非洲猪瘟病毒Georgia 2007/1株CD2v蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2019.12.029

摘要/Abstract

摘要： 本研究旨在通过原核表达系统表达非洲猪瘟病毒（African swine fever virus，ASFV）Georgia 2007/1株EP402R基因，获得其编码的CD2v重组蛋白，并针对纯化的CD2v重组蛋白制备多克隆抗体。将ASFV EP402R全长基因进行密码子优化后连入pET-28a（+）表达载体，构建原核重组表达质粒，经1 mmol/L IPTG于16 ℃诱导12 h后，利用SDS-PAGE和Western blotting对重组蛋白进行表达鉴定和反应原性分析。以纯化的CD2v重组蛋白为免疫原制备鼠源抗CD2v多克隆抗体，随后以间接ELISA方法、间接免疫荧光试验及Western blotting分别检测多克隆抗体的效价和特异性。结果显示，ASFV EP402R基因克隆至pET-28a（+）获得pET-28a-EP402R重组质粒，转化大肠杆菌BL21（DE3）感受态细胞后经诱导表达获得CD2v重组蛋白，其大小约为47 ku，重组蛋白主要以包涵体形式存在，部分也可以融合表达蛋白形式存在。Western blotting结果显示，其可溶性上清经镍柱纯化后能被ASFV阳性血清识别，具有良好的反应原性。间接ELISA检测该多克隆抗体效价可达1：512 000，间接免疫荧光试验和Western blotting表明该多克隆抗体可特异性识别真核表达的CD2v蛋白。以上结果表明，通过原核表达的ASFV CD2v重组蛋白具有较好的免疫原性，利用重组蛋白制备的多克隆抗体具有较高的抗体效价和特异性，为进一步研究ASFV EP402R生物学功能及基因缺失毒株的鉴别诊断和疫苗开发提供技术储备。

关键词: 非洲猪瘟病毒(ASFV); EP402R基因; CD2v重组蛋白; 原核表达; 多克隆抗体

Abstract: The study was aimed to express the EP402R gene of African swine fever virus (ASFV) Georgia 2007/1 strain via prokaryotic expression system,obtain the recombinant CD2v protein,and prepare polyclonal antibodies against the purified recombinant CD2v protein.After codon optimization,ASFV EP402R full-length gene was linked into pET-28a(+) expression vector to construct prokaryotic recombinant expression plasmid.After induction by 1 mmol/L IPTG at 16 ℃ for 12 h,the recombinant protein was identified by SDS-PAGE and Western blotting.The purified recombinant CD2v protein was used as immunogen to prepare mouse anti-CD2v polyclonal antibodies.The antibody titer was measured by indirect ELISA and the specificity was further analyzed by indirect immunofluorescence assay (IFA) and Western blotting.The results showed that ASFV EP402R gene was successfully cloned into pET-28a(+),and pET-28a-EP402R was obtained.The recombinant plasmid was transformed into E.coli BL21(DE3) for expression,the recombinant protein was expressed mainly in the form of inclusion bodies,with molecular mass at about 47 ku,while some of the recombinant protein could also exist in a soluble form.Western blotting results showed that the purified protein had good immunoreactivity.The indirect ELISA result showed that the polyclonal antibodies had a high titer of 1:512 000,IFA and Western blotting results indicated that it could specifically recognize recombinant CD2v protein.These results confirmed the recombinant CD2v protein expressed via prokaryotic system had good immunogenicity,and the prepared polyclonal antibodies had high titer and specificity.This research provided technical support for further study of ASFV EP402R biological function,as well as its gene-deletion based vaccine development.

Key words: African swine fever virus (ASFV); EP402R gene; CD2v recombinant protein; prokaryotic expression; polyclonal antibodies

中图分类号:

S852.65⁺1

任肖, 吴竞, 于海男, 林伟东, 侯绍华, 郭晓宇, 朱鸿飞. 非洲猪瘟病毒Georgia 2007/1株CD2v蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2019, 46(12): 3698-3706.

REN Xiao, WU Jing, YU Hainan, LIN Weidong, HOU Shaohua, GUO Xiaoyu, ZHU Hongfei. Prokaryotic Expression and Polyclonal Antibody Preparation of African Swine Fever Virus Georgia 2007/1 Strain CD2v Protein[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3698-3706.

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