重组抗HER2 ScFv/FDT/caspase-6基因的构建、表达及其活性鉴定

目的:构建重组抗HER2 ScFv/FDT/caspase-6 基因的表达载体,转染SGC7901胃癌细胞后观察其促凋亡作用. 方法:采用重组PCR法,将抗HER2的单链抗体(e23sFv)通过白喉毒素的Furin识别位点的编码序列(FDT)与人重构型caspase-6(RC6)基因重组,构建融合蛋白表达基因e23sFv-FDT-RC6. 将所获得的重组基因克隆入真核表达载体pCMV,以脂质体法瞬时转染HER2阳性的SGC7901细胞和HER2阴性的HeLa细胞. MTT法检测目的基因转染后细胞的增殖情况. 间接免疫荧光染色观察目的基因的表达对SGC7901细胞的促凋亡作用. 结果:把e23sFv-RC6,e23sFv-FDT-RC6基因克隆入真核表达载体pCMV,酶切鉴定和基因测序证明目的基因序列正确. 转染SGC7901和HeLa细胞后,Western Blot检测到目的蛋白的表达. MTT实验观察到转染pCMV-e23sFv-FDT-RC6的SGC7901细胞的增殖被明显抑制,间接免疫荧光染色可以观察到表达e23sFv-FDT-RC6融合蛋白的SGC7901细胞形态发生改变,部分细胞呈现典型凋亡特征. 结论:重组抗HER2 ScFv/FDT/caspase-6基因的表达可促进HER2阳性SGC7901胃癌细胞的凋亡.

Gene construction, expression and activity identification of recombinant anti-HER2 ScFv/FDT/caspase-6

AIM: To construct the recombinant expression vector of anti-HER2 ScFv/FDT/caspase-6 and investigate its pro-apop- tosisactivity after transfected into gastric cancer SGC7901 cells. METHODS: Anti-HER2 ScFv (e23sFv) was attached to human recombinant caspase-6 (RC6) gene by recombinant PCR, and the Furin recognition site of diphtheria toxin (FDT) served as the linkerto construct e23sFv-FDT-RC6 recombinant gene. The recombinant gene was cloned into the vector pCMV, and the recombinant plasmid was transiently transfected into HER2 positive SGC7901 cells and HER2 negative HeLa cells via lipofectamine mediation. MTT assay was used to show how the cell living status was affected by the gene transfection. The pro-apoptotic activity of recombinant plasmid was detected by indirect immunofluorescent staining. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that e23sFv-RC6 and e23sFv-FDT-RC6 genes had been cloned into vector pCMV. After transfection, the expression of fusion protein was found through Western Blot. MTT assays showed that the growth of SGC7901 cells was inhibited. Typical apoptotic changes of the SGC7901 cells transfected with pCMV-e23sFv-FDT-RC6 were seen by indirect immunofluorescent staining. CONCLUSION: The expression of recombinant anti-HER2 ScFv/FDT/caspase-6 gene can induce the apoptosis of HER2 positive SGC7901 cells.