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

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

作　　者：任君琳[1] 王涛[2] 许彦鸣[2] 孟艳玲[1] 温伟红[1] 张瑞[2] 张巍[1] 杨安钢[1] 

机构地区：[1]第四军医大学基础部免疫学教研室,陕西西安710033 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2007年第3期193-196,共4页Journal of the Fourth Military Medical University

基　　金：国家重点基础研究发展计划(2004CB518805);教育部长江学者和创新团队发展计划(IRT0459);国家自然科学基金(30370314;30400403)

摘　　要：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.AIM： To construct the recombinant expression vector of anti-HER2 ScFv/FDT/caspase-6 and investigate its pro-apoptosis activity after transfected into gastric cancer SGC7901 cells. METHODS： Anti-HER2 SeFv （e23sFv） was attached to human recombinant caspase-6 （RC6） gene by recombinant PCR, and the Fufin recognition site of diphtheria toxin （FDT） served as the linker to construct e23sFv-FDT-RC6 recombinant gene. The recombinant gene was cloned into the vector pCMV, and the recombinant plasmid was transiently transfeeted into HER2 positive SGC7901 cells and HER2 negative HeLa cells via lipofectamine mediation. MTY assay was used to show how the cell living status was affected by the gene transfeetion. The pro-apoptotie activity of recombinant plasmid was detected by indirect immunofluorescent staining. RESULTS： Restriction endonuelease digestion and DNA sequencing proved that e23sFv-RC6 and e23sFv-FDT-RC6 genes had been cloned into vector pCMV. After transfeetion, the expression of fusion protein was found through Western Blot. MTY 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.

关 键 词：ScFv抗体 caspase-6基因 细胞凋亡 

分 类 号：Q786[生物学—分子生物学]