Production of Egg Yolk Antibody (IgY) against Recombinant ... 1029.pdf · Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Pro-tein. Acta Scientiae

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S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Pro-tein. Acta Scientiae Veterinariae. 40(2): 1029. Acta Scientiae Veterinariae, 2012. 40(2): 1029.

ORIGINAL ARTICLEPub. 1029

ISSN 1679-9216 (Online)

Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein

Shuizhong Han, Xiaoying Zhang & Jinzi Zhao

ABSTRACT

Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hem-orrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The isolate was defined as CPV-2a (JN403045) subtype by sequencing. The VP2 ORF was inserted into pET-32a by T4 ligase and introduced into the E. coli Bal21. VP2 protein was produced by the induction of the E. coli Bal21 containing pET-32a-VP2 with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant VP2 protein (rVP2) fused with His-tag was analyzed by SDS-PAGE and detected by western blotting using anti-His monoclonal antibody. The rVP2 was purified by Ni+-affinity purification chromatog-raphy under denature condition and dialyzed against PBS. The concentration of the rVP2 was determined by Bradford method. After immunizing the chickens with rVP2, anti-VP2 IgY was isolated by PEG 6000 precipitation and analyzed by SDS-PAGE. The activity and specificity of the IgY antibody were analyzed by indirect ELISA and western blotting. SDS-PAGE analysis showed that the rVP2 fused with His-tag had a molecular of 85 kDa, accompanied with the low mo-lecular fractions around 50 kDa and 40 kDa. The rVP2 could be recognized be the anti-His monoclonal antibody. The IgY antibody isolated by PEG 6000 method and analyzed by SDS-PAGE showed the IgY mainly contained two parts, 23 kDa and 67 kDa, which corresponded to light and heavy chain, respectively. In additional, some lower bands around 40 kDa were presented on the gel. The anti-VP2 IgY reached to 1:40960 after the fourth immunization. The anti-VP2-IgY could recognize the VP2 specially in Western blotting, while no reaction was seen with the low molecular.Discussion: The emergence of the low molecular proteins in 50 kDa and 40 kDa in the pellets of the bacterial lysates may be due to the degradation of the rVP2 by endogenous proteases in E. coli. The fact that IgY could recognize the entire VP2 fraction suggests the lost of the antigenic sites of the low molecular proteins.

Keywords: canine parvovirus (CPV), VP2 protein, egg yolk antibody (IgY).

Received: October 2011 www.ufrgs.br/actavet Accepted: December 2011

College of Veterinary Medicine, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China. CORRESPONDENCE: X.Y Zhang [ [email protected] - Fax & Tel: 86-29-8709 1239].

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S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Pro-tein. Acta Scientiae Veterinariae. 40(2): 1029.

INTRODUCTION

Canine parvovirus (CPV) disease is a serious infectious disease among canines featured by myo-carditis and hemorrhagic enteritis [11]. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the proteins in the capsid are VP2 protein [13]. A series of B cell epitopes have been found on the VP2 protein and could be used as a vaccine for the prevention of CPV [3, 9].

After first isolation of CPV-2 in the US in 1978 [2], its variants, including CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide [4, 7]. In China, most isolates were identified as CPV-2a [18]. Although the antibody induced by the CPV-2/CPV-2b vaccine may offer cer-tain protection effect, it may not be sufficient [5, 12, 14]. The morbidity of CPV disease is considerable high among canines and has leaded to great economic loss in China according to our previous survey [8]. Tradi-tionally, monoclonal antibody has been used for CPV treatment, here we present a relatively inexpensive and simple method to develop high qualified antibody based on chicken egg yolk antibody (IgY) technology.

IgY antibody is recognized as an excellent source of polyclonal antibodies with relatively sim-ple, noninvasive method and large-scale production advantage compared with the mammalian antibodies [19]. IgY antibody was used for the passive protection of the pathogen infections, especially the intestinal infections, such as rotavirus [15], Salmonella [10], and exhibited good efficiencies.

