteins. All nine

clones produced GST-fusion proteins in E. coli cells which were well detectable by

s W g the gel with Coomassie blue R-250.

All the nine GST-fusion proteins were tested for their immunoreactivity towards human

anti-E. granulosur antibodies by EITB. The four GST-fusion proteins which were found

10 be expressed in larger quantities (HSPZ, HSP4, CWPS and CFP7) gave strong positlve

~ ~ m l s with all 10 sera collected from surgically confmed patients w~th CE. Other GST-

fusion proteins (HSP9. CWP4, C W 9 , CFPZ and CFP3) though recognized by anti-E.

sranulosvr antibodies, showed weak sigoals with the surgically conf~rmed patient sera in

WWtemblot analysis.

~n the pnrssnt study, therefore, the clones HSPZ. HSP4. CWPS and CPP7 producmg

rccombii E grunularus antlgens were selected to test thm specificity to use as

magnostrc antigens for the dagnosls of CE The four clones, HSP2. HSP4. CWPS and

CF'P7 p d N d expnsslon pmtems with a MI of about 40 kDa. 24 kDa, 12 kDa and 10

rcspodivdy, afta clcav~ng the GST morety (Mr of about 27 ma).

In the prssmt study, E grnnulosus recombinant anbgen B812 was also produced by

clomg and expression of correspanbg genomlc DNA hgment The E granulosm

recombmant antigen BE12 showed a Mr of about 8 kDa, after cleaving the GST molety

For fwthcr immunological evaluation of these E. granularus wombinant antigens, a

of sera collected from LO surgicaly confirmed patients with CE, 10 patients with

NCC, 3 patients each with filariasis, schistosom~asis, malaria, amoebiasis, and 10 healthy

individuals, were tested by both EITB and EL1SA.Both ElTB and ELISA using GST-

fusion proteins produced by HSP4, CWP5 clones and Eg-AgB812 (designated as Eg-

1CW24, Eg-rCW12 and Rec Eg-AgB81Z respectively) showed 100 % sensitivity and

specificity in initial screening.

The recombinant proteins, Eg-rCW24. Eg-CW12 and Rec Eg-AgB812 were further

evaluated fpr their potential use as immunodiagnostic antigens by ELISA and Dot-ELISA

for diagnosis of CE.