重组tBid融合蛋白基因的构建及其在HeLa细胞中的分泌表达

Construction of recombinant immuno-tBid gene and expression of a secreted fusion protein in HeLa cells

目的:构建重组的Immuno-tBid基因,并检测其在HeLa细胞中的分泌表达。方法:将肿瘤表面抗原HER2的单链抗体基因与绿脓杆菌外毒素PE的转膜结构域基因(PE253-364aa)和tBid基因连接,并在5'端连接前导肽基因,构建成Immuno-tBid(e23sFv-PEII-tBid61/tBid76)基因。将其克隆到pCMV载体并转染HeLa细胞,G418筛选获得阳性克隆,Genomic-PCR和RT-PCR分别扩增相应的重组基因片段。建株的HeLa细胞培养上清进行蛋白G琼脂糖凝胶免疫沉淀,Western Blot检测。并通过间接免疫荧光染色和细胞计数观察建株的HeLa细胞生长和形态变化。结果:成功构建了重组的Immuno-tBid基因的真核表达载体(pCMV-e23sFv-PEII(253-364)-tBid61/tBid76)。稳定转染HeLa细胞,获得G418抗性的阳性HeLa细胞克隆,Genomic-PCR检测证实目的基因已整合到细胞基因组中。RT-PCR检测表明目的基因在RNA水平上的表达。细胞培养上清进行蛋白G琼脂糖凝胶免疫沉淀,Western Blot检测,证实建株的HeLa细胞可以通过分泌途径产生Immuno-tBid蛋白。间接免疫荧光染色和核染色结果显示,分泌Immuno-tBid蛋白的HeLa细胞呈标准的S型曲线生长,形态正常。结论:成功构建了重组的Immuno-tBid基因,建株的HeLa细胞可以通过分泌途径产生Immuno-tBid蛋白。

Objective：To construct recombinant immuno - tBid genes and to test if targeted pro - apoptotic fusion protein, immuno- tBid, can be secreted in HeLa cells. Methods： Immuno- tBid gene was produced by sequential fusion of the following genes ： a signal peptide, a single - chain HER2 antibody （ sFv23e）, a PE translocation domain （aa253 -364） and tBid . The immuno- tBid genes were cloned into a pCMV plasmid, stably tranfected into HeLa cells,and selected by G418 . Then the muhiclone mixture of the viable cells were collected for Genomic -PCR and RT - PCR detection. The supernatant of the transduced cells were subjected to Western Blot analysis. Changes of the morphology and growth curve of transduced HeLa cells were studied by imm, unofluorescence staining and viable cell counting. Results： Recombinant immuno- tBid genes were successfully reconstructed. The G418 -resistant cells were subcloned, and confirmed that the target genes were integrated into cell genome by genomic - PCR. RT - PCR was used to demonstrate the expression of the target genes in RNA level. Western Blot of culture medium confirmed that transfected HeLa cells could express and secret immuno - tBid protein stably. Immuno - tBid - secreting HeLa cell exhibited similar growth curve and morphology to non -transfected HeLa cells. Concliusion： Recombinant immuno - tBid genes have been successfully constructed and its fusion protein can be expressed in the secreted form in HeLa cells