【摘要】 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更多还原

【Abstract】 Aim To increase the stability and activity of humanized hepatocellular carcinoma (HCC) specific single-chain Fv (hscFv 25 ) targeting human tumor necrosis factor alpha (hTNFα and to explore the soluble expression of the novel targeting cytokine in E.coli . Methods The expression vector pGEX-hFvT of recombinant disulfide-stabilized hscFv 25 -hTNFα mutant gene was constructed via site-mutation by PCR and overlapping PCR and expressed by IPTG induction in E coli . Then the antibody activity and hTNFα mutant activity of the recombinant protein were detected by immunohistochemical staining and MTT colorimetry, respectively. Results Recombinant gene could be expressed effectively in E.coli in soluble form and the expressed product had dual activities of antibody and hTNFα mutant. After stored at 4℃ for 3 months, the antibody activity of the fusion recombinart protein did not change apparently. Anti-tumor activity of the fusion protein increased by four times than that of natural hTNFα. Conclusion The recombinant gene is expressed successfully and effectively in E.coli , and the stability and the anti-tumor activity of the expressed product increase significantly. These lay the foundation for the further research on its clinical application.更多还原