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Investigation On The Surface Display System Of Lactococcus Lactis For Expressing The Antigen Fragment Of UreB Gene Of Helicobacter Pylori

Posted on:2008-05-25Degree:MasterType:Thesis
Country:ChinaCandidate:G Z GuFull Text:PDF
GTID:2120360215991270Subject:Biochemical Engineering
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Helicobacter pylori(Hp) is an etiologic agent colonizing the gastic mucosa of human beings, is hightly associated with chronic gastritis, peptic ulcer and gastric neoplasm. Current therapies for eradicating Hp depend on the use of combined antibiotics. However, the high cost, low patient compliance, and increasing of resistant strains make these therapies impractical on a large scale. Therefore, there is an urgent need for effective vaccines against Hp infection.Urease is one of the primary antigens of Hp. And Urease is protective, exposed to cell surface and highly conservative, and predominates in the the Hp vaccines. In particular, it was demonstrated that UreB, the urease B subunit, could stimulate the body to raise immunoresponse against the challenge from Hp. But, UreB expression has not been easy for its high molecular weight. In addition, it was confirmed that recombinant UreB as an immunogen can induce dominant production of conformational structure specific antibodies which might not abolish the enzymatic activity. In contrast, when some linear epitopes were used as the immunogen, a good amount of epitope-specific antibodies can significantly inhibit the activity. And some researches report that Hp can be eliminated and prevented by inhibiting the enzymatic activity of Hp urease. We speculated that a vaccine developed with an eptitop or short fragment of UreB as an antigen would generate more protection for the organism. Moreover, Hp infects the organism via oral route by means of attacking gastric mucosal epithelia. During this process, the entry of this pathogen allows the exposition of antigens to the mucosa, inducing the secretion of IgAs, the main class of antibodies that can be efficiently secreted by epithelium, neutralizing the toxicity of this agent. Therefore, the most effective way to induce mucosal immunity is to inoculate an antigen directly to the mucosal surface which can come true by oral route. Over the decade, L. lactis has been confirmed to be efficient for the presentation of antigens to the mucosal immune system, to elicit a specific response. Note that the protective immune response depends not only on antigen and the delivery vehicle, but also on the location of the antigen. Antigens anchored to the surface of L. lactis can directly stimulate the Peyer's patches in the intestinal tract to induce more protection.The investigation uses the E fragment of UreB as an antigen, tries to construct two surface disyplay systems, L. lactis MG1363(pAMJ399-spax-e) and L. lactis MG1363(pAMJ399-ca-e), aims to discuss whether the E fragment exposed to the surface of L. lactis MG1363 can induce more protection for the body or not, and hopes to establish the oral vaccine base by means of the above-mentioned systems. The main results of this investigation were summarized as follows:1. Two pairs of primers were designed for PCR amplification. And the SPA anchor(spax) from S. aureus Cowanâ… and the AcmA anchor (ca)from L. lactis MG1363 were cloned from genomic DNA.2. The covalent surface display vector pAMJ399-spax was constructed by fusing the spax fragment into the Speâ… -digested site of the secretion-type expression vector pAMJ399. And the non-covalent surface display vector pAMJ399-ca-e was performed by integration of the ca fragment into the Pstâ… -Salâ… -digested sites of the pAMJ399 vector.3,Two e fragments of ureB gene were obtained with PCR amplification. And One was digested with Bglâ…¡and ligated with Bglâ…¡-digested pAMJ399-spax-e, resulting in plasmid pAMJ399-spax-e, the other was digested with Bglâ…¡and Pstâ… , and cloned into the corresponding sites of pAMJ399-ca, leading to plasmid pAMJ399-ca-e.4,Total cellular proteins from E. coli JM109(pAMJ399-spax-e) and E. coli JM109(pAMJ399-ca-e) after inducement were analyzed by SDS-PAGE. And a target protein of approximately 42kDa from the former was visible as a faint band. Subsequently, the two plasmids, pAMJ399-spax-e and pAMJ399-ca-e, were electroporated respectively into the conjugation recipient of L. lactis MG1363. As a result, two surface display systems, L. lactis MG1363(pAMJ399-spax-e) and L. lactis MG1363 (pAMJ399-ca-e), generated and was expected to be capable of anchoring the E fragment of UreB to L. lactis MG1363.
Keywords/Search Tags:Helicobacter pylori, L. lactis, Urease, ureB gene, Surface display
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