Gene Cloning, Expression, Monoclonal Antibody Preparation And B Cell Epitope Determination Of Major Protective Antigens Of Helicobacter Pylori

HeIicobacter pylori (H.pylori, Hp), a curved microaerophilic Gram-negativebacterium, colonizes long-term in the gastric mucosa of human. Many studiesdemonstrated that Hp infection was the main cause of various chronic gastroduodenaldiseases such gastritis, peptic ulceration, mucosa-associated lymphoid tissue (MALT)and peptic cancer. WHO has already considered it as classâ… carcinogen.Approximately half of the world's populations are infected with Hp. Even thoughantibiotics are still the most effective treatment for the Hp infection, the use ofdrugs for treatment is limited because of expansive infection, side-effects andbacterial résistance, the frequency of re-infection. With people comprehensivelyunderstanding of the genome and proteome of Hp, vaccination is an alternative wayto control Hp infection and become one of the most promising approaches. Previousstudies have proved that the immunization had prevented and treated Hp infection,providing people's long-lasting and strong immunity to the disease. Thus, it is ofgreat interests to develop Hp vaccine.Commonly, Hp natural infection induces intensive immune responses in thehuman body, but the infection is chronic and persistent, and even leads to infectionfor life. It is indicated that the human body exists immune tolerance and the immune response can not pay a role. So the protective antigens must be chosen and reshapedto induce effective immune response in the design of Hp vaccine. With thedevelopment of genome, proteome, immunome and vaccinome of Hp, manyimportant protective antigens in Hp infection were discovered, which lay foundationfor the understanding of Hp invading the human, causing infection, immunemechanism and the design of a new generation vaccine. Previous studiesdemonstrated that VacA, Lpp20, HspA, CagA, UreaseA, UreaseB, Catalase, NapA,adhesion et. al antigens were alone used as antigens in the animal experiment and hadcertain immune protective effect. But the protective effect had the limitation, in whichthe weak immunity did not induce effective immune response and the individual didnot response to the re-infection of Hp. Telford et al demonstrated that the protectiveratio of alone Hp antigen was under 80% and two or more Hp combination antigenshad the ideal protective effect. Though whole molecular antigens remarkably improvevaccine protection, it does not been avoided that the side-effects harm to the humanbody. To further improve the security and validity of vaccine, epitope vaccines basedon major protective antigens of Hp become a new study aspect.The immune mechanism of Hp infection demonstrated that the host producesspecific and non-specific immune response against the infection. In the specificimmune response, antigens inducing antibodies may play an important role in the Hpimmune protection because Hp infection is mainly extracelluar. So the Identificationof B epitopes within major protective antigens is extremely important. The presentstudy determined B epitopes of major protective antigens of Hp by phage randompeptide library technology.According to corresponding reference, Lpp20, HspA, UreaseA, CagA, UreaseBand Catalase antigens were chosen as our study target. At first, specific PCR primersof six genes are designed according to the sequence of Hp standard strain 26695 on Genbank. The genes encoding Lpp20, HspA, UreaseA, CagA, UreaseB and Catalaseof helicobacter pylori (Hp) were amplified from Hp NCTC11639 chromosomal DNAby PCR .They were first operated T-A clone and sequenced. The amplified Lpp20,HspA,UreaseA,CagA,UreaseB fragments were composed of 528bp, 351bp,675bp,855bp, 1704bp,1515bp and accessed on GenBank(accession No DQ106902,DQ141574, DQ141577, DQ141575, DQ141576 and DQ333889).The nucleotide andamino acid sequences were high homology compared with other Hp strains onGenBank. Then the genes of Lpp20, HspA, UreaseA, CagA, UreaseB and Catalasecloned into pGEX-4T-1 fusion expression vector were expressed in E.coIi Top10 with48000Da, 41000Da, 52000Da, 60000Da, and 91000Da and 85000Da fusion proteinrespectively. Expression products were ultrasonicated and the target proteins weresoluble in the supernatants, which were purified by Glutathione Sepharose 4B affinitychromatography. Western blotting proved that six recombinant proteins werespecifically recognized by the serum of Hp -infected patients. This proved that sixrecombinant proteins preserved original antigenicity.To prepare monoclonal antibodies (mAbs) against Hp, the BALB/c mice wereimmunized with supernatant and precipitation of culture combination of Hp standardand clinical strains after ultra sonication. Thirty-four mAbs were obtained byhybridoma technology and the subtype of identified mAbs were all IgG1 (κ). The titerof culture supernatant was 1:32～1:128 and the titer of ascites was 1:32000～1:128000 with affinity constants (K_aff) from 1×10⁸ L/M to 1×10¹⁰L/M. Recombinantexpression Lpp20,HspA,UreaseA,CagA,UreaseB,Catalase proteins were usedto identify mAbs by ELISA and Western-blotting and specific mAbs were obtainedrespectively against these six antigens. We labeled mAbs with HRP and identifiedepitopes by competitive ELISA. Three mAbs against Lpp20 were the same epitope,two mAbs against HspA were the same epiotpe, four mAbs against UreaseA were thesame epiotpe, six mAbs against UreaseB were two epitopes and two mAbs againstCatalase were the same epitope. The preparation and determination of these mAbs laid foundation for the screening and identification of antigenic determinant by phagerandom display library.In order to map the epitope of UreaseB antigen, the Ph.D.