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目的 提高抗肝癌抗体靶向人肿瘤坏死因子(hscFv2 5-hTNFα)的稳定性和杀伤活性 ,并探讨该新型靶向细胞因子在大肠杆菌中的可溶性表达。方法 以引物PCR法和重叠延伸PCR法 ,对人源化抗肝癌hscFv2 5及hTNFα基因进行定点突变 ,构建重组表达载体 pGEX -hFvT ,并以IPTG在大肠杆菌中诱导表达。用免疫组化染色和MTT比色法 ,分别检测重组蛋白的抗体和hTNFα突变体的双重活性。结果 重组基因在大肠杆菌中获得高效可溶性表达。表达产物具有抗体和hTNFα突变体的双重活性 ,重组蛋白的稳定性得到大幅度提高 ,抗体活性于 4℃放置 3个月活性无明显下降 ,抗肿瘤活性较天然hTNFα高 4倍。结论 重组基因在大肠杆菌中获得了高效功能性表达 ;重组蛋白稳定性及抗肿瘤活性得到大幅度提高 ,为进一步的临床应用研究奠定了基础
Objective To improve the stability and killing activity of anti-hepatoma antibody targeting human tumor necrosis factor (hscFv2 5-hTNFα) and to explore the soluble expression of the novel targeting cytokine in E. coli. Methods The humanized anti-hepatocarcinoma hscFv2 5 and hTNFα genes were mutated by primer PCR and overlap extension PCR. The recombinant expression vector pGEX-hFvT was constructed and induced by IPTG in E. coli. Immunohistochemistry and MTT assay were used to detect the dual activity of the recombinant protein and hTNFα mutant, respectively. Results The recombinant plasmid was highly efficient and soluble in E. coli. The expressed product had the dual activity of the antibody and hTNFα mutant, and the stability of the recombinant protein was greatly improved. The activity of the antibody was not significantly decreased after 3 months of storage at 4 ° C, and the anti-tumor activity was 4 times higher than that of the natural hTNFα. Conclusion The recombinant protein was highly expressed in Escherichia coli. The stability and antitumor activity of the recombinant protein were greatly enhanced, which laid the foundation for further clinical application