The present study was conducted to characteri-ze the specific IgY antibody produced by immunizing the hens with CPV-VP2 expressed in the E. coli.

MATERIALS AND METHODS

Materials

CPV strain was isolated from puppies that had a history of vomiting and diarrhea in the Xi’an Pet Hospital. The animals were confirmed with CPV infections by a colloidal gold test kit1. Fecal samples were obtained by rectal swabs and homogenized (10%, w/v) in phosphate buffered saline (PBS, pH 7.2) containing streptomycin (100 μg/mL) and penicillin (100,000 IU/L).

DNA cloning of the VP2 gene

Full length of VP2 ORF (1~1755nt) was ampli-fied by PCR. The supernatant was boiled as template. The primers (F 5’-ATGGATCCCCAATGAGTGATG-GAGCAG-3’; R 5’-CCGCTCGAGATATAATTTTC-TAGGTGCTAGTTG-3’) including a BamH I and Xho I restriction site represented underline respectively, were designed using primer 5.0 based on the CPV reference strain (GenBank Accession No.M19296) and custom-synthesized by the Genscript2. The template was denatured at 94oCfor 5 min, followed by 35 cycles of 94oC for 30 s, 55oC for 30 s and 72oC for 1 min and final extension at 72oC for 10 min.

The amplified fragments were purified and ligated into pMD18-T plasmid vector3 by T-A clone. The resulted plasmid was designated pMD18-T-VP2 and introduced into E. coli DH5α. The positive colony was further confirmed by restriction enzyme digestion and sequencing.

Expression of the His-tagged recombinant CPV-VP2 protein

The plasmids pMD18-T-VP2 and pET-32a were digested with BamH I and Xho I, respectively, eluted by gel purification, and then ligated with T4 DNA ligase to yield pET-32a-VP2 recombinant plas-mid. The plasmid was transferred into E. coli BL21 (DE3), inoculated into LB agar medium containing ampicillin (100 μg/mL) and grown overnight at 37oC. A single colony was obtained and further cultured at 37oC in LB liquid medium, supplemented with ampicillin (100 μg/mL) until the OD

630 reached 0.8,

then induced by adding IPTG at a final concentration of 1.0 mmol/L for 5 h. After harvesting the cells by centrifugation (7,000 g, 20 min, 4oC), the pellets were lysed by sonication (10 s each, 10 cycles with 5 s in-terval) in iced water bath. After centrifugation (16,000 g, 20 min), the supernatant and pellet of the lysate were collected separately. Both of the supernatant and pellet were analyzed on 12% SDS-PAGE followed by Western blotting to detect 6×His-tagged recombinant proteins. Diluted mouse anti-His monoclonal antibody4 was used as primary antibody (1:1000, v/v), and HRP conjugated rabbit anti-mouse antibody4 was used as secondary antibody (1:5,000 v/v). The reactivity was detected using a chemiluminescent substrate with luminol and H

2O

25.

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Purification of the His-tagged CPV-VP2 fusion proteins

After passing through 0.22 μm filter, the VP2 protein was purified from the dissolved pellet using His Trap affinity column6 under denaturing conditions (8 mol/L Urea) as described in the manual instruction. After washing the column with the binding buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, 30 mmol/L imi-dazole, pH 7.4), the VP2 protein was eluted using elu-tion buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, 500 mmol/L Imidazole, pH 7.4). 12% SDS-PAGE was used to analyze the relative purification efficiency of VP2 protein followed by Commassie blue staining. The concentration of the VP2 protein was determined by Bradford method after dialyzing against PBS (100 mmol/L, pH 7.4) containing different amounts of urea.

Immunization

Twenty-week-old Hy-line Brown hens were kept in individual cages with food and water ad libitum. They were immunized intramuscularly with VP2 pro-tein mixed with Freund’s adjuvant7 at different sites of the breast. 1000 μg of freeze-drying VP2 protein were suspended in 500 μL of PBS and emulsified with an equal volume of complete Freund’s adjuvant for the first immunization. Three booster immunizations were followed up using incomplete Freund’s adjuvant with two weeks interval. Eggs were collected daily, marked and stored at 4oC°until they were further processed.

