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目的:构建HER2靶向免疫促凋亡蛋白e23sFv-Fdt-tBid真核表达载体,并运用Flp-InTM定点整合表达系统建立稳定表达该细胞的CHO工程细胞株。方法:应用PCR技术以pcMV-e23sFv-Fdt-tBid为模板扩增e23sFv-Fdt-tBid片段,并将其克隆入pcDNA5/FRT载体中;通过Flp-InTM定点整合表达系统将e23sFv-Fdt-tBid整合入Flp-lnTM CHO细胞的基因组中一个单拷贝位点上,以适当的潮霉素B筛选定点整合e23sFv-Fdt-tBid cDNA的细胞克隆;通过基因组PCR技术和Westernblot技术检测e23sFv-Fdt-tBid基因的整合及该蛋白表达。结果:成功构建了pcDNA5/FRT-e23sFv-Fdt-tBid表达载体;成功通过Flp-InTM定点整合表达系统建立稳定表达e23sFv-Fdt-tBid蛋白的CHO工程细胞株;成功通过基因组PCR技术及基因组PCR技术和Western blot技术检测了e23sFv-Fdt-tBid基因的整合及该蛋白表达。结论:成功地构建了HER2靶向免疫促凋亡蛋白e23sFv-Fdt-tBid真核表达载体并建立了稳定表达该蛋白的CHO工程细胞株,为该蛋白的应用研究奠定了重要基础。
OBJECTIVE: To construct eukaryotic expression vector of HER2 targeting pro-apoptotic protein e23sFv-Fdt-tBid and to establish CHO cell line stably expressing the cell line using Flp-InTM site-specific integrated expression system. METHODS: The e23sFv-Fdt-tBid fragment was amplified by PCR from pcMV-e23sFv-Fdt-tBid and cloned into pcDNA5 / FRT vector. The e23sFv-Fdt-tBid fragment was integrated by Flp-InTM site- One single-copy site in the genome of Flp-1nTM CHO cells was screened with suitable hygromycin B for the site-specific integration of the cell clones of e23sFv-Fdt-tBid cDNA; the e23sFv-Fdt-tBid gene was detected by genomic PCR and Western blot The integration and expression of this protein. Results: The pcDNA5 / FRT-e23sFv-Fdt-tBid expression vector was successfully constructed. The CHO cell line stably expressing e23sFv-Fdt-tBid protein was successfully established by Flp-InTM site-specific integrated expression system. Genomic PCR and genomic PCR Western blot was used to detect the integration of e23sFv-Fdt-tBid gene and its expression. CONCLUSION: The eukaryotic expression vector for HER2 targeting pro-apoptotic protein e23sFv-Fdt-tBid has been successfully constructed and a CHO cell line stably expressing the protein has been established, which laid an important foundation for the application of this protein.