Prokaryotic expression of HCV NS5ATP1 gene and preparation of polyclonal antibody / 解放军医学杂志

ABSTRACT

Objective To construct prokaryotic expression vector of hepatitis C virus NS5ATP1 gene, and to induce its expression in E. coli. To purify the fusion protein and obtain its polyclonal antibody from immunized New Zealand rabbits. Methods The NS5ATP1 gene, which was cut from the vector pGBKT7-NS5ATP1, which was self-constructed by the authors, was cloned into plasmid pET32a(+) to construct the pET32a(+)-NS5ATP1 prokaryotic expression vector. It was proved that the recombinant plasmid was constructed correctly by sequencing. And then the expression vector was transformed into the competent E. coli DH5? and BL21. After being induced with IPTG, the NS5ATP1 fusion protein was expressed and analyzed with SDS-PAGE and Western blot. The transformed bacteria were fragmented by ultrasonic and then separated by SDS-PAGE. The fusion protein formed inclusion body. They were then purified and re-natured through Ni+ affinity column chromatography. The purified pET32a(+)-NS5ATP1 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and immunizing potence of polyclonal antibody were evaluated by Western blot and ELISA. Results After transferring pET32a(+)-NS5ATP1 plasmid into DH5? and BL21 and induced with IPTG, the NS5ATP1 fusion protein of about 56kD was highly expressed. SDS-PAGE analysis showed that the fusion protein products were mainly in inclusion body and expressed in the highest level at 4.5h of induction. The purified protein and polyclonal antibody were obtained successfully. ELISA manifested the titer of polyclonal antibody was over 1∶512 000. The high specificity was testified by Western blot. Conclusions The successful expression and purification of NS5ATP1 fusion protein and the preparation of NS5ATP1 specific polyclonal antibody will be valuable for the study on the biological function of NS5ATP1.