牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备

doi:10.3969/j.issn.1004-1524.2019.11.07

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (11): 1819-1824.DOI: 10.3969/j.issn.1004-1524.2019.11.07

牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备

张康¹, 张璇², 闫遵祥¹, 王磊¹, 张凯¹, 张景艳¹, 罗永江¹, 仇正英¹, 薛欢³, 李建喜^1,*

1.中国农业科学院兰州畜牧与兽药研究所/农业农村部兽用药物创新重点实验室/甘肃省新兽药工程重点实验室,甘肃兰州730050;
2.兰州理工大学生命科学与工程学院,甘肃兰州 730050;
3.西安交通大学基础医学院,陕西西安 710061

收稿日期:2019-04-20 出版日期:2019-11-25 发布日期:2019-12-04
通讯作者: *,李建喜,E-mail: Lzjianxil@163.com
作者简介:张康(1987—),男,甘肃兰州人,助理研究员,主要从事奶牛疾病免疫及预防。E-mail: 467863181@qq.com
基金资助:
国家奶牛产业技术体系(CARS36); 中央级公益性科研院所基本科研业务费专项(1610322019005); 中国农业科学院科技创新工程协同创新(CAAS-XTCX2016011-01-09); 中兽医与临床科技创新工程(CAAS-ASTIP-2015-LIHPS)

Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies

ZHANG Kang¹, ZHANG Xuan², YAN Zunxiang¹, WANG Lei¹, ZHANG Kai¹, ZHANG Jingyan¹, LUO Yongjiang¹, QIU Zhengying¹, XUE Huan³, LI Jianxi^1,*

1. Key Laboratory of New Animal Drug Project of Gansu Province, Key Laboratory of Veterinary Drug Innovation of Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, China;
2. College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China;
3. School of Basic Medical Science, Xi'an Jiaotong University, Xi'an 710061, China

Received:2019-04-20 Online:2019-11-25 Published:2019-12-04

摘要/Abstract

摘要： 为制备牛病毒性腹泻病毒(BVDV)的Core蛋白及其多克隆抗体,根据Core蛋白的编码基因序列,设计合成一对特异性引物,利用RT-PCR扩增BVDV Core基因并定向插入Pet30a载体中,构建重组质粒Pet30a-Core,经酶切鉴定后转化大肠埃希菌TOP 10感受态细胞,筛选获得阳性重组菌,以IPTG进行诱导后成功表达出分子质量为20 ku的重组Core蛋白。将诱导表达的蛋白产物经融合蛋白的Ni柱亲和法进行纯化,利用纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,并利用间接ELISA法测出多克隆抗体效价达到1∶512 000。蛋白质印迹法(Western blot)和间接免疫荧光试验(IFA)证实,多克隆抗体可与细胞中的BVDV抗原发生反应,具有良好的免疫原性和特异性。本实验成功制备了BVDV重组Core蛋白的兔源多克隆抗体,为BVDV的检测及其Core蛋白功能研究奠定了基础。

关键词: 牛病毒性腹泻病毒, Core蛋白, 表达, 多克隆抗体

Abstract: In order to obtain the Core protein of bovine viral diarrhea virus (BVDV) and its polyclonal antibody, a pair of specific primers was designed and synthesized according to the coding sequence of the Core protein, and the BVDV Core gene was amplified by RT-PCR and inserted into the Pet30a vector. The recombinant plasmid Pet30a-Core was constructed and transformed into Escherichia coli TOP 10 competent cells by enzyme digestion. The positive recombinant bacteria were screened and the recombinant Core protein with molecular mass of 20 ku was induced to express with IPTG. The protein was purified by Ni column affinity method of the fusion protein, and the polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purified recombinant protein, and the polyclonal antibody titer was determined by indirect ELISA to reach 1∶512 000. Western blot and indirect immunofluorescence assay (IFA) confirmed that polyclonal antibodies reacted with BVDV antigens in cells and had good immunogenicity and specificity. The rabbit polyclonal antibody against BVDV recombinant Core protein was successfully prepared, which laid a foundation for the detection of BVDV and the study of Core protein function.

Key words: bovine viral diarrhoea viruses (BVDV), Core protein, expression, polyclonal antibodies

中图分类号:

S858.23

张康, 张璇, 闫遵祥, 王磊, 张凯, 张景艳, 罗永江, 仇正英, 薛欢, 李建喜. 牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备[J]. 浙江农业学报, 2019, 31(11): 1819-1824.

ZHANG Kang, ZHANG Xuan, YAN Zunxiang, WANG Lei, ZHANG Kai, ZHANG Jingyan, LUO Yongjiang, QIU Zhengying, XUE Huan, LI Jianxi. Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies[J]. , 2019, 31(11): 1819-1824.

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牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备

Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies

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