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为了构建可在哺乳动物细胞内高效表达人次级淋巴组织趋化性细胞因子(hCCL21)的真核表达质粒pVAX1-hCCL21,以便用于hCCL21基因治疗研究。以BamHI和XhoI双酶切重组克隆pSK-hCCL21,胶回收约540bp的hCCL21目的基因片段,以亚克隆法构建于真核表达载体pVAX1的相应酶切位点,转化后进行菌落PCR分析和BamHI、XhoI双酶切鉴定。阳性克隆提取质粒,体外电穿孔法转染COS-7细胞,Westernblot法检测转染细胞中hCCL21的瞬时表达,趋化小室法检测表达产物对淋巴细胞的免疫趋化作用。结果显示,菌落PCR和酶切鉴定证实,目的基因hCCL21正确插入到真核表达载体pVAX1中,转染COS-7细胞后,细胞培养上清及裂解产物中可检测到重组蛋白hCCL21的表达,并且表达产物对人外周血单个核细胞有明显的趋化活性。已成功构建了可在哺乳动物细胞内高效表达hCCL21基因的真核表达质粒pVAX1-hCCL21,为进一步研究hCCL21基因治疗肿瘤奠定了基础。
To construct an eukaryotic expression plasmid pVAX1-hCCL21 that is highly efficient for expression of human secondary lymphoid chemotactic cytokines (hCCL21) in mammalian cells for use in hCCL21 gene therapy studies. The recombinant plasmid pSK-hCCL21 was digested with BamHI and XhoI. The hCCL21 gene fragment of about 540 bp was recovered and subcloned into the corresponding restriction sites of the eukaryotic expression vector pVAX1. After transformation, colony PCR analysis and BamHI, XhoI double digestion identification. The positive clones were used to extract the plasmids. The transfection of COS-7 cells was performed by electroporation in vitro. The transient expression of hCCL21 in transfected cells was detected by Western blot. The chemotaxis of lymphocytes was detected by chemotactic chamber assay. The results showed that colony PCR and restriction enzyme digestion confirmed that the target gene hCCL21 was correctly inserted into the eukaryotic expression vector pVAX1 and the expression of the recombinant protein hCCL21 was detected in the cell culture supernatant and the lysate after transfection of COS-7 cells The expressed product has obvious chemotactic activity on human peripheral blood mononuclear cells. The eukaryotic expression plasmid pVAX1-hCCL21, which can efficiently express hCCL21 gene in mammalian cells, has been successfully constructed, which lays the foundation for further study of hCCL21 gene therapy for tumors.