【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)構成嚴重威脅。為改變這一嚴重形勢,BVDV病毒研究刻不容緩。本文致力于穩(wěn)定表達牛病毒性腹瀉病毒衣殼蛋白的牛腎細胞系的構建,這是為后期牛病毒性腹瀉-黏膜病毒疫苗的研究奠定基礎,進而為食源性危害問題的防御提供一道保障。本研究采用質粒構建的方法得到表達衣殼蛋白的重組質粒,通過電穿孔法進行細胞轉染,通過對穩(wěn)定細胞系的篩選及蛋白免疫印跡的鑒定,最終獲得一株穩(wěn)定表達牛病毒性腹瀉病毒衣殼蛋白的牛腎細胞系。主要結果如下:(1)以含有BVDV SD-1毒株基因的質粒pUC19-SD1-1為模板,通過PCR得到目的基因Npro-C-Ems; (2)以真核細胞表達載體pcDNA3.1(+)為載體,構建表達病毒蛋白Npro-C-Ems的重組質粒pc-Npro-C-Ems; (3)通過電轉的方法,將質粒pc-Npro-C-Ems轉入MDBK細胞中進行穩(wěn)定篩選;(4)用Western blotting的方法驗證篩選出能夠穩(wěn)定地表達病毒BVDV衣殼蛋白的細胞克隆。本研究最終得到了一株能夠穩(wěn)定表達牛病毒性腹瀉病毒BVDV衣殼蛋白的牛腎細胞系pc- Npro-C-ms-457，該細胞的成功構建將為進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。
[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.
【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.653

【參考文獻】

相關期刊論文 前7條

1 劉嬡;馮將;鮮思美;劉宗勝;李鵬飛;羅代兵;;羊口瘡病毒F1L和B2L基因真核表達質粒構建及其在MDBK細胞中的表達[J];中國人獸共患病學報;2015年12期

2 范璐;王麗;史西保;樊劍鳴;王愛萍;張改平;;穩(wěn)定表達靶向BVDV shRNA細胞系的建立及其對BVDV復制的影響[J];河南農業(yè)科學;2014年12期

3 駱延波,張紹學,柴家前,王新平;牛病毒性腹瀉病毒免疫機理研究進展[J];山東農業(yè)大學學報(自然科學版);2000年02期

4 杜銳,王新平,宣華,王全凱,楊林,王樹志;從幼鹿頑固性腹瀉病料中檢出牛病毒性腹瀉——粘膜病病毒[J];經濟動物學報;1998年01期

5 孫序新，江煥賢，，陳永　;牦牛病毒性腹瀉—粘膜病病毒的分離及鑒定[J];中國獸藥雜志;1994年04期

6 申之義,王克恭,關平原,李平安;牛病毒性腹瀉/粘膜病病毒的分離鑒定 Ⅰ.病毒分離培養(yǎng)、理化特性測定、中和試驗[J];中國獸醫(yī)雜志;1993年03期

7 李佑民,劉振潤,武銀蓮;牛病毒性腹瀉—粘膜病病毒株(長春184)的分離與鑒定[J];獸醫(yī)大學學報;1983年02期

相關博士學位論文 前1條

1 付強;牛病毒性腹瀉病毒持續(xù)性感染的分子機制研究及其定點突變株的構建[D];石河子大學;2015年

相關碩士學位論文 前1條

1 王銀;表達牛病毒性腹瀉病毒E2基因的重組I型牛皰疹病毒的構建[D];揚州大學;2013年

本文編號：2366688