The Research On The Eukaryotic Expression Of Gene PorB And Fusion Gene PorB-cfxB Of Neisseria Gonorrhoeae

ObjectivesThis research is to construct eukaryotie expression vector pcDNA3.1-porB andpcDNA3.1-porB-ctxB of Neisseria gonorrhoeae and to express the recombinant protein inNIH3T3cells respectively.It will be very helpful for the further research of new vaccine ofNeisseria gonorrhoeae.Methods1To construct eukaryotie expression vector pcDNA3.1-porB and pcDNA3.1-porB-ctxB of Neisseria gonorrhoeae: Epsilon extra-cting recombinantplasmid pGEX4T-porB with alkaline lysis method.With pGEX4T-porB as thetemplate, PCR amplification of porB gene, recovering the purified and usingBamHⅠand XhoⅠ double enzyme porB gene; epsilon extracting recombinantplasmid pGEX4T-porB-ctxB with alkaline lysis method, then doubleenzyming the slice porB-ctxB with BamHⅠand XhoⅠ.The porB andporB-ctxB were cloned into pcDNA3.1(+) eukaryotic expression vector,construction of eukaryotic expression recombinant plasmid pcDNA3.1-porBand pcDNA3.1-porB-ctxB.2The eukaryotic expression of recombinant plasmid pcDNA3.1-porB andpcDNA3.1-porB-ctxB:The recombinate expression plasmids were transfectedinto NIH3T3cells in vitro using liposome and the expression products in the cells would be detected by indirect immunofluorescence assay.Results1The recombinant expression plasmid pcDNA3.1-porB and pcDNA3.1-porB-ctxB was successfully constructed by double enzymed， PCR andsequencing analysis.2The recombinate expression plasmids were transfected into NIH3T3cellsusing liposome and two protein expression were detected in NIH3T3cells byindirect immunofluorescence assay.ConclusionsThe recombinant expression plasmid pcDNA3.1-porB and pcDNA3.1-porB-ctxBwere successfully constructed and efficiently expressed in NIH3T3cells. It lays thefoundation for the follow-up study of immunological activity of the PorB protein andPorB-CTB fusion protein of Neisseria gonorrhoeae,and it will be very helpful for thefurther research of new vaccine of Neisseria gonorrhoeae．...