IgY antibody purification

IgY antibody was extracted and purified from eggs using PEG precipitation procedure as described before [19]. Briefly, the yolk of a single egg was sepa-rated from the white by an egg separator and diluted 1:2 with sterile PBS with vortex. The PEG 6000 (3.5%, w/v) was added to precipitate abundance of lipids and lipoproteins. After gentle shaking at room temperature for 10 min followed by centrifugation (16,000 g, 20 min, 4oC), the supernatant was harvested and filtered. PEG 6000 was added to a final concentration of 8% (w/v), the mixture was centrifuged again under the above conditions after gentle shaking at room tempe-rature for 10 min. The precipitation was dissolved in 10 mL PBS and 12% (w/v) PEG 6000 was added and centrifuged as described above. Finally, the pellets were dissolved in 1.2 mL PBS and dialyzed against bulks of PBS over night at 4oC. The purity of the isolated IgY antibody was determined by 12% SDS-

-PAGE under reducing conditions, and stained with Coomassie Brillant Blue.

Indirect Enzyme-linked immune sorbent assay (iELISA)

Specific activity of IgY antibody against VP2 protein was determined by iELISA. Briefly, a 96-well microtiter plate8 was coated with 100 μL of VP2 antigen (10 μg/mL) in sodium bicarbonate buffer (pH 9.6), and incubated at 4oC overnight. After washing three times with PBS containing 0.05% (v/v) Tween 20 (PBST), the nonspecific binding sites were blocked with 5% (w/v) nonfat milk powder in PBST for 4 h at 37oC. After three times washing, 100 μL of a serially diluted IgY antibody in PBST was added to the wells as the primary antibody and then incubated for 2 h at 37oC. The plate was washed again and incubated with 100 μL of HRP labeled goat-anti-chicken IgG (1:6000) as the secondary antibody for 1 h at 37oC. Finally, the activity was detected with 100 μL of 3,3’,5,5’-tetra-methylbenzidine (TMB, 1%, w/v) as substrate for 15 min at room temperature, The reaction was stopped by adding 50 μL of 2 mol/L H

2SO

4 to the wells. Optical

density (OD) at 450nm

was read on microtiter plate reader9. While OD

Sample/OD

Negative≥> 2.1, the maximum

dilution multiple of the sample was determined as IgY antibody titer.

Western blotting

The purified VP2 protein was separated using 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer western blotting apparatus10. The unreacted sites were blocked with 5% (w/v) nonfat milk power in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 (v/v), (pH 8.0) buffer for 1 h and then incubated with anti-VP2 IgY antibody (1:1000) for 1 h at room temperature. After washing three times with TBST, HRP-conjugated goat-anti- chicken antibody11 diluted in TBST (1:5000) was added and incubated for 1 h at room temperature. The membrane was washed three times with TBST and the specific binding of IgY an-tibody to the VP2 protein was detected as described above.

RESULTS

Expression of the recombinant VP2 protein

The coding region (1-1755 nt) of the VP2 protein was amplified by PCR and inserted into the

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cloning vector to construct pMD18-T-VP2. After res-triction enzyme digestion analysis and sequencing, the clinical sample was defined as CPV-2a type (GenBank Accession No. JN403045). After 5 h induction with IPTG, a prominent band with the expected molecu-lar weight of 85 kDa was observed in the insoluble fraction of the bacterial containing the pET-32a-VP2 plasmid, while no proteins bands at the approximate size were observed in both none-IPTG induction and IPTG-treated culture containing pET-32a only (Figure 1). It revealed that the recombinant proteins were expressed in inclusion body format. In addition to the expected molecular weight proteins, two lower bands, around 50 kDa and 40 kDa were expressed in the insoluble fraction.

Western blotting showed that the bands around 85 kDa and 50 kDa were recognized by mouse-anti-His monoclonal antibody.

