LyGDI真核表达载体构建及稳定转染A549细胞株的建立

Construction of Eukaryotic Expressing Vector of LyGDI and Establishment of Stable Transfectant A549 Cell Line

目的构建Rho蛋白鸟苷解离抑制因子(LyGDI)真核表达载体并建立稳定转染A549细胞株。方法PCR扩增LyGDI基因片段,构建pEGFP-C1-LyGDI真核表达载体,经酶切、PCR、测序验证其正确性。脂质体法转染真核细胞A549,G418筛选建立稳定转染的细胞株,RT-PCR、免疫印迹检测稳定转染的细胞株。结果构建了真核表达载体并建立了稳定转染的A549细胞株,成功表达LyGDI蛋白。结论LyGDI真核表达载体成功构建及稳定转染A549细胞株的建立,为研究LyGDI过度表达对肿瘤侵袭性和转移的影响奠定了实验基础。

Objective To construct the eukaryotic plasmid of Rho guanine nucleotide dissociation inhibitors-2 （LyGDI） and transfect A549 cell so as to establish stable cell line. Methods The gene of LyGDI was amplified by PCR . Eukaryotic vector pEGFP-Cl-LyGDI was constructed and confirmed by enzyme digestion,PCR,DNA sequencing. We transfected the recombinant vector into A549 cell by lipofectamineTM 2000.Stable transfected A549 cell line was established after screening culture by G418 and was identified by RT-PCR and Western blot. Results The eukaryotic expression vector pEGFP-Cl- LyGDI was constructed,stable transfected A549 cell line was established and LyGDI protein was expressed successfully. Conclusion The construction of the eukaryotie expression vector pEGFP-Cl- LyGDI and the establishment of stable transfected A549 cell line have laid the experiment foundation for further studies on the effect of over expression on invasion and metastasis of cancer.