稳定表达牛病毒性腹泻病毒衣壳蛋白牛肾细胞株构建
发布时间：2018-12-06 21:29
【摘要】：牛病毒性腹泻-黏膜病毒(BVDV)是一种引起牛病毒性腹泻-黏膜病的病毒,对畜牧养殖业构成严重威胁。为改变这一严重形势,BVDV病毒研究刻不容缓。本文致力于稳定表达牛病毒性腹泻病毒衣壳蛋白的牛肾细胞系的构建,这是为后期牛病毒性腹泻-黏膜病毒疫苗的研究奠定基础,进而为食源性危害问题的防御提供一道保障。本研究采用质粒构建的方法得到表达衣壳蛋白的重组质粒,通过电穿孔法进行细胞转染,通过对稳定细胞系的筛选及蛋白免疫印迹的鉴定,最终获得一株稳定表达牛病毒性腹泻病毒衣壳蛋白的牛肾细胞系。主要结果如下:(1)以含有BVDV SD-1毒株基因的质粒pUC19-SD1-1为模板,通过PCR得到目的基因Npro-C-Ems; (2)以真核细胞表达载体pcDNA3.1(+)为载体,构建表达病毒蛋白Npro-C-Ems的重组质粒pc-Npro-C-Ems; (3)通过电转的方法,将质粒pc-Npro-C-Ems转入MDBK细胞中进行稳定筛选;(4)用Western blotting的方法验证筛选出能够稳定地表达病毒BVDV衣壳蛋白的细胞克隆。本研究最终得到了一株能够稳定表达牛病毒性腹泻病毒BVDV衣壳蛋白的牛肾细胞系pc- Npro-C-ms-457，该细胞的成功构建将为进一步的牛病毒性腹泻-黏膜病毒疫苗的开发研究提供辅助原料,奠定坚实的研究基础。
[Abstract]:Bovine viral diarrhea-mucosal virus (BVDV) is a kind of virus that causes bovine viral diarrhea-mucosal disease, which poses a serious threat to animal husbandry. In order to change this serious situation, BVDV virus research is urgent. This paper is devoted to the construction of bovine kidney cell line expressing bovine viral diarrhea virus capsid protein stably, which lays a foundation for the research of bovine viral diarrhea mucosal virus vaccine in the later stage, and provides a guarantee for the defense of foodborne hazard. In this study, recombinant plasmids expressing capsid protein were obtained by the method of plasmid construction. The cells were transfected by electroporation, and the stable cell lines were screened and identified by Western blotting. A stable bovine kidney cell line expressing bovine viral diarrhea virus capsid protein was obtained. The main results are as follows: (1) using plasmid pUC19-SD1-1 containing BVDV SD-1 strain gene as template, the target gene Npro-C-Ems; was obtained by PCR. (2) using eukaryotic cell expression vector pcDNA3.1 () as vector, the recombinant plasmid pc-Npro-C-Ems; expressing viral protein Npro-C-Ems was constructed. (3) the plasmid pc-Npro-C-Ems was transferred into MDBK cells for stable screening by electroporation. (4) the cell clones which could stably express the capsid protein of virus BVDV were screened by Western blotting method. In this study, a bovine kidney cell line pc- Npro-C-ms-457, which can stably express bovine viral diarrhea virus BVDV capsid protein, was obtained. The successful construction of this cell will provide auxiliary raw materials for further development of bovine viral diarrhea mucosal virus vaccine and lay a solid foundation for research.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.653
本文编号：2366688
[Abstract]:Bovine viral diarrhea-mucosal virus (BVDV) is a kind of virus that causes bovine viral diarrhea-mucosal disease, which poses a serious threat to animal husbandry. In order to change this serious situation, BVDV virus research is urgent. This paper is devoted to the construction of bovine kidney cell line expressing bovine viral diarrhea virus capsid protein stably, which lays a foundation for the research of bovine viral diarrhea mucosal virus vaccine in the later stage, and provides a guarantee for the defense of foodborne hazard. In this study, recombinant plasmids expressing capsid protein were obtained by the method of plasmid construction. The cells were transfected by electroporation, and the stable cell lines were screened and identified by Western blotting. A stable bovine kidney cell line expressing bovine viral diarrhea virus capsid protein was obtained. The main results are as follows: (1) using plasmid pUC19-SD1-1 containing BVDV SD-1 strain gene as template, the target gene Npro-C-Ems; was obtained by PCR. (2) using eukaryotic cell expression vector pcDNA3.1 () as vector, the recombinant plasmid pc-Npro-C-Ems; expressing viral protein Npro-C-Ems was constructed. (3) the plasmid pc-Npro-C-Ems was transferred into MDBK cells for stable screening by electroporation. (4) the cell clones which could stably express the capsid protein of virus BVDV were screened by Western blotting method. In this study, a bovine kidney cell line pc- Npro-C-ms-457, which can stably express bovine viral diarrhea virus BVDV capsid protein, was obtained. The successful construction of this cell will provide auxiliary raw materials for further development of bovine viral diarrhea mucosal virus vaccine and lay a solid foundation for research.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.653
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