二硫键稳定的抗肝癌人源化单链抗体-突变体人论著TNFα融合基因在CHO(dhf)细胞中的表达

Expression of disulfide-stabilized humanized ScFv fused to mutant hTNFα against hepatocellular carcinoma in CHO(dhfr )cells

目的 :提高抗肝癌靶向人肿瘤坏死因子 (hScFv2 5 hTNFα)的稳定性和杀伤活性 ,构建二硫键稳定的抗肝癌人源化单链抗体 突变体人TNFα融合基因 (ds ScFv hTNFα) ,并在CHO细胞中表达。方法 :常规构建ds ScFv hTNFα融合基因 ,并克隆入真核表达载体pCI(dhfr1) ,磷酸钙共沉淀法转染中国仓鼠卵巢细胞株 (CHO dhfr ) ,甲氨蝶呤 (MTX)高压筛选高表达细胞株 ,免疫荧光染色和MTT法检测重组蛋白的抗体和突变体hTNFα的双重活性。结果 :目的基因在CHO(dhfr )中获得了表达 ,表达产物具有抗体和突变体hTNFα的双重活性 ,且稳定性得到大幅度提高 ,达到 4℃放置 4个月活性无明显下降。结论 :抗肝癌人源化ds ScFv hTNFα融合基因在CHO细胞中获得功能性表达 ,为进一步的临床应用研究奠定了基础。

Objective:To increase the stability and the activity of tumor necrosis factor targeting to hepatocellular carcinoma and to study the soluble expression of the new type targeting tumor necrosis factor in CHO cells. Methods:The expression vector of fusion gene of recombined disulfide stabilized hScFv 25 and mutant hTNFα was constructed and expressed in CHO cells.Site directed mutation via primer PCR and overlap extended PCR was achieved in humanized ScFv against hepatocellular carcinoma and the mutant hTNFα gene was amplified through PCR.The double activity of recombined protein of the antibody and mutant hTNFα were examined by immunofluorescence stain and MTT.Results:Expression of the object gene with double activity of the antibody and the mutant hTNFα was achieved in CHO cells. The activity of expressed products did not decline obviously at 4℃ for 4 months. Conclusions:Recombined gene was functionally expressed in CHO cells, in the meantime the stability of the product was increased dramatically. These lay a foundation for the further research on its clinical application.