重组抗HER2 ScFv/tbid基因的表达及其对乳腺癌SKBr-3细胞的促凋亡作用

目的:观察构建的重组抗HER2 ScFv /tbid基因在SKBr-3细胞中的表达及其对转染的SKBr-3细胞的作用. 方法:用限制性内切酶双酶切已构建的pcDNA-bid质粒,获得带有PE转膜结构域和tbid的重组基因,将所获基因插入到真核表达载体pCMV单链抗体基因ScFv23e的下游,转染SKBr-3细胞. 间接免疫荧光法检测目的蛋白的表达,MTT法检测目的基因转染后细胞的增殖情况. 结果:转染SKBr-3细胞后,检测出目的蛋白的表达. MTT实验发现细胞的增殖被明显抑制. 间接免疫荧光双标记染色检测tBid的表达,并可观察到Cyt c在细胞质中存在, SKBr-3细胞出现凋亡. 结论:重组抗HER2 ScFv /tBid分子可以在转染的SKBr-3细胞中表达,并且可抑制转染细胞的生长,诱导细胞发生凋亡.

Expression and proapoptotic activity of recombinant anti-HER2 ScFv/ tbid gene in SKBr-3 cells

AIM: To observe the expression and the effect of recombinant anti-HER2 ScFv/tBid gene in the transfected SKBr-3 cell line. METHODS: Recombinant gene of PE transmembrane domain and tbid was obtained from pcDNA-bid plasmid by restriction enzyme digestion, and then cloned into vector pCMV and the recombinant plasmid pCMV-rpb was transfected into SKBr-3 cells. Expression of recombinant plasmid and morphologic changes of SKBr-3 cells were detected by immunofluorescent staining. MTT test was used to show how the cells' living status was affected by the gene transfection. RESULTS: The recombinant plasmid was constructed and transfected into SKBr-3 cells successfully. The fusion protein was found in the cytoplasm of transfected SKBr-3 cells. MTT test and indirect immunofluorescent staining showed the apoptosis of transfected SKBr-3 cells. CONCLUSION: Recombinant proapoptotic protein of tBid can be expressed in transfected SKBr-3 cells and the expressed fusion protein can induce apoptosis.