人GRB2基因真核表达及其在293T细胞中的定位研究

Human GRB2 eukaryotic expression and cellular localization in 293T cells

[目的]构建人GRB2蛋白真核表达质粒p Enter-GRB2,并观察其在293T细胞中的定位。[方法]从Hela细胞中提取总RNA,通过RT-PCR获得GRB2全长c DNA序列,并将其克隆到真核表达载体p Enter中,构建重组质粒p Enter-GRB2。转染293T细胞24 h、36 h和48 h后通过Western Blot检测其表达情况,通过免疫荧光观察其在细胞中的定位情况。[结果]经双酶切及测序鉴定,GRB2开放阅读框正确地构建到了真核表达质粒p Enter中,免疫印迹检测可见大小约为25 k Da的特异性条带,免疫荧光实验表明GRB2蛋白在细胞质和细胞核中均存在。[结论]成功构建了真核表达质粒p Enter-GRB2并在293T细胞中表达,证实GRB2在293T细胞中不仅存在于细胞质中,也存在于细胞核中,为更加深入地研究其生物学功能及机制奠定了理论基础。

[ Objective] To construct the recombinant plasmid pEnter- GRB2 expressing GRB2 protein in eukaryotic ceils and observe the cellular localization of GRB2 in 293T cells. [ Methods ] The cDNA fragment encoding GRB2 was amplified by RT - PCR with the total RNA extracted from Hela cells and inserted into MCS of pEnter. After identification by dual - enzyme digestion and sequencing,the positive recombinant plasmid was transiently transfected into 293T cells for 24 hours or 48 hours and the cellular localization of GRB2 was observed with the help of confocal laser scanning microscope. [ Results ] The ORF of GRB2 was inserted into the right place of pEnter. There was a specific band at 25 kDa and red fluorescence could be seen in both cytoplasm and nucleus. [ Conclusion] The eukaryotic expression plasmid pEnter- GRB2 was successfully constructed and expressed effectively in both cytoplasm and nucleus of 293T cells, Which lay the basis for further research of biological function and mechanism of GRB2.