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Study On Clone And Initial Function Of CagX Gene In The Helicobacter Pylori

Posted on:2009-02-14Degree:MasterType:Thesis
Country:ChinaCandidate:S R DongFull Text:PDF
GTID:2120360245977987Subject:Clinical Laboratory Science
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Objective:1.To Clone and detect cagX gene of typeⅣsecretion system(T4SS)in helicobacter pylori;2.To express CagX protein by prokaryotic expression system in E.coli3.To investigate the initial biological function of Purify CagX protein by cocultivated with SGC 7901 cells,which posed a basis for further researching on its biological function and pathogenic mechanism.Materials and Methods:1.H.pylori NCTC 11637 was inoculated onto Columbia-selective agar plates at 37℃for 4 days,under microaerophilic condition.Organisms were identified as H.pylori by colony morphology,Gram staining,rapid urease test and oxidase test;2.H.pylori cagX gene was amplified by PCR using the genome DNA.The PCR product was inserted into pGEM-T vector and then transformed into E.coli DH5α.The positive recombinant clone was analyzed by digestion of restriction endonuclease.Then the cagX gene fragment was inserted directionally into vector pET32a(+)to construct recombinant clone of cagX.After they were expressed by BL-21 E.coli cells,the PCR products were sequenced and analyzed;3.CagX protein was purified by Ni2+-NTA column and detected by SDS-PAGE and Western-blot;4.The purified CagX protein was cocultivated with SGC 7901 cells in different concentrations of CagX protein induced at different induction times.The contributions of cell multiplication were observed by morphology and MTT.Results:1.A 1569 base pairs long cagX gene was obtained by PCR method. GenBank accession number is EF608160.The cagX gene is a species heightly conservative prokaryon gene.The sequence analysis for cagX showed that it shares 99%~96%homology with other prokaryon strains but none with eukaryote in GenBank.The result of analysis revesled that there were certain differences in secondary structure,but the antigenicity was identical.The Phylogenic tree shows that the sequence similarity of NCTC 11637 is highly with 22695 to NCTC 11638 and cosmid clone 36;2.The prokaryotic expression vector pET32a-cagX was obtained.The fusion CagX protein about 80.5kDa was inducted under the induction environment with IPTG concentration as 1mmol/L,30℃for 4h and purified with Ni2+-NTA column;3.The purified CagX protein was cocultivated with SGC 7901 cells and found that the CagX have little influence to SGC 7901 multiplication.Conclusions:1.The recombinant plasmid pET32a-cagX was constructed successfully which were confirmed by sequencing.The homogenicity of nucleic acid and that of amino acid was high;2.The CagX protein was expressed successfully which facilitated further research;3.The CagX protein has influence on SGC 7901 multiplication.
Keywords/Search Tags:Helicobacter pylori, T4SS, cagX, sequence analysis
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