人apoA-I分泌型表达调控细胞模型构建

分别从pMD18-T质粒和人基因组DNA扩增出人apoA-I CDS区序列和apoA-I启动子(702bp片段),与pEGFP-N1重组,构成受apoA-I启动子调控的pEGFP-N1质粒和融合蛋白表达质粒,分别转染人肝癌HepG2细胞,以绿色荧光为标志筛选稳定转染系列克隆。用RT-PCR、荧光显微镜、免疫荧光术等鉴定其中一个克隆融合蛋白的表达;分别以胰岛素和葡萄糖刺激物鉴定该克隆的外源apoA-I启动子调控。结果表明:人apoA-I分泌型表达调控肝细胞模型初步建成。

The Cell Pattern Construction of Human apoA-I Gene Regulation and Secretory Expression

Amplified apoA-I CDS(region)from pMD18-T pl as mid and apoA-I promoter(702bp)DNA segment from human genomic DNA were cloned in pEGFP-N1 plasmid to generate a series of fusion protein expression plasmids. The expression of the fusion proteins are under the regulation of apoA-I promo ter. HepG2 cells were transfected with these fusion protein expression plasmids, and a series of stable fusion protein expression cell clones were screened by e nhanced green fluorescent protein(EGFP)marker. With RT-PCR,fluorescent micro scope,immunity fluorescent and other methods, one stable fusion protein express ion clone was identified. Insulin and glucose regulate the fusion protein expres sion. The results demonstrate that human apoA-I promoter can regulate gene expr ession of human liver cell in response to glucose and insulin in a similar way a s endogenous apoA-I,and the liver cell pattern of human apoA-I gene regulatio n and secretory expression has been established primarily.