| Helicobacter pylori (H. pylori) is one of the most common bacteria infections agents to human being. Many studies have shown that it is themain cause of chronic gastritis and peptic ulcers, and it also has relationship with the gastric cancer and the gastric MALT lymphoma. In 1994, WHO classified H. pylori as a definite (class I) carcinogen. Center for Diseases Control and Prevent has declareded H. pylori infection as one of the new emerging infectious diseases.It is evaluated that the infection rate of H. pylori is almost 80%. Among them 50% migjt develop gastritis; 10-15% may might develop peptic ulcer, and a small part might develop gastric cancer. Obviously, it is important to prevent Hp infection. Due to lacking of suitable diagnostic method and effective antibiotics against the bacteria, the more effective wayof preventing the infection of H. pylori is to be immunized with the vaccine against Hp.In the late years, most vaccines studied were on the base of virulent factors of H. pylori, such as Ure, Hsp, VacA, CagA and so on. As to Vac A and CagA, they could prevent and cure the infection caused by thetype I as antigen, but they were invalid on the type II; Urease could induce immunizingreaction of body, but it damaged the mucosa of stomach. So, it is essential to develope new protective antigen.Motility is one of the essential virulence, and it is showed that flagellin gene is conservative in much sduty. The flagellin protein, which flagellin gene codes, is not only very necessary for the pathogenic mechanism of Hp, but also has immunogenecity. So it is an important candidate component of gene engineering vaccine. Therefore, firstly,flaA(1533bp),flaB(l545bp) gene was amplified by PCR, molecular clone was used to ligate flaA, flaB gene with pNEB193 to construct recombinant plasmid. Then the recombinant plasmid was transformed into E.coli TBl competent cells, and positive clones were obtained after being screened by blue-white trial and cutted by two enzymes. Then flaA ,flaB gene was sequenced and homology comparison were done by using DNASIS software. Then flaA genewas cutted from positive recombinant plasmid and was inserted into expression vector pMAL-C2x and then transformed into TB1 competent cells. After IPTG-induced incubation, FlaA fusion protein(called MFA) was expressed in E.coli strain TB1, split products of culture was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the gelobtained was stained by Coomassie brilliant and scanned to qualify the recombinant protein. Meanwhile, Western-blot is applied to testify the truth of expression product and to determine immunoreactivity and immunogenicity of the fusion protein. METHODS1 Isolation and identification of H. pylori Gastric mucosalbiopsyspecimens were obtained at routine endoscope, and then daubed on the medium. After incubation at 37C in microaerobic conditions for 5 to 7 days, suspect colonies which exhibited typical colonial morphology, strainingmethod and biochemical relations were positive were subcultured. These isolates were kept at -80C.2 Clone of flaA, flaB gene flaA, flaB gene was amplified by PCR method, then digested flaA PCR products with restriction endonuclease Sal I, Pst I and digestedy flaB PCR products with restriction endonuclease Pst I, EcoRI, and meanwhile the cloning vector pNEB193 was digested by the same enzyme. The sticky ends were produced. Then target gene was ligated directionally to the polylinker cloning sites of pNEB193 byT4ligase. Then the recombinant plasmid was transformed into E.coli TB1.The positive clone was identified by the blue-white trial, special PCR andrestriction enzyme digestion.3 Sequence analysis of objective gene The positive clone was sequenced and compared with the reported corresponding sequences to calculate the homologues.4 Expression of fusion protein flaA gene was cutted from positive recombined plasmid and was inserted to expression vector pMAL-C2x. FlaA fusion pr...
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