人生长分化因子-9(GDF-9)基因真核表达载体的构建及表达

Construction and expression of the eukaryotic expression vector of human growth differentiation factor-9(GDF-9) gene

目的:构建人生长分化因子-9(GDF-9)基因真核表达载体并分离纯化获得重组GDF-9蛋白。方法:以cDNA克隆为模板,PCR法扩增出人GDF-9基因,将目的基因连接到pcDNA3.1(+)真核表达载体,构建重组质粒pcDNA3.1(+)/GDF-9,脂质体法将重组质粒转染到HEK293T细胞,通过Western blotting检测GDF-9蛋白表达。结果:以双酶切及基因测序鉴定构建的重组真核质粒,Western blotting方法证实人GDF-9基因正确表达。结论:成功构建人GDF-9基因pcDNA3.1(+)/GDF-9重组质粒,并在真核细胞系表达,获得重组GDF-9蛋白,为进一步研究GDF-9基因提供便利工具。

Objective： To construct the eukaryotic expression vector of human growth differentiation factor-9 （GDF-9） gene and obtain purified recombinant GDF-9 protein. Methods： Human GDF-9 gene was cloned by PCR method using the complementary DNA, and was inserted into pcDNA3.1（＋） vector to construct recombinant eukaryotic expression plasmid pcDNA3.1 （＋）/GDF-9, which was transfected into human embryonic kidney （HEK） 293T cell line by liposome transfecting technique. The expression of GDF-9 was detected by Western blotting. Results： Recombinant plasmid constructed successfully was confirmed by double enzyme digestion and gene sequencing result. Recombinant protein GDF-9 successfully was verified by Western blotting. Conclusion： The recombinant eukaryotic expression plasmid pcDNA3.1 （＋）/GDF-9 was successfully constructed. Recombinant protein GDF-9 was successfully obtained, which is conducive for the further research of the function of GDF-9.