Cloning and Sequence Analysis of Adhesion Gene of J · Life Science Journal, 3 (4), 2006, Huang, et al ,Cloning and Sequence Analysis of Adhesion Gene hpaA ligase, DNA gel extraction

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JCloning and Sequence Analysis of Adhesion Gene hpaA

of Helicobacter pylori~

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Xueyong HuangI.2, Yi Ren3, Guangcai DuanI.2, Qingtang Fan2, Yuanlin XiI,

Zhigang HuangI.2, Chunhua Songi

1. Department of Epidemiology, College of Public Health, Zhengzhou University,

Zhengzhou, Henan 450052 , China

2. Henan Key Laboratory of Molecular Medicine, Zhengzhou, Henan 450052, China

3. Department of Labor and Environmental Health, College of Public Health,

Zhengzhou University, Zhengzhou, Henan 450052 , China

Abstract: Objective. To clone the adhesion gene hpaA of Helicobacter pylori strain rvIEL-Hp27 isolated from a pa-tient in Zhengzhou City, and analyze the hpaA gene nucleotide and putative amino acid sequences. Methods. hpaAgene of the Helicobacter pylori rvIEL-Hp27 was amplified by PCR. After purified, the target fragment was clonedinto plasmid pBluescriptb II and subject to nucleotide sequenced. The homologies of the nucleotide and putativeamino acid sequences of hpaA were respectively analyzed. Results. hpaA gene of 783 bp, encoding the polypeptidesof 260 amino acids, was obtained from the Helicobacter pylori strain rvIEL-HP27 genomic DNA. The homologiesof the nucleotide and putative amino acid sequences compared with the published hpaA gene sequences were94. 76% - 97. 19% and 95. 38% - 98. 46 0/0, respectively. Conclusions. The recombined plasmid carring hpaA

gene has been successfully constructed, and sequence analysis indicates that hpaA is a highly cotL'ierved prokaryoticgene and might be a potential candidate for Helicobacter pylori vaccine development. [Life Science Journal. 2006;3(4):42-48J (ISSN: 1097-8135).

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Keywords: Helicobacter pylori; hpaA gene; cloning; sequence analysis

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Abbreviations: HpaA: Helicobacter pylori adhesion; MALT: mucosa associated lymphoid tissue

1 Introduction

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Helicobacter pylori is one of the commongram-negative bacteria causing chronic infection,which infects more than 50 % of the human popula-tion. Infection of the gastric mucosa with Heli-cobacter pylori results in a number of disease out-comes including gastritis, which precedes the de-velopment of peptic ulcer disease, gastric cancerand lymphomas of the mucosa associated lymphoidtissue ( MALT) [1,2J. AIthough significant progresshas been made in treating Helicobacter pylori in-fection with current triple or quadruple therapybased on antibiotics, given in conjunction with bis-muth compounds and proton pump inhibitor, thelimitations of pharmacological therapy such as sideeffects, poor compliance, high cost, and most im-portantly, rapid emergence of antibiotic resistancehave set the stage for the development of less costlyand more efficient means to prevent and controlHelicobacter pylori infections[3.4J. Immunizationagainst the bacterium represents a cost-effective

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strategy to prevent Helicobacter pylori infection,the selection of antigenic targets is critical in, thedesign of Helicobacter pylori vaccine[5J. Helicobac-ter pylori adhesion(HpaA) is a flagellar sheath pro-tein with approximately 29 kDa located in the bac-terialouter membrane[6J, So in this study, the re-combinant plasmid inserted with hpaA of Heli-cobacter pylori was constructed and the homologiesof the nucleotide and putative amino acid sequenceswere respectively analyzed, which will be helpfulfor determining whether the HpaA becomes one ofthe good candidates as an antigen in Helicobacterpylori vaccine.

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2 JMaterials and Methods

2. 1 MaterialsThe strain MEL-HP27 of Helicobacter pylori

and cloning pBluescriptb II were preserved by ourlaboratory, E. coli strains JM1O9 were purchasedfrom New England Biolabs (Beijing) LTD (BeijingChina). Pyrobest DNA polymerase, restriction en-donuclease enzymes (BamHI, Hind ill), T4 DNA

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ligase, DNA gel extraction kit and. 100 bp DNAmarker were provided by TaKaRa Company(Dalian, China).2.2 Bacterial culture and preparation of DNAtemplate

Helicobacter pylori MEL-HP27 strains weregrown on solid Columbia agar with 100 mIlLfrozen-melting sheep blood, 50 mllL fetal bovineserum, and antibiotic supplement (vancomycin 10mg/L, polymyxin B O.33 mg/L, amphotericin A 5mgIL, trimethoprim 5 mgIL) in a microaerophilicatmosphere for 3 days to 4 days at 37°C.

