小鼠4-1BBLcDNA的克隆及其在COS-7细胞中的表达

目的：克隆小鼠4-1 BBL基因，构建其真核表达载体，并观察其在哺乳动物细胞中的表达。方法：取C57BL/6小鼠脾细胞，经PHA诱导后，以RT-PCR克隆4-1 BBLcDNA，测序，构建pCDNA3．1( )-m4-1BBL真核表达质粒，转染COS-7细胞，RT-PCR检测m4-1BBLmRNA表达，间接免疫荧光法和流式细胞仪检测4-1 BBL蛋白的表达，结果：从小鼠脾细胞中克隆到m4-1 BBL cDNA，经测序完全正确，所构建的m4-1BBL质粒在COS-7细胞中获得高效表达。结论：小鼠4-1BBLcDNA基因克隆、真核表达载体构建及其在COS-7中的表达均获成功，为进一步研究其功能奠定了基础。

Cloning of mouse 4-1BBL gene and its expression in COS-7 cells

AIM: To obtain mouse 4 1BBL gene and construct its eukaryotic expression vector and express it in mammalian cells. METHODS: RT PCR was applied to amplify mouse 4 1BBL gene from total RNA of C57BL/6 mouse spleen cells after stimulating by PHA. Then cloning vector of m4 1BBL was constructed by gene recombination technique. After sequencing, the cDNA was recombinated into eukaryotic expression vector pCDNA3.1(+) and transfected into COS 7 cells. m4 1BBLmRNA of transfected cells was detected by RT PCR. Indirect immunofluorescent methods and flow cytometry were applied to test the expression of m4 1BBL protein. RESULTS: The m4 1BBL cDNA was obtained from C57BL/6 mouse spleen cells and was confirmed correct by sequence analysis. The COS 7 cells transfected with pCDNA3.1(+) m4 1BBL could efficiently express m4 1BBL mRNA and protein. CONCLUSION: The cloning of mouse 4 1BBL gene and the construction of its eukaryotic expression vector were all successful. This study lay the foundation for further research on m4 1BBL.