人酪氨酸蛋白激酶Lyn的真核表达、纯化及鉴定  

Eukaryotic expression,protein purification and identification of human tyrosine-protein kinase Lyn

作　　者：张佳凤[1] 郝文波[1] 熊玉锋 徐岚[1] 庄斯慧 罗树红[1] 

机构地区：[1]南方医科大学生物技术学院抗体工程研究所,广东广州510515

出　　处：《生物技术》2016年第1期42-47,共6页Biotechnology

基　　金：国家高技术研究发展技术(863)计划项目("蛋白质测序新技术新装备及配套试剂国产化";No.2014AA020904)资助

摘　　要：[目的]构建含人酪氨酸蛋白激酶Lyn基因的载体并进行真核表达、纯化和研究其对细胞增殖的影响。[方法]提取人Hela细胞总RNA,用RT-PCR方法获得Lyn基因并克隆至pcDNA3.1(-)载体。经双酶切、PCR和测序方法鉴定后,将重组质粒瞬时转染HEK 293T细胞表达目的蛋白,应用组氨酸标签镍离子螯合磁珠纯化融合蛋白,通过Western Blot检测蛋白的表达及纯化,并用CCK-8法检测过表达Lyn后细胞增殖能力的变化。[结果]成功构建真核表达质粒pcDNA3.1(-)-Lyn并进行瞬时表达和蛋白纯化,CCK-8法检测过表达Lyn的HEK 293T细胞的增殖能力显著性下降(P<0.01)。[结论]Lyn在HEK 293T细胞中成功瞬时表达及纯化,并可以使细胞的增殖能力受到明显抑制,为稳定表达和深入研究其生物学功能及作用机制奠定基础。[ Objective] To construct a eukaryotic expression plasmid of human tyrosineprotein kinase Lyn, obtain the His/ Mye -Lyn fusion protein and study its effect on cell proliferation. [ Methods] The full -length Lyn gene was amplified from total RNA in Hela cells by RT - PCR, and cloned into express vector pcDNA3.1 （ - ） - His/Myc to construct pcDNA3.1 （ - ） - Lyn. After transfeeting the recombinant plasmid transiently into HEK 293T cells, Western Blot was performed fordetecting the protein expression and the products purified by BeaverBeadsTMHis -tag Kit followed by CCK- 8 to measure cell proliferation. [ Results] The eukaryotic expression plasmid was constructed successfully and was expressed in HEK 293T cells. The results of CCK- 8 showed that the overexpression of Lyn in HEK 293T cells could significantly inhibit cell proliferation （P 〈 0. 01 ）. [ Conclusion] Lyn was expressed in HEK 293T cell and CCK -8 results suggest that overexpressed Lyn obviously inhibit cell proliferation which provides a foundation for further investigation of biological function and metabolic characteristics of Lyn.

关 键 词：酪氨酸蛋白激酶Lyn 真核表达 蛋白纯化 细胞增殖 

分 类 号：Q291[生物学—细胞生物学]