斜带石斑鱼IgZ重链基因的表达纯化和抗体制备

Prokaryotic expression, purification of orange-spotted grouper (Epinephelus coioides) IgZ heavy-chain protein and preparation of polyclonal antibody against IgZ

为获得斜带石斑鱼IgZ重链基因的多克隆抗体,成功构建了斜带石斑鱼免疫球蛋白IgZ重链重组蛋白表达质粒pQE30/IgZ,将其转化到大肠杆菌表达菌株M15中,确定了最佳诱导表达条件:IPTG 0.2 mmol/L,诱导温度30℃,诱导时间6 h。所获得的IgZ重链重组蛋白经Ni-NTA亲和层析后,纯度达到85%以上;以纯化的IgZ重链重组蛋白为抗原免疫新西兰兔,制备出相应的多克隆抗体,经ELISA测定抗血清效价约为1∶320 000。

The recombinant expression plasmid was constructed by means of linking the grouper IgZ cDNA with prokaryotic expression vector pQE30. It was identified by endonuclease digestion and DNA sequencing of the recombinant plasmid. Then, the recombinant plasmid pQE30/IgZ was transformed into E. coli M15. And the most optimum induced expression conditions were determined： the IPTG was 0.2 mmol/L, the induced temperature was 30℃, the induction time was 6 h. The recombinant IgZ heavy-chain protein was gained by means of Ni-affinity chromatography. The purity of the recombinant IgZ heavy-chain protein was more than 85%. The anti-IgZ polyclonal antibody was gotten by means of immuning New Zealand white rabbit with the recombinant IgZ heavy chain protein. The result of Enzyme-linked Immunosorbent Assay （ELISA） showed that the highest titer of anti-serum was 1：320 000 for IgZ.