The Helicobacter pylori strains were harvestedand suspended in 1 ml sterile normal saline and pel-leted by centrifugation at 10,000 g for 5 minutes.The precipitate was resuspended in lysis buffer (10mM Tris-HCl, pH 8.0, 0.1 M EDTA, pH 8.0,0.5 % (w/v) SDS, 20 !ig/ml RNase), and then,protease K was added in to a final concentration of100 !ig/ml, the lysate was incubated in a waterbath at 42°C for 2 hours. The solution was cooledto room temperature, and mixed with an equal vol-ume of phenol equilibrated. The two phases wereseparated by centrifugation at 10,000 g for 10 min-utes at room temperature, and the aqueous phasewas extracted with phenol twice again. After-wards, O. 1 volume of 2.5 M ammonium acetateand 2 volume of ethanol were added to the aqueousphase. The precipitate was collected by centrifuga-tion at 10,000 g for 2 minutes, washed twice with70 % ethanol, and dissolved in an appropriate vol-ume of TE buffer (pH 8.0)[7,8]. The DNA con-centration was measured by ultraviolet spectropho-tometry.2.3 Synthetic primers and PeR

Oligonucleotide primers were designed to am-plify hpaA gene from Helicobacter pylori strainMEL-HP27 based on the published correspondinggenome sequence of 26695 and J99. The sequenceof sense primer with a restriction endonuclease siteof Bam HI was: 5'-CGGGATCCATGAAAGCAAAT AA TC-3'. The sequence of antisense primerwith a restriction endonuclease site of Hind ill was:5' -CGCAAGCTTTTATCGGTTTCT-3'. PCR wasperformed in a 50 !il reaction mixture in O. 6 mltube in an automatic thermal cycler. The PCRmixture contained5 !il of 10 X PCRbuffer, 2. 5 !ilof sample DNA, 4 !il of 2.5 mmol/L deoxynucleo-side triphoshpate, 2 !il of O. 25 !imollL oligonu-cleotide primers, O. 5 !il Pyrbest DNA polymerase(1. 25 U), 34 !il of MilliQ H2O. The parametersfor PCR were as follows: 95°C for 5 minutes, 1cycle; 94°C for 60 seconds, 45°C for 50 seconds,72 °C for 50 seconds, 30 cycles; 72 °C for 10 min-utes, 1 cycle. The amplified products (3 !il) were

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observed by electrophoresis on 10 g/L agarose gelcontaining O. 1 !ig of ethidium bromide per ml inTBE buffer. The PCR product was visualized un-der UV light and photographed.2.4 Construction of recombinant plasmids

PCR products were digested by restriction en-donucleases Barn HI and Hind ill, meanwhilepBluescriptb II plasmid was digested by Bam HIand Hind ill, too. The target fragments of hpaAand pBluescriptb II were recovered by DNA gel ex-traction kit, and then these two fragments were lig-ated by using T4 DNA ligase at a molar ratio of 6:1 at 16°C for 12 hours. The recombinant plasmidwas transformed into E. coli JM1O9. The E. coliJM1O9 containing the recombinant plasmid wasamplified in LB solid medium containing ampicillin(100 mgIL). Clones were picked out randomlythrough blue/white screening and cultivated in 4 mlLB medium containing 100 mg/L of ampicillin, at200 rlmin at 37°C overnight. Finally the recombi-nant plasmids were extracted by Sambrook' smethod and identified by PCR and restriction endo-muclease enzyme digestion.2.5 Sequence determination and homology analy-

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sisThe sequence determination of hpaA gene of

recombinant plasmid was carried out by ShanghaiDNA Biotechnologies Company (China), in themeantime, the sequence of hpaA gene and aminoacid were analyzed by software Omiga. 2. 0 andDNAmen, and compared the homology based onthe GenBank (No. NCOO0915, strain 26695; No.NCOO0921, strain J99 j No. X92502, strain11637; No. AF479028 , strain CH-TX1 ; No.U35455, strain CCUG 17874; No. X615.74,strain 8826; No. DQ115385, strain K51; No.AY714223 , strain Y06).

3 Results

3.1 PCR amplification of hpaA encoding se-quence

The hpaA of MEL-HP27 strain was amplifiedby PCR from the above primers. The PCR productwas electrophoresed and visualized by 10 g/L a-garose gel (Figure 1). It revealed that the size ofhpaA DNA fragment amplified by PCR was 783bp, and was compatible with the expectant size.3.2 Construction and identification of recombi-nant plasmids .