-12 library wasscreened with specific anti- UreaseB mAb U001 as selective molecular. After threerounds of biopanning, the phages bound to mAb were well enriched and phage cloneswere randomly selected to test for their immunoreactivity with mAb L001. Thesequences of the DNA of 15 positive clones were determined and the deduced aminoacid sequences of the corresponding peptides were analyzed. The most positive phageclones presented the sequence of. EHWSHDMFSPGD. Compared with the originalamino acid sequence of UreaseB protein, E, H, D, M of this 12 peptides sequence in1,5,6,7 positions had high homology with amino acid residues (347-353) of UreaseBprotein. E, H, D, M may determine the property of antigenic epitope and may be keyamino acids to form a motif of UreaseB protein. This notion was further confirmed bycompetitive ELISA that the positive phage clone inhibited UreaseB antigen binding tomAb. To identify the immunigenity of this peptide, the positive phage clone was usedto immunize BALB/c mice and ELISA and Western-blotting proved that the UreaseBantigen was recognized by antiserum. The epitope postion was adjacent to aneutralizing epitope of UreaseB at 321-339 aa reported by a foreign researcherpreviously. It suggested that there might be predominant antigenic epitopes onUreaseB protein. Still, more investigation is needed.In order to map the epitope of Lpp20 antigen, the Ph.D.-12 library was screenedwith specific anti- Lpp20 mAb L001 as selective molecular. After three rounds ofbiopanning, the phages bound to mAb were well enriched and phage clones wererandomly selected to test for their immunoreactivity with mAb L001. The sequencesof the DNA of 15 positive clones were determined and the deduced amino acidsequences of the corresponding peptides were analyzed. The most positive phageclones presented the sequence of SWPLYSDASGLG. Compared with the original amino acid sequence of Lpp20, D, A, S, G of this 12 peptide sequence had the samehomology with Lpp20 amino acid residues (114-117). D, A, S, G may determine theproperty of antigenic epitope and may be key amino acids to form a motif of Lpp20protein. It is speculated that this epitope may be liner. This notion was furtherconfirmed by competitive ELISA that the positive phage clone inhibited Lpp20antigen binding to mAb. To identify the immunigenity of this peptide, the positivephage clone was used to immunize BALB/c mice and ELISA and Western-blottingproved that the Lpp20 antigen was recognized by antiserum.In order to map the epitope of Catalase antigen, the Ph.D.-12 library wasscreened with specific anti- Catalase mAb C001 as selective molecular. After threerounds of biopanning, the phages bound to mAb were well enriched and phage cloneswere randomly selected to test for their immunoreactivity with mAb C001. Thesequences of the DNA of 15 positive clones were determined and the deduced aminoacid sequences of the corresponding peptides were analyzed. The most positive phageclones presented the sequence of SVSLPYANLAHI. Compared with the originalamino acid sequence of Catalase, S, P, N, L, A of this 12 peptide sequence in 1, 5,8,9,10 positions had high homology with amino acid residues (394-405) of Catalaseprotein. S, P, N, L, A may determine the property of antigenic epitope and may be keyamino acids to form a motif of Catalase. This notion was further confirmed bycompetitive ELISA that the positive phage clone inhibited Catalase antigen binding tomAb. To identify the immunigenity of this peptide, the positive phage clone was usedto immunize BALB/c mice and ELISA and Western-blotting analysis proved that theCatalase antigen was recognized by antiserum.In conclusion, the present study screened and identified B cell epitopes ofUreaseB, Lpp20, Catalase of Hp major protective antigens by phage random peptidelibrary technology .From the above results, the study platform of B cell epitope was set up and laid theoretic foundation for the development vaccine for Hp vaccine. Atthe same time, the results also indicated the potential of using phagotopes asalternative vaccine components. The comparison of mimotopes selected from apeptide library with mAb and the corresponding antigen sequence may lead to abetter understanding of the epitope of Hp from molecular level and to the design ofspecific peptides for vaccines.