Purification of the His-tagged CPV-VP2 fusion protein

To purify the CPV-VP2 protein, the crude ex-tract was filtered and loaded onto the Ni+- resin purify column. The recombinant proteins eluted from the column were analyzed by 12% SDS-PAGE. It showed

Figure 1. SDS-PAGE analysis of the soluble and insoluble frac-tions from lysates for recombinant CPV-VP2 protein expression.Lane 1: pET-32a(+) without induction with IPTG; Lane 2: pET-32a(+) induction with IPTG; Lane 3: solubility of pET-32a(+) without induction with IPTG; Lane 4: solubility of pET-32a-VP2 without induction with IPTG; Lane 5: solubility of pET-32a in-duction with IPTG; Lane 6: solubility of pET-32a-VP2 induction with IPTG; Lane 7: insolubility of pET-32a without induction with IPTG; Lane 8: insolubility of pET-32a-VP2 without induction with IPTG; Lane 9: insolubility of pET-32a induction with IPTG; Lane 10: insolubility of pET-32a-VP2 induction with IPTG; M: Marker.

Figure 2. Western blotting analysis of the re-combinant proteins using anti-His monoclonal antibody. Lane 1: Marker; Lane 2: insolubility of pET-32a-VP2 induction with IPTG.

that two bands, around 85 kDa and 50 kDa were eluted from the column.

Purification of IgY antibody

IgY antibody was isolated and purified from the egg yolk by PEG 6000 precipitation method. The effectively was estimated by SDS-PAGE under redu-cing condition (Figure 3). It showed that IgY antibody contained two major proteins 23 kDa and 67 kDa that correspond to light and heavy chain, respectively. In additional, some lower bands around 40 kDa were presented on the gel.

Specific activity of IgY antibody against VP2 protein determined by ELISA

The levels of specific activities of anti-VP2 IgY antibody were determined by iELISA. The IgY antibody titer increased from initial immunization

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and arrived the peak (1:40960) after the fourth im-munization.

Detection of VP2 antigen with anti-VP2 IgY

Binding activity of the IgY antibody to the VP2 protein was further determined by western blotting. As shown in Figure 5, isolated IgY antibody could bind the rVP2 protein (85 kDa) at a dilution of 1:1000, while could not recognize the fraction around 50 kDa.

DISCUSSION

The CPV isolated from Shaanxi was defined as New-CPV-2a subtype by sequencing, which posed an amino mutation at the position 297 (Ser-Ala) of the VP2 protein. While using the prokaryotic expression vector pET-32a, the histidine tags at both amino and carbon terminal of the VP2 protein will be added, which would increase its molecular mass to 85 kDa. SDS-PAGE analysis of the soluble and insoluble of the lysate, VP2 protein was found mainly as insoluble inclusion body (Figure 1, lane 10). In addition to the expect VP2 protein, two other bands, around 50 kDa and 40 kDa existed in the insoluble fractions (Figure 1, lane 10). Parvovirus capsids had a tendency to be cleaved in vivo infection as described in the Aleutian disease virus (ADV) infection [1]. The nature cle-avage of VP2 to VP3 was observed in the full CPV particles only [16]. Therefore, the emergence of the lower proteins may be not due to the degradation of

Figure 3. SDS-PAGE analysis of the recombinant proteins puri-fied using Ni+ affinity column. Lane 1: pET-32a-VP2 without induction with IPTG; Lane 2: pET-32a-VP2 induction with IPTG; Lane 3: solubility of pET-32a-VP2 induction with IPTG; Lane 4: insolubility of pET-32a-VP2 induction with IPTG; Lane 5: flow-through; Lane 6: elutes in 20 mmol/L imidazole; Lane 7: elutes in 500 mmol/L imidazole; M: marker.

