RNA干扰体外抑制HBeAg表达的实验研究

发布时间：2018-01-16 21:36

本文关键词：RNA干扰体外抑制HBeAg表达的实验研究　出处：《重庆医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的及意义:构建带有绿色荧光蛋白报告基因的哺乳动物 shRNA表达载体和 HBeAg 真核表达载体,两者共转染 Hela 细胞,探讨 shRNA表达载体对 HBeAg 表达的抑制作用,为应用 RNAi 技术治疗乙型肝炎奠定一定的理论基础。方法:本研究采用 PCR 方法从含有人 U6 启动子的载体 PTZU6+1 中扩增 U6 启动子,将其克隆到 pEGFP-C1 载体中,通过限制性内切酶、PCR 及测序对构建的载体进行鉴定,并转染 Hela 细胞,用倒置荧光显微镜初步观察绿色荧光蛋白的表达;采用 PCR 的方法从质粒 PHBV1.5 中扩增乙肝病毒前 C/C 基因,克隆到 pCDNA3.1 的多克隆位点区 EcoRI 和 BamHI 位点,构建 HBeAg 表达载体,通过酶切、PCR及测序鉴定,把质粒转染 Hela 细胞,用 RT-PCR 检测 mRNA 的转录,免疫细胞化学检测HBeAg前体的表达,ELISA和MEIA分别检测细胞裂解液HBeAg 前体和培养上清 HBeAg 的表达;把构建的两载体按 7∶1 的比例共转染 Hela 细胞,用半定量 PCR 和 MEIA 分析 shRNA 表达载体对 HBeAg mRMA 和蛋白质的抑制情况;结果:经限制性内切酶、PCR 及测序证实已成功构建哺乳动物 shRNA 表达载体,该载体可以表达绿色荧光蛋白;成功构建了 HBeAg 表达载体,该载体可以在 Hela 细胞中表达 HBeAg 前体,并且可以分泌表达成熟的 HBeAg;初步筛选到 psiHBV2 载体对 HBeAg的表达有一定的抑制作用,其余两序列无抑制作用;结论:1、成功构建带有绿色荧光蛋白报告基因的哺乳动物 shRNA 表达载体,为应用该载体进行 RNAi 相关研究奠定一定的基础;2、成功构建乙肝病毒 HBeAg表达载体,该载体可以分泌表达 HBeAg,可以胜任 RNA 干扰的靶载体;
[Abstract]:Objective and significance: to construct mammalian shRNA expression vector and HBeAg eukaryotic expression vector with green fluorescent protein reporter gene and co-transfect them into Hela cells. To investigate the inhibitory effect of shRNA expression vector on HBeAg expression. Methods: in this study, PCR was used to amplify human U6 promoter from the vector PTZU6 1 containing human U6 promoter. U6 promoter. The recombinant plasmid was cloned into pEGFP-C1 vector and identified by restriction endonuclease polymerase chain reaction and sequencing. The vector was transfected into Hela cells. The expression of green fluorescent protein was preliminarily observed by inverted fluorescence microscope. The pre-C / C gene of hepatitis B virus was amplified from plasmid PHBV1.5 by PCR. The polyclonal region of pCDNA3.1 was cloned into EcoRI and BamHI sites. The expression vector of HBeAg was constructed and identified by restriction endonuclease digestion and sequencing. The plasmid was transfected into Hela cells. The transcription of mRNA was detected by RT-PCR and the expression of HBeAg precursor was detected by immunocytochemistry. The expression of HBeAg precursor and culture supernatant HBeAg were detected by ELISA and MEIA, respectively. The two vectors were co-transfected into Hela cells at the ratio of 7: 1. The inhibition of HBeAg mRMA and protein by shRNA expression vector was analyzed by semi-quantitative PCR and MEIA. Results: the mammalian shRNA expression vector was successfully constructed by restriction endonuclease polymerase chain reaction and sequencing, which could express green fluorescent protein. HBeAg expression vector was successfully constructed, which can express HBeAg precursor in Hela cells and secrete mature HBeAg. The results showed that psiHBV2 vector could inhibit the expression of HBeAg to some extent, but the other two sequences had no inhibitory effect on the expression of HBeAg. ConclusionThe mammalian shRNA expression vector with green fluorescent protein reporter gene was successfully constructed, which laid a foundation for the application of the vector in the study of RNAi. 2. The HBeAg expression vector of hepatitis B virus was constructed successfully. The vector can secrete HBeAgand be competent for the target vector of RNA interference.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【共引文献】