SARS冠状病毒N蛋白的细胞定位及其RNA干扰研究

发布时间：2018-01-21 04:49

本文关键词： SARS 冠状病毒核衣壳蛋白细胞定位短发夹状 RNA RNA 干扰抗病毒基因治疗　出处：《重庆医科大学》2005年博士论文　论文类型：学位论文

【摘要】：目的: 构建增强型绿色荧光蛋白(EGFP)与 SARS 冠状病毒不同长度核衣壳蛋白区段融合表达的 pEGFP-C1 重组载体,对 293 细胞进行瞬时转染,显微镜下观察核衣壳蛋白不同区段在细胞内的定位;构建增强型绿色荧光蛋白(EGFP)与 SARS 冠状病毒(SARS-CoV)各结构蛋白的融合表达载体以及各结构基因的短发夹状 RNA(shRNA)表达载体,将两种重组载体共转染 293 细胞后观察 shRNA 对 SARS 冠状病毒结构蛋白表达的影响。方法: 首先用两种蛋白分析软件 Prosite 和 PredictNLS server (prediction and analysis of nuclear localization signals , Columbia University Bioinformatics Center)分析 SARS 冠状病毒核衣壳蛋白(nucleocapsid protein,N 蛋白)序列,找出其中的核定位信号(nuclear localization signals, NLS)序列。 PCR 扩增或人工合成 SARS 冠状病毒不同长度核衣壳蛋白基因片段,分别与载体 pEGFP-C1 连接,得到不同长度 N 蛋白的重组表达载体。将重组的 pEGFP-C1 载体转染 293 细胞,分别在荧光显微镜和激光共聚焦显微镜下观察不同长度核衣壳蛋白在细胞内的定位。同时,将带有 N 基因全长的重组 pcDNA-3.1(-)载体转染 293 细胞,应用免疫
[Abstract]:Objective: to construct a recombinant pEGFP-C1 vector expressing enhanced green fluorescent protein (EGFP) and SARS coronavirus nucleocapsid protein with different length. The transient transfection of 293 cells was carried out and the localization of different regions of nucleocapsid protein in the cells was observed under microscope. Construction of fusion expression vector of enhanced green fluorescent protein (EGFP) with SARS coronavirus SARS-CoV and short hairpin RNAs of each structural gene. ShRNA) expression vector. The effects of shRNA on the expression of structural protein of SARS coronavirus were observed after co-transfection of two recombinant vectors into 293 cells. Methods: first, two protein analysis softwares, Prosite and PredictNLS server, were used. Prediction and analysis of nuclear localization signals. Analysis of SARS Coronavirus nucleocapsid protein by Columbia University Bioinformatics Center. Nucleocapsid protein. The sequence of nuclear localization signal (NLS) was found. The nucleocapsid protein gene fragments of SARS coronavirus were amplified by PCR and ligated with the vector pEGFP-C1. The recombinant expression vector of N protein with different length was obtained. The recombinant pEGFP-C1 vector was transfected into 293 cells. The localization of nucleocapsid proteins of different lengths was observed under fluorescence microscope and confocal laser microscope. Transfection of recombinant pcDNA-3.1 ~ (1 +) vector containing N gene into 293 cells was carried out and immunized.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373.1

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