摘要
目的构建人pUB6/V5-HisB-SPOP真核表达质粒及稳定表达外源性人SPOP基因的人胃癌AGS细胞系。方法采用基因重组技术将人SPOP cDNA插入真核表达载体pUB6/V5-HisB,构建人pUB6/V5-HisB真核表达质粒。脂质体介导空载体pUB6/V5-HisB和表达质粒pUB6/V5-HisB-SPOP分别转染人胃癌AGS细胞株,杀稻瘟菌素筛选阳性克隆,扩大培养,逆转录-聚合酶链式反应(RT-PCR)和蛋白免疫印迹法(Western blot)分别检测SPOP mRNA和蛋白表达。结果双酶切和基因测序结果均证实人pUB6/V5-HisB-SPOP真核表达质粒构建成功。SPOP转染组与空白对照组、空载体组比较,SPOP蛋白表达显著上调(P<0.05)。结论成功构建人SPOP真核表达质粒并获得稳定表达SPOP基因的胃癌AGS细胞系,为进一步研究SPOP基因在胃癌发生发展中作用奠定实验基础。
Objective To construct human speckle-type POZ protein（SPOP） expression plasmid SPOP-V5/His and cell model with up-regulation of SPOP in human gastric cancer cell line AGS. Methods SPOP-VS/His was constructed by use of recombinant DNA technique and was demonstrated by restriction endonuelease mapping. SPOP-VS/His and pUB6/V5-HisB vector as control were transfected into human gastric cell line AGS by using Lipofectamine 2000 for screening stable cell lines. The positive clonies were selected by blastieidin,Western blot was used to detected the expression of SPOP. Results Eukaryotic expression plasmid SPOP-VS/His was successfully constructed and confirmed by restriction endonuclease digestion and sequencing. Western blot showed the level of SPOP in gastric cancer cell line AGS transfected with SPOP-V5/His was significantly increased compared with control. Conclusion We successfully constructed cell models with up-regulation of SPOP in human gastric cancer cell line AGS which is important for our further study on the role of SPOP in the development of gastric cancer.
出处
《重庆医学》
CAS
北大核心
2015年第17期2335-2337,共3页
Chongqing medicine
基金
江西省研究生创新专项资金(YC2011-B007)
