Prokaryotic Expression of Vero Toxin Ⅱ B Subunit from Escherichia coli O157 and Preparation of Monoclonal Antibodies

Abstract:

The segment of VT2-B subunit was amplified by PCR，then purified and digested with the endonucleases EcoRⅠand XhoⅠ.The digested segment was inserted into the double-cleavaged expression vector pGEX_4T-2.The recombinant expression vector was named as pGEX_4T-2-VT2-B.The expression vector was transformed into E.coli BL21 compotent cells,and highly expressed a VT2B-GST fusion protein after IPTG inducing.The target fusion protein occupied 35% of total cell protein and in soluble form.The high-purified fusion protein was obtained by GSH-Sepharose Resin.The BALB/C mice were immunized with the purified recombinant protein.Two strains of monoclonal antibody 4B×F10×D4 and 4B×8G×11F were obtained.Ascitic monoclonal antibodies were generated by using cells 4B×8G×11F.Western blot analysis showed that the recombinant protein had a specific affinity for monoclonal antibody,while not for vector protein.The results showed that the McAb generated was against the VT2-B subunit,not against the GST.