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目的:构建小鼠次级淋巴组织趋化因子(mSLC)高效真核细胞表达载体pVAX1-mSLC。方法:用BamHⅠ和XbaⅠ双酶切重组克隆pMD-mSLC,胶回收约410bp的SLC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HinDⅢ酶切鉴定。阳性克隆提取质粒,体外电穿孔法转染COS-7细胞,用Westernblot检测转染细胞中mSLC的瞬时表达。结果:菌落PCR和HinDⅢ酶切鉴定证实,mSLC基因片段正确插入到真核表达载体pVAX1中。Westernblot检测表明,以重组质粒pVAX1-mSLC转染的COS-7细胞能表达mSLC,表达产物的相对分子量同预期的结果相一致。结论:成功地构建小鼠SLC基因的真核表达质粒pVAX1-mSLC,用它转染COS-7细胞后,能瞬时表达mSLC,为下一步mSLC的肿瘤基因治疗研究奠定了基础。
Objective: To construct mouse eukaryotic expression vector pVAX1-mSLC for secondary lymphoid tissue chemokine (mSLC). Methods: The recombinant plasmid pMD-mSLC was digested with BamHⅠ and XbaⅠ. The fragment of about 410bp was amplified by Sj-PCR and subcloned into the corresponding restriction site of eukaryotic expression vector pVAX1. After transformation, colony PCR analysis and HinD Ⅲ digestion were carried out. The positive clones were used to extract plasmids. COS-7 cells were transfected by electroporation in vitro. The transient expression of mSLC in transfected cells was detected by Western blot. Results: The colony PCR and HinD Ⅲ digestion confirmed that the mSLC gene fragment was correctly inserted into the eukaryotic expression vector pVAX1. Western blot analysis showed that COS-7 cells transfected with the recombinant plasmid pVAX1-mSLC could express mSLC, and the relative molecular weight of the expressed product was consistent with the expected results. CONCLUSION: The eukaryotic expression plasmid pVAX1-mSLC of mouse SLC gene is successfully constructed. After transfection with COS-7 cells, it can transiently express mSLC, which lays the foundation for the further study of tumor gene therapy in mSLC.