Figure 4. SDS-PAGE analysis of the iso-lated IgY antibody purified by PEG 6000.

the VP2 protein. The second possibility may be the rare codons in the amino terminal of VP2 ORF, which may be likely to hamper the expression efficiency in E. coli. However, elimination of the rare codons at both the amino and carbon terminal would not avoid the emergence of the lower molecular fractions [17]. The third possibility of the emergence of the lower bands may be the degradation of the high molecular weight proteins by endogenous proteases in E. coli. Over expression of proteins with high molecular weight in a high rate may have an effect on the protein folding. Improper folding of the proteins may be degraded by the endogenous proteases to conserve cellular resour-ces. Analysis of the recombinant proteins by western blotting showed that 87 kDa and 50 kDa proteins could be recognized by the anti-His monoclonal antibody,

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revealing that His-tag existed at the terminal of the proteins, while the 40 kDa one lost the tag and could not be recognized (Figure 2). Purification of the rVP2 protein from the insoluble fraction by Ni+ affinity co-

Figure 5. Development of IgY antibody titer against CPV-VP2 protein.

Figure 6. Western blotting analysis of the binding activity of the anti-VP2 IgY. Lane 1: Marker; Lane 2: insolubility of pET-32a-VP2 induction with IPTG.

lumn, two proteins including the 85 kDa and 50 kDa were purified together, which was in accordance with the result analyzed by the western blotting (Figure 3).

The chickens were first immunized with rVP2 protein in the dosage of 300 μg per immunization, yet it produced a poor antibody titers response in iELISA analysis (data not shown). Thus, the antigen dosage of 1mg rVP2 protein per injection was chosen as des-cribed above. The antibody titers reached the peak of 1:40560 after the fourth immunization (Figure 5). This revealed that dose of the antigen had an effect on the production of antibody in chicken. It had been reported that antigen among 10~1000 μg may more efficiently induce the antibody response [6].

The VP2 inclusion body washed by 0.05% Tween-20 was applied on the SDS-PAGE and was analyzed by western blotting using the purified IgY antibody as the primary antibody. It revealed that the 85 kDa band was recognized by the anti-VP2-IgY anti-body, however, the 50 kDa was not (Figure 6). Most of the antigen sites were presented in the amino terminal region of VP2 [9]. Anti-VP2-IgY antibody may not recognize the degraded production if they have lost the required N-terminal sequences from VP2 protein.

The entire virus particles may often induce to considerable protection efficiency in different IgY antibody studies [15]. However, choosing target virus protein as antigen could lead to better effect on pathogen detection. Comparison of the sequences of VP2 ORF revealed that CPV isolated in China had

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high identities (98.3%) [18]. In this study, Anti-VP2 IgY antibody was produced by immunized the chicken with the rVP2 protein, which had never been reported before according to our survey. Compared the anti-body production between chicken and mammalian, an extraordinary amount of antibody (approximately 17-35 g of total IgY/chicken/year) can be produced from only one hen and almost 10% is antigen-specific [19]. Anti-VP2 IgY antibody may be applied into the immunotherapy and immunoprophylaxis for the CPV infection. Furthermore, IgY antibody doesn’t react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays [19]. Therefore, the anti-VP2-IgY could be developed into the diagnosis and treatment reagents for CPV infection, but the efficiency should be investigated further.

SOURCES AND MANUFACTURERS1Bioland, Chungnam, Korean.

2Genscript, Nanjing, China. 3TakaRa, Dalian, China.4ZSGB-BIO, Beijing, China.5Thermo scientific, Waltham, Massachusetts, USA.6GE Healthcare, Wauwatosa, Wisconsin, USA.7Sigma-Aldrich Co., St. Louis, Missouri, USA.8NUNC, Roskilde, Denmark.9Biotek, Winooski, Vermont, USA.10Junyi Company, Beijing, China.11Abcam, Cambridge, Massachusetts, USA.

Acknowledgements. This work was supported by Ministry of Education and State Administration of Foreign Experts Affairs “overseas teacher” project (No. MS2011XBNL057) and a grant (No. 01140407) for returned oversea Chinese Scholars of Northwest A & F University, China.

Ethical approval. All of the experiments were performed ac-cording to Experimental Animal Management Law of China and approved by Animal Ethics Committee of Northwest Sci-Tech University of Agriculture and Forestry.

Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Production of Egg Yolk Antibody (IgY) against Recombinant ... 1029.pdf · Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Pro-tein. Acta Scientiae