【作者】 陶鹏；

【导师】 黄爱龙；

【作者基本信息】 重庆医科大学 ， 内科学， 2005， 博士

【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,应用免疫更多还原

【Abstract】 Objective : To study the cellular localization of Severe Acute Respiratory Syndrome Associated Coronavirus (SARS-CoV) nucleocapsid protein, a series of fusion expression plasmids , containing EGFP (enhanced green fluorescent protein)genes and different length SARS-CoV nucleocapsid gene ,were constructed. To observe the effect of RNA interference on SARS-CoV structural gene expression, the following plasmids were constructed respectively: 1. the plasmids containing the structural genes of SARS-CoV and EGFP gene; 2. the shRNA plasmids targeting the structural genes of SARS-CoV. Methods:The sequence of SARS-CoV nucleocapsid protein was analyzed by Prosite and PredictNLS server (prediction and analysis of nuclear localization signals,Columbia University Bioinformatics Center) to identify nuclear localization signals(NLS). Different length fragments of SARS-CoV nucleocapsid gene were obtained by PCR or chemical synthesis. These fragments were cloned into pEGFP-C1 vector to form plasmids pEGFP-C1-N、pEGFP-C1-N1 and pEGFP-C1-N2 respectively. 293 cells were examined by fluorescence microscope and laser confocal microscope after being transfected with pEGFP-C1-N、pEGFP-C1-N1 and pEGFP-C1-N2 plasmid. At the same time, the cellular localization of SARS-CoV nucleocapsid protein was investigated by cell immuno-fluorescence, using FITC-labelled second antibody. The EGFP fusion expression plasmids were constructed by cloning synthesized SARS-CoV structural gene fragments into pEGFP-C1. The designed and synthesized shRNA sequences,containing a 19bp reverse repeated motif of target sequence with 4bp spacer ,were ligated into vector pTZU6+1 to generate shRNA plasmids. The shRNA plasmids were cotransfected with the EGFP fusion expression plasmids encoding the corresponding SARS-CoV proteins at a 5:1 mass ratio. Fluorescence was observed 48h after transfection. The expression levels of GFP and nucleocapsid protein were determined by Western blot respectively. The transcription of N gene was detected by RT-PCR. Results:By using PROSITE, we identified a nuclear localization signal sequence from amino acids 373 to 389 or from amino acids 374 to 390 . However, the analysis of PredictNLS server revealed that the nuclear localization signal region was located between amino acids 36 and 44. Agarose gel electrophoresis of PCR product showed that SARS-CoV Ngene was amplified. Identification of pEGFP-C1-N、pEGFP-C1-N1 and pEGFP-C1-N2 plasmid by restriction enzyme digest showed that different length fragments of SARS-CoV nucleocapsid gene had been cloned into pEGFP-C1. In addition, sequencing of the recombinant plasmids indicated that the inserted sequences were correct. Both the GFP fusion expression strategy and the immune fluorescence showed the following findings: N1 protein(1-187aa) was located in both cytoplasm and cell nucleus;N protein(1-422aa) and N2 protein(161-422aa) were located in cytoplasms . Restriction enzyme digest and DNA sequencing showed that the EGFP fusion expression plasmids(pEGFP-C1-N1 、 pEGFP-C1-E 、pEGFP-C1-M、and pEGFP-C1-S) and the shRNA plasmids (pshRNA-N1、 pshRNA-N2、pshRNA-E、pshRNA-M and pshRNA-S)were constructed successfully. Among a total of 5 shRNA plasmids,we obtained one shRNA plasmid which could sequence-specifically reduce target gene expression. The introduction of pshRNA-N1 plasmid efficiently and specifically inhibited the synthesis of protein N by western blot analysis of GFP fusion protein. RT-PCR showed that RNAs of N gene were clearly reduced when pEGFP-C1-N1 was cotransfected with pshRNA-N1, whereas the control vector did not exhibit inhibitory effect on N gene transcription. Conclusion : Successfully constructed the recombinant plasmidscontaining different structural genes of SARS-CoV. N1 protein was located in both cytoplasm and nucleus;N protein and N2 protein were located in cytoplasms. shRNAs targeting different SARS-CoV sequences have significantly various inhibitory effects on SARS-CoV gene expression. The introduction of pshRNA-N1 was shown to efficiently and更多还原