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目的:观察构建的重组抗HER2ScFv/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Cytc在细胞质中存在,SKBr-3细胞出现凋亡.结论:重组抗HER2ScFv/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.
OBJECTIVE: To observe the expression of recombinant anti-HER2ScFv / tbid gene in SKBr-3 cells and its effect on transfected SKBr-3 cells.Methods: The constructed pcDNA-bid plasmid was digested with restriction enzymes , And the recombinant gene with PE transmembrane domain and tbid was obtained.The gene was inserted into the downstream of the ScFv23e single-chain antibody gene of eukaryotic expression vector pCMV and transfected into SKBr-3 cells.The expression of the target protein was detected by indirect immunofluorescence MTT assay was used to detect the proliferation of the transfected cells.Results: After transfection of SKBr-3 cells, the expression of the target protein was detected.MTT assay showed that the proliferation of cells was significantly inhibited.Indirect immunofluorescence double labeling staining for the detection of tBid The results showed that the recombinant anti-HER2ScFv / tBid could be expressed in transfected SKBr-3 cells and could inhibit the growth of transfected cells and induce the expression of Cytc in the cytoplasm.Conclusion: Cell apoptosis.