HuR基因克隆及其在肝癌细胞中的表达

HuR gene clone and construction of its eukaryotic expressional vector

目的:克隆人抗原R基因(HuR)的cDNA,构建重组真核表达载体pEGFP-HuR,并稳定转染肝癌细胞株。方法:应用逆转录聚合酶链反应技术,从肝癌细胞中扩增得到人HuR基因的cDNA序列,克隆至增强型绿色荧光蛋白表达载体中,经Xho I和EcoR I双酶切和DNA测序鉴定后,将重组真核表达载体通过脂质体法导入肝癌细胞中,利用新霉素(G418)筛选出稳定转染pEGFP-HuR的肝癌细胞株,并用Western blot技术检测HuR基因的表达。结果:Xho I和EcoR I双酶切和PCR结果证实真核表达载体pEGFP-HuR构建成功。Western blot结果显示,在稳定转染重组真核表达载体的肝癌细胞中HuR基因表达水平上调。结论:成功构建了真核表达载体pEGFP-HuR,并筛选获得其稳定转染后HuR表达上调的肝癌细胞株,作为进一步研究HuR基因生物学功能的前期工作。

Objective: To construct the eukaryotic expression vector pEGFP-HuR and screen its stable transfected hepatocellular carcinoma cells. Methods: The coding region of HuR gene sequence was amplified from SMMC-7721 cells by reversing transcription polymerase chain reaction( RT-PCR). The fragment was inserted into green fluorescent protein( GFP) expression vector pEGFP-C1 digested by Xho I and EcoR I enzymes,the same as PCR product digestion. The recombinant plasmid was transfected into SMMC-7721 cells and the pEGFP-HuR stable transfected hepatocellular carcinoma cells were screened by G418 drug. The expression of HuR gene was detected by Western blot. Results: Both double digestion analysis by Xho I and EcoR I and DNA sequencing result verified that the pEGFP-HuR eukaryotic expression vector was successfully constructed,the HuR protein level in pEGFP-HuR stable transfection cells was obviously raised. Conclusion: The pEGFP-HuR eukaryotic expression vector was successfully constructed and HuR gene was significantly expressed in stable transfection cells,which provides the help for the study of HuR gene.