【摘要】：牛病毒性腹瀉-黏膜病毒(BVDV)是一種引起牛病毒性腹瀉-黏膜病的病毒,對畜牧養(yǎng)殖業(yè)構(gòu)成嚴(yán)重威脅。為改變這一嚴(yán)重形勢,BVDV病毒研究刻不容緩。本文致力于穩(wěn)定表達(dá)牛病毒性腹瀉病毒衣殼蛋白的牛腎細(xì)胞系的構(gòu)建,這是為后期牛病毒性腹瀉-黏膜病毒疫苗的研究奠定基礎(chǔ),進(jìn)而為食源性危害問題的防御提供一道保障。本研究采用質(zhì)粒構(gòu)建的方法得到表達(dá)衣殼蛋白的重組質(zhì)粒,通過電穿孔法進(jìn)行細(xì)胞轉(zhuǎn)染,通過對穩(wěn)定細(xì)胞系的篩選及蛋白免疫印跡的鑒定,最終獲得一株穩(wěn)定表達(dá)牛病毒性腹瀉病毒衣殼蛋白的牛腎細(xì)胞系。主要結(jié)果如下:(1)以含有BVDV SD-1毒株基因的質(zhì)粒pUC19-SD1-1為模板,通過PCR得到目的基因Npro-C-Ems; (2)以真核細(xì)胞表達(dá)載體pcDNA3.1(+)為載體,構(gòu)建表達(dá)病毒蛋白Npro-C-Ems的重組質(zhì)粒pc-Npro-C-Ems; (3)通過電轉(zhuǎn)的方法,將質(zhì)粒pc-Npro-C-Ems轉(zhuǎn)入MDBK細(xì)胞中進(jìn)行穩(wěn)定篩選;(4)用Western blotting的方法驗證篩選出能夠穩(wěn)定地表達(dá)病毒BVDV衣殼蛋白的細(xì)胞克隆。本研究最終得到了一株能夠穩(wěn)定表達(dá)牛病毒性腹瀉病毒BVDV衣殼蛋白的牛腎細(xì)胞系pc- Npro-C-ms-457，該細(xì)胞的成功構(gòu)建將為進(jìn)一步的牛病毒性腹瀉-黏膜病毒疫苗的開發(fā)研究提供輔助原料,奠定堅實的研究基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.653

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