SARS-CoV結(jié)構(gòu)蛋白的表達(dá)、純化和鑒定及中國家養(yǎng)果子貍類SARS-CoV病毒的血清學(xué)檢測
發(fā)布時間：2018-07-25 06:15
【摘要】：本論文在原核表達(dá)系統(tǒng)中對SARS-CoV 的主要結(jié)構(gòu)蛋白基因進(jìn)行了克隆、表達(dá)與多克隆抗體制備,同時對中國家養(yǎng)果子貍類SARS-CoV 病毒流行情況在血清學(xué)檢測方面做了初步研究,論文包括四個章節(jié)。 第一章對SARS-CoV 近年的研究進(jìn)展作了簡要綜述,并對本研究的背景和目的意義作了簡要介紹。 第二章對SARS-CoV 的主要結(jié)構(gòu)蛋白基因基因進(jìn)行了克隆、原核表達(dá)、純化與多克隆抗體制備。根據(jù)SARS-CoV 基因組全序列,設(shè)計相應(yīng)的引物,用RT-PCR 的方法擴(kuò)增得到S, S_1, S_2, S_△,M, E, N,ORF03,ORF08 基因。將這些結(jié)構(gòu)蛋白基因分別克隆至原核表達(dá)載體pGEX-KG,經(jīng)IPTG 誘導(dǎo),S_1,S_2, S_△,M,E, N,ORF08 基因在E.coli BL21 (DE3)菌株中得到了高效表達(dá)。表達(dá)的目的蛋白大小分別為98 kDa,86 kDa, 58 kDa,52 kDa,34 kDa,74 kDa,38 kDa,我們純化了其中的5 種蛋白GST-S2, GST-S△, GST-E, GST-N , GST-08,并用純化了的蛋白免疫新西蘭大白兔制備多克隆抗體,1:2000 倍稀釋后用于Western Blot 分析,獲得特異性顯色信號,所得重組蛋白和抗體將用于SARS-CoV 的診斷、亞單位疫苗的研究。另外我們對所純化蛋白做免疫原性的初步分析,發(fā)現(xiàn)GST-N 蛋白免疫小鼠可以產(chǎn)生較強(qiáng)的免疫應(yīng)答,其抗體與滅活SARS-CoV 病毒產(chǎn)生特異性的反應(yīng),表明該蛋白具有較強(qiáng)的免疫原性,適用于SARS 的早期檢測。 第三章主要內(nèi)容為家養(yǎng)果子貍類SARS-CoV病毒的血清學(xué)檢測。我們在2003年和2004 年從不同的養(yǎng)殖場收集了110 份家養(yǎng)果子貍的血清,這些血清來自于中國四個省五個地區(qū)不同的養(yǎng)殖場,這些養(yǎng)殖場都在較為偏遠(yuǎn)的山區(qū)且沒有受2003 年SARS 流行的影響。我們用兩種不同的方法(Western blot 和ELISA)檢測中國家養(yǎng)果子貍血清中的抗體,結(jié)果顯示84%的樣品含有抗SARS-CoV 的抗體,ELISA 檢測還發(fā)現(xiàn)來自于不同地區(qū)的家養(yǎng)果子貍血清樣品含有抗SARS-CoV
[Abstract]:In this paper, the major structural protein genes of SARS-CoV were cloned in prokaryotic expression system, expressed with polyclonal antibody, and the serological detection of SARS-CoV virus in Chinese house-bred civets was preliminarily studied. The paper includes four chapters. In the first chapter, the research progress of SARS-CoV in recent years is briefly reviewed, and the background and purpose significance of this study are briefly introduced. In chapter 2, the main structural protein genes of SARS-CoV were cloned, expressed in prokaryotic, purified and prepared polyclonal antibody. According to the whole sequence of SARS-CoV genome, the corresponding primers were designed and amplified by RT-PCR. These structural protein genes were cloned into the prokaryotic expression vector pGEX-KG and were highly expressed in E.coli BL21 (DE3) strain by IPTG induction. The expressed target proteins were 98 kDa-86 kDa, 58 kDa-52-kDa-34 kDa-74 kDa-38kDa. five proteins, GST-S2, GST-S, GST-E, GST-N, GST-08, were purified. The purified proteins were used to immunize New Zealand white rabbits to prepare polyclonal antibodies, which were diluted by 12: 2000 times and then analyzed by Western Blot. Specific chromogenic signals were obtained and recombinant proteins and antibodies were obtained for the diagnosis of SARS-CoV and the study of subunit vaccines. In addition, we made a preliminary analysis of the immunogenicity of the purified protein, and found that the GST-N protein immunized mice could produce a strong immune response, and its antibody had a specific reaction with inactivated SARS-CoV virus, which indicated that the protein had strong immunogenicity. Suitable for early detection of SARS. The third chapter focuses on the serological detection of SARS-CoV virus in the family of civets. We collected 110 sera from different farms in 2003 and 2004 from four provinces and five regions in China. These farms are in remote mountain areas and are not affected by the 2003 SARS epidemic. Two different methods, (Western blot and ELISA, were used to detect the antibodies in the sera of Chinese house-grown civets. The results showed that 84% of the samples contained antibodies against SARS-CoV and the samples from different regions contained anti-SARS-CoV.
【學(xué)位授予單位】：中國科學(xué)院研究生院（武漢病毒研究所）
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R373
本文編號：2142883
[Abstract]:In this paper, the major structural protein genes of SARS-CoV were cloned in prokaryotic expression system, expressed with polyclonal antibody, and the serological detection of SARS-CoV virus in Chinese house-bred civets was preliminarily studied. The paper includes four chapters. In the first chapter, the research progress of SARS-CoV in recent years is briefly reviewed, and the background and purpose significance of this study are briefly introduced. In chapter 2, the main structural protein genes of SARS-CoV were cloned, expressed in prokaryotic, purified and prepared polyclonal antibody. According to the whole sequence of SARS-CoV genome, the corresponding primers were designed and amplified by RT-PCR. These structural protein genes were cloned into the prokaryotic expression vector pGEX-KG and were highly expressed in E.coli BL21 (DE3) strain by IPTG induction. The expressed target proteins were 98 kDa-86 kDa, 58 kDa-52-kDa-34 kDa-74 kDa-38kDa. five proteins, GST-S2, GST-S, GST-E, GST-N, GST-08, were purified. The purified proteins were used to immunize New Zealand white rabbits to prepare polyclonal antibodies, which were diluted by 12: 2000 times and then analyzed by Western Blot. Specific chromogenic signals were obtained and recombinant proteins and antibodies were obtained for the diagnosis of SARS-CoV and the study of subunit vaccines. In addition, we made a preliminary analysis of the immunogenicity of the purified protein, and found that the GST-N protein immunized mice could produce a strong immune response, and its antibody had a specific reaction with inactivated SARS-CoV virus, which indicated that the protein had strong immunogenicity. Suitable for early detection of SARS. The third chapter focuses on the serological detection of SARS-CoV virus in the family of civets. We collected 110 sera from different farms in 2003 and 2004 from four provinces and five regions in China. These farms are in remote mountain areas and are not affected by the 2003 SARS epidemic. Two different methods, (Western blot and ELISA, were used to detect the antibodies in the sera of Chinese house-grown civets. The results showed that 84% of the samples contained antibodies against SARS-CoV and the samples from different regions contained anti-SARS-CoV.
【學(xué)位授予單位】：中國科學(xué)院研究生院（武漢病毒研究所）
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R373
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