Clone-expression And Preparation Of Polyclonal Antibody Of Helicobacter Pylori Sod Gene

It is konwn that Cytotoxin—associated gene A(CagA) is one of the most important virulence factor of H.pylori.To'further study on the biological function of CagA,The research group of H.pylori in Zhengzhou university constructed CagA negative mutants-Hp27△cagA via homologous recombination.Some different functional protein of H.pylori related with CagA was screened out and identified by comparing the biological property of wild strains and mutans with the aid of proteomics methods.One of Some different functional protein is superoxide dismutase(SOD) protein.The study on the relationship of SOD with CagA is not reported yet.The first important thing is to construct polyclonal antibody of SOD,and then confirm that it is different of the establishment of SOD between wild and CagA deleted mutants.Methods:1.Target gene was cloned by PCR and it was through primer primier 5.0 system that the primers was desigend.2.The amplified gene fragment were connected with vector pMD19-T,and transformed into DH5a.3.Double digestion with Nde I,Xho I restriction endonuelease,the target fragment were connected with pET30(a) and transformed into BL21.4.Target protein was purified by Ni-NTA column according to the charcter of the fusion protein with with 6×His-tag affinity.5.The purified fusion SOD protein as antigen was injected into rabbit.6.The titer of the polyclonal antiserum was measured by the methods of ELISA. Results:1.Sod gene fragment was amplified by using total DNA of Hp27 as the template.2.After amplified products was purified and colleded,connected with vector pMD19-T,and the transformed into DH5a,the recombinant plasmid pMD19-sod was constructed.3.We got the he recombinant plasmid pET30-sod confirmed by DNA sequence analysis.4.The fusion protein with 6×His-tag affinity was got via affinity chromatography.5.The titer of the polyclonal antiserum was more than 1:3200,it meant the success of the preparation of polyclonal antiserum.Conclusion:1.Sod gene fragrrtent could be cloned from Hp27 for the first time.2.Recombinant plasmid pET30-sod was induced to express.The studies suggest that the suitable inducement condition was 0.1mmol/1 IPTG for 4.5 hours.3.The solubility analysis shows that most of the fusion proteins of SOD exists as soluble protein and the expression is much higher measured by SDS-PAGE.4.Antiserum with high titer could be obtained in rabbit immunized by purified fusion SOD protein.These work will lay a foundation for further study on sod protein.