本文關(guān)鍵詞： SARS 冠狀病毒 核衣殼蛋白 細(xì)胞定位 短發(fā)夾狀 RNA RNA 干擾 抗病毒 基因治療　出處：《重慶醫(yī)科大學(xué)》2005年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的: 構(gòu)建增強(qiáng)型綠色熒光蛋白(EGFP)與 SARS 冠狀病毒不同長(zhǎng)度核衣殼蛋白區(qū)段融合表達(dá)的 pEGFP-C1 重組載體,對(duì) 293 細(xì)胞進(jìn)行瞬時(shí)轉(zhuǎn)染,顯微鏡下觀察核衣殼蛋白不同區(qū)段在細(xì)胞內(nèi)的定位;構(gòu)建增強(qiáng)型綠色熒光蛋白(EGFP)與 SARS 冠狀病毒(SARS-CoV)各結(jié)構(gòu)蛋白的融合表達(dá)載體以及各結(jié)構(gòu)基因的短發(fā)夾狀 RNA(shRNA)表達(dá)載體,將兩種重組載體共轉(zhuǎn)染 293 細(xì)胞后觀察 shRNA 對(duì) SARS 冠狀病毒結(jié)構(gòu)蛋白表達(dá)的影響。 方法: 首先用兩種蛋白分析軟件 Prosite 和 PredictNLS server (prediction and analysis of nuclear localization signals , Columbia University Bioinformatics Center)分析 SARS 冠狀病毒核衣殼蛋白(nucleocapsid protein,N 蛋白)序列,找出其中的核定位信號(hào)(nuclear localization signals, NLS)序列。 PCR 擴(kuò)增或人工合成 SARS 冠狀病毒不同長(zhǎng)度核衣殼蛋白基因片段,分別與載體 pEGFP-C1 連接,得到不同長(zhǎng)度 N 蛋白的重組表達(dá)載體。 將重組的 pEGFP-C1 載體轉(zhuǎn)染 293 細(xì)胞,分別在熒光顯微鏡和激光共聚焦顯微鏡下觀察不同長(zhǎng)度核衣殼蛋白在細(xì)胞內(nèi)的定位。同時(shí),將帶有 N 基因全長(zhǎng)的重組 pcDNA-3.1(-)載體轉(zhuǎn)染 293 細(xì)胞,應(yīng)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R373.1

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本文編號(hào)：1450518