小鼠4-1BBL基因真核表达载体构建及其在COS-7细胞中的表达

目的：构建含小鼠4-1BBL编码区cDNA序列的真核表达载体，并检测其在COS-7细胞中的表达。方法：将目的基因4-1BBL克隆人pUCl9载体中并进行酶切鉴定和DNA序列测定。亚克隆入真核质粒pIRES2-EGFP，构建真核表达载体pIRES2-EGFP-m4-1BBL。用脂质体法将重组质粒转入COS-7细胞，以RT-PER和观察细胞内荧光的方法检测m4-1BBL的表达。结果：酶切鉴定和序列分析证实，重组质粒含有人4-lBBL全长cDNA编码序列，转染实验表明4-lBBL基因能在COS-7细胞中表达。结论：鼠4-1BBL全长cDNA基因真核表达载体构建及其在COS-7中的表达均获成功，为进一步研究奠定了基础。

Construction of eukaryotic expression vectors containing mouse 4-1BBL gene and its expression in mammalian cells

Objective:To construct an eukaryotic expression vector containing the coding region of mouse 4-1BBL cDNA and detect its expression in COS-7 cells. Methods:The m4-1BBL gene was ligated into the multiple clone site of pUC19 vector by gene recombination technique. After sequencing, the cDNA was recombinated into a eukaryotic expression vector pIRES 2-EGFP. Then the recombinated plasmid pIRES 2-EGFP-m4-1BBL was transfected into COS-7 cells using Lipofectamine 2000. The m4-1BBL mRNA of transfected cells was detected by RT-PCR. Green fluorescence was observed by fluorescence microscope. Results:Restriction analysis and DNA sequence analysis showed that the m4-1BBL cDNA has been successfully inserted into pIRES 2-EGFP eukaryotic expression vector. The COS-7 cells transfected with pIRES 2-EGFP-m4-1BBL could be detected expressing m4-1BBL.Conclusion: The eukaryotic expression vector containing m4-1BBL gene was successfully constructed and expressed. This study lay the foundation for further research on m4-1BBL.