抗人肝癌基因工程嵌合Fab抗体的原核表达及复性

Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma

目的 分别在大肠杆菌中高效表达嵌合Fd及嵌合轻链 ,并进行嵌合Fab复性的研究。方法 分别将嵌合Fd及嵌合轻链基因插入到原核表达载体pET32a中 ,构建成重组载体pET32a/cFd和pET32a/cL。转化大肠杆菌后诱导其表达。大量制备表达的嵌合Fd及嵌合轻链后 ,按二者绝对量等摩尔比混合后进行透析复性。然后 ,分别利用SDS PAGE、Westernblot和ELISA进行分析和鉴定。结果 成功获得了嵌合Fd及嵌合轻链的原核非融合高效表达 ,表达蛋白相对分子质量 (Mr)均在 2 4×10 3 左右。表达量分别约占菌体总蛋白的 2 8.3%和 32 .3% ,2种目的蛋白均主要以不溶性的包涵体形式存在。另外 ,嵌合Fd与嵌合轻链在缓慢透析的条件下成功地复性为嵌合Fab抗体 ,Mr 约为 4 2×10 3 ,具有特异性抗原结合活性和良好的亲和力。在起始总蛋白浓度为 10 0 μg/ml时 ,复性效率约为4 9 %。结论 成功实现了嵌合Fab抗体的高效表达及高效复性 。

Objective To separately express chimeric Fd and chimeric light chain in E.coli high efficiently and refold them into chimeric Fab. Methods The chimeric Fd and light chain genes were inserted into prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then, recombinant vectors were transformed into E.coli and induced by IPTG. Moreover, a large amount of chimeric light and chimeric Fd expression products were prepared. And then, they were mixed to refold into cFab by gradient dialysis . The refolded product was identified and analyzed by SDS PAGE electrophoresis, Western blot assay and ELISA. Results The high efficient non fusion prokaryotic expression of both chimeric Fd and chimeric light chain were fulfilled with the expression level of 28.3% and 32.3% of total bacteria proteins, respectively. and the relative molecular mass of them were 24kD or so and mainly existed in inclusion bodies. In addition, Chimeric Fd and chimeric light chain were successfully refolded together into chimeric Fab by means of gradient dialysis, with about 59.45% of refolding recovery when the concentration of initial total protein was 100μg/ml. The renatured protein could specifically bind to relative antigen with high affinity. Conclusion High efficient expression and renaturation of chimeric Fab were successfully fulfilled, which lay a solid foundation for potential application in clinical treatment of hepatoma.