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目的:构建以寡聚精氨酸(R9)为蛋白质转导结构域的嵌合抗体/caspase-3的真核表达载体,并检测瞬时转染该载体对HER2阳性SGC-7901肿瘤细胞及HER2阴性HeLa肿瘤细胞生长的影响。方法:用PCR法获得融合了R9的活性caspase-3基因片段,并将其克隆入含有HER2单链抗体e23sFv基因的真核表达载体,以脂质体法转染SGC-7901细胞及HeLa细胞,用间接免疫荧光、细胞计数等方法,检测其在两种细胞中的表达及其对两种细胞生长的影响。结果:成功构建了以R9为蛋白质转导结构域的嵌合抗体/caspase-3基因真核表达载体pCMV-e23sFv-R9-casp3,瞬时转染该质粒发现SGC-7901细胞和HeLa细胞均以分泌形式表达e23sFv-R9-casp3蛋白,但SGC-7901细胞生长受到明显抑制,细胞形态发生改变,出现核浓缩等凋亡特征;而HeLa细胞生长未受抑制,细胞形态无明显变化。结论:以分泌形式表达的嵌合蛋白,其HER2抗体部分能够介导靶向识别,R9可以辅助效应分子活化、转位至细胞液,活性caspase-3能高效杀伤细胞。
OBJECTIVE: To construct an eukaryotic expression vector of chimeric antibody / caspase-3 with oligo-arginine (R9) as the protein transduction domain and detect the expression of HER2-positive SGC-7901 tumor cells and HER2-negative HeLa tumor cell growth. Methods: The active caspase-3 gene fragment fused with R9 was obtained by PCR and cloned into the eukaryotic expression vector containing e23sFv gene of HER2. The recombinant plasmid was transfected into SGC-7901 cells and HeLa cells by lipofectamine. Indirect immunofluorescence, cell counting and other methods to detect its expression in two kinds of cells and its effect on the growth of two kinds of cells. Results: The eukaryotic expression vector pCMV-e23sFv-R9-casp3 of chimeric antibody / caspase-3 gene with R9 as the protein transduction domain was successfully constructed. Transient transfection of this plasmid revealed that both SGC-7901 cells and HeLa cells were secreted The expression of e23sFv-R9-casp3 protein was observed in SGC-7901 cells. However, the growth of SGC-7901 cells was inhibited obviously. The morphological changes of cells were observed. The apoptosis of HepG2 cells was not inhibited and the cell morphology was not changed. Conclusion: The chimeric protein expressed in secretory form can partially recognize the HER2 antibody. R9 can assist in the activation of effector molecules and translocation into the cytosol. The active caspase-3 can kill cells efficiently.