Cloning And Prokaryotic Expression Of Surface Epitope Gene In The Ferric Enterobactin Receptor Protein FepA Of Escherichia Coli

Objective: In order to make the Monoclonal antibody which is used for detecting E.coli in raw milk, we studied about Cloning, Construction of prokaryotic expression vector for the gene encoding the surface epitope region of Ferric Enterobactin Receptor Protein FepA. These results were expected to lay foundation for further studies on making Monoclonal antibody and detecting E.coli in raw milk.Methods: A pair of primers were designed according to digestion sites in plasmid pGEX-6P-1 and the fepA gene sequence published by GeneBank. The Target fragment was amplified from E. coli genomic DNA by PCR, then cloned into pGEX-6P-1 and transformed into the host E. coli strain JM109. The Prokaryotic expression vector was constructed successfully. The recombinant plasmid was taken and transformed into DH5a for expression. Induced by IPTG at 37℃, the recombinant E.coli was analyzed by SDS-PAGE.Results:(1)In this study, we established the PCR system which was used for amplifying the gene encoding the surface epitope region of Ferric Enterobactin Receptor Protein FepA and obtained 598 bp amplified fragment.(2)Using T-A cloning technique, the PCR product was cloned into Cloning Vector pMD19-T Simple Vector successfully and the Cloning Vector containing the target gene was thus constructed successfully. Sequence analysis demonstrated that its homology with the Ferric Enterobactin Receptor Protein FepA of most other E.coli strains on GeneBank was between 95%～99%.(3)By the strategy of directional cloning, the target gene was cloned directional into expression vector pGEX-6P-1 and the Prokaryotic expression vector containing the target gene was thus constructed successfully.(4)Induced by IPTG at 37℃, competent cell containing recombinant plasmid expressed the 48 KD fusion protein. The fusion protein existed in the form of soluble and inclusion body protein and its main form was inclusion body, which was demonstrated through SDS-PAGE analysis of Lysates from recombinant E.coli.Conclusion: The gene encoding the surface epitope region of Ferric Enterobactin Receptor Protein FepA had been expressed successfully in the competent cell E.coli DH5αand the expression protein which was 48 KD existed in the form of soluble and inclusion body protein.