Recombinant plasmid pBluescriptb II-hpaAwas digested with BamHI and Hind ill and con-firmed by PCR, then digestive product and PCRproduct were visualized on 10 g/L agarose gel (Fig-ure 2). It demonstrated that recombinant plasmidwas digested to 3,000 bp and 783 bp DNA frag-ment, which contained the objective gene, and

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The outer membrane is a continuous structure onthe surface of gram-negative bacteria, which havebilateral particular significance as a potential targetfor protective immunity and bacterialpathogens[9,lO]. In other studies, outer membranevaccines have been used with considerable success

to induce protection against a number of organ-isms[4,1l]. The hpaA gene is located in genomeDNA of Helicobacter pylori and considerably con-servative for its nucleotide and amino acid se-quences. HpaA is one of the major structural outermembrane proteins of Helicobacter pylori and playsan important role in adhesion of the microbe[12,13].Furthermore, antibody against HpaA almost couldbe found in all Helicobacter pylori infected patientssera, which will be an ideal antigen candidate forHelicobacter pylori vaccine. In this study, thehpaA gene was cloned from strain MEL-HP27,which consists of 783 base pairs and encodes thepolypeptides of 260 amino acids. The homologies,ofthe nucleotide and putative amino acid sequences ofhpaA gene from Helicobacter pylori strain MEL-HP27 compared with the 8 published hpaA genesequenceswere as high as 94. 25% - 97. 32% and95.38 % - 98.46 %, respectively. These data indi-cate that the mutation level of the hpaA gene ofHelicobacter pylori strain MEL-HP27 is within therange reported by GenBank, and suggest thatHpaA is an excellent and ideal antigen for develop-ing Helicobacter pylori vaccine.

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hpaA gene was amplified from the recombinantplasmid by PCR.3.3 Sequence analysis

Sequencing results showed that the hpaA geneconsists of 783 base pairs and encodes the polypep-tides of 260 amino acids. The sequencing results ofhpaA from strain MEB-HP27 are published in the

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Figure1. The result of hpaA gene amplification using PCRLane 1: 100 bp DNA ladder; Lane 2 and Lane 3: PeR prod-uct of hpaA gene.

GenBank, the accession number is DQ353891. Thehomologies of the nucleotide and putative aminoacid sequences compared with eight publishedhpaA gene sequences were 94.76% - 97.19% and95.38 % - 98. 46% , respectively(Figures 3, 4).The strain MEL-HP27 was quite identical toNCTO1637 than the others with nucleotide ho-mologies of 97. 19%, and the amino acid identitywas 97. 31 % against NCTC11637. There are only22 base pairs different between MEL- HP27 andNCTC11637, at 62nd site codon AAGIN- AGG/R, at 1O0th site codon AAT IN-AGC/S, at 112thsite codon GCG/A-TCG/S, at 124th site codonAGTiS-AATIN at 137th site codon ACA/T-ATAll, at 164th site codon ATCII-GCTN, at256th site codon AACIN-GGC/G(codon/amnioacid). These analysis indicated that the hpaA genesequence and the putative amino acid sequence werequite conservative and might be a potential antigencandidate for Helicobacter pylori vaccine develop-ment.

~4 Discussion

Helicobacter pylori adhesion is a flagellarsheath potein locatedin the bacterialouter membrane.

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gested by &mHI and Hind ill; Lane 3: pBluescript-hpaAdigested by Hind ill; Lane 4: pBluescript-hpaA digested by&mHI and Hind ill ; Lane 5: hpaA gene amplified by PeRfrom recombinant plasmid pBluescript-hpaA,

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Cancer 2004; 109(1): 138 - 43.2. Kauser F, Hussain MA, Ahmed I, et al. Comparing

genomesof Helicobacterpylori strains from the high-alti-tude desert of Ladakh, India. J Clin Microbiol 2005; 43(4) :1538 - 45.

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10. Keenan U, Allardyce AR, Bagshaw FP. Lack of protec-tion following immunization with Helicobacter pylorimembrane vesicles highlight santi genic differences be-tween H. felis and Helicobacter pylori. FEMS Microbiol-ogy Letters 1998;161:21-7.

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13.Alm RA, Ling SL, Moir DT, et al. Genomic-sequencecomparison of two unrelated isolates of the human gastricpathogen Helicobacter pylori. Nature 1999; 397: 176-80.

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6. Valkonen KH, Wadstrom T, Moran AP. Identificationof the N-acetylneuraminyllactose-specific laminin-bindingprotein of Helicobacter pylori. Infect Immun 1997; 65(3):916-23.

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pylori vacuolating cytotoxin gene by introduction of di-rected mutagenesis. World J Gastroenterol 2003; 9( 10):2251-7.

8. Chen XJ, Yan J, Shen YF. Dominant cagA/vacA geno-types and coinfection frequency of H. pylori in peptic ul-cer or chronic gastritis patients in Zhejiang Province andcorrelations among different genotypes, coinfection andseverity of the diseases. Chinese Medical Journal 2005;118(6):460-7.

9. Richard AA, James B, Beth M, et al. Comparative ge-nomics of Helicobacter pylori : analysis of the outer mem-brane protein families. Infection and Immunity 2000; 68(7) :4155 - 68.

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ReceivedJune 18, 2006

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Documents

Cloning and Sequence Analysis of Adhesion Gene of J · Life Science Journal, 3 (4), 2006, Huang, et al ,Cloning and Sequence Analysis of Adhesion Gene hpaA ligase, DNA gel extraction