GRP78基因短发夹RNA表达载体的构建和鉴定

目的构建葡萄糖调节蛋白78(GRP78)基因短发夹RNA表达载体并鉴定,为探讨GRP78对胃癌发生发展作用及机制提供前期基础。方法运用核糖核酸(RNA)干扰技术构建靶向干扰GRP78蛋白短发夹RNA(GRP78-shRNA)载体,载体经转化、抽提及测序,用脂质体将GRP78-shRNA载体瞬时转染至SGC-7901胃癌细胞中,应用荧光显微镜、反转录定量PCR(qRT-PCR)及免疫印迹(Western blot)观察GRP78表达情况。结果GRP78-shRNA载体测序显示序列正确；转染GRP78-shRNA载体的SGC-7901胃癌细胞可见绿色荧光；qRT-PCR和Western blot结果显示,转染GRP78-shRNA载体的SGC-7901胃癌细胞中GRP78基因和蛋白表达降低。结论成功构建并鉴定GRP78基因短发夹RNA表达载体。

Construction and identification of short hairpin RNA expression vector of GRP78 gene

The short hairpin RNA expression vector of glucose regulatory protein 78 (GRP78) gene was constructed and identified, to provide a preliminary basis for exploring the effect and mechanism of GRP78 on the development of gastric cancer. MethodsThe GRP78 short hairpin Ribonucleic acid (GRP78-shRNA) vector was constructed by RNA interference technology. The vector was transformed, extracted and sequenced. The GRP78-shRNA vector was transiently transfected into SGC-7901 gastric cancer cell by liposome of lipofectamine 2000. The expression of GRP78 was observed by fluorescence microscopy, Quantitative Real-time PCR (qRT-PCR) and immunoblot(Western blot). ResultsThe sequence of GRP78-shRNA vector was correct. The green fluorescence was observed in SGC-7901 gastric cancer cell transfected with GRP78-shRNA vector. The qRT-PCR and Western blot results showed that the expression of GRP78 gene and protein was decreased in SGC-7901 gastric cancer cell transfected with GRP78-shRNA vector. ConclusionThe short hairpin RNA expression vector of GRP78 gene was successfully constructed and identified.