非洲猪瘟病毒B438L蛋白的原核表达及其多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2021.03.023

摘要/Abstract

摘要： 本研究旨在通过构建非洲猪瘟病毒（ASFV）B438L基因原核表达系统表达p49重组蛋白，并制备抗p49蛋白的多克隆抗体。根据ASFV Georgia 2007/1（GenBank登录号：FR682468.1）公布的基因序列合成B438L基因，并将其插入pET-28a （+）载体，构建pET-28a-B438L重组质粒，转化大肠杆菌BL21（DE3）感受态细胞，在16 ℃经1 mmol/L IPTG诱导12 h，通过SDS-PAGE对重组蛋白表达形式进行分析；利用串联质谱技术鉴定重组蛋白是否正确表达；将纯化的p49重组蛋白免疫小鼠制备鼠源抗p49多克隆抗体，利用间接ELISA方法测定该多克隆抗体的效价，并以间接免疫荧光试验及Western blotting检测该多克隆抗体的特异性。结果显示，试验成功构建了pET-28a-B438L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了p49重组蛋白，重组蛋白主要以包涵体形式表达，大小约为60 ku；串联质谱技术进一步证实该蛋白为p49。间接ELISA检测制备的鼠源抗p49多克隆抗体效价达1：64 000，间接免疫荧光试验及Western blotting结果表明制备的鼠源抗p49多克隆抗体能特异性识别p49蛋白。以上结果表明，该原核表达的ASFV p49重组蛋白具有良好的免疫原性，利用表达的p49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 p49蛋白的结构与功能、研制相关的ASFV诊断试剂及疫苗提供了生物材料。

关键词: 非洲猪瘟病毒(ASFV); B438L基因; p49重组蛋白; 原核表达; 多克隆抗体

Abstract: The purpose of this study was to express the recombinant protein p49 via constructing the prokaryotic expression system of African swine fever virus (ASFV) B438L gene,then prepare the polyclonal antibody against p49 protein.B438L gene was synthesized according the gene sequence of ASFV Georgia 2007/1 (GenBank accession No.:FR682468.1),then inserted into pET-28a(+) vector to construct pET-28a-B438L recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,then induced by 1 mmol/L IPTG at 16 ℃ for 12 h,the recombinant protein expression form was identified by SDS-PAGE.MALDI-TOF/TOF was used to determine whether the recombinant protein was expressed correctly.The polyclonal antibody against p49 was prepared by immunizing mice with the purified recombinant protein p49,the antibody titer was detected by indirect ELISA,and the specificity was identified by indirect immunofluorescence assay (IFA) and Western blotting.The results showed the pET-28a-B438L was successfully constructed.Then the recombinant plasmid was transformed into E.coli BL21(DE3),and the recombinant protein was expressed mainly in the form of inclusion bodies,with mass at about 60 ku.The recombinant protein was further confirmed by tandem mass spectrometry.The indirect ELISA result showed that the titer of polyclonal antibodies was as high as 1:64 000.The results of IFA and Western blotting indicated that the polyclonal antibody could specifically recognize the recombinant protein p49.These results confirmed the recombinant protein p49 expressed via prokaryotic system had good immunogenicity,and the prepared polyclonal antibodies had high titer and specificity.This study provided technical support for further study of the biological structure and function of ASFV p49 protein for the further ASFV related diagnosis and vaccine development.

Key words: African swine fever virus (ASFV); B438L gene; p49 recombinant protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺1

刘雪婷, 王召阳, 鑫婷, 郭晓宇, 姜一曈, 崔帅, 于海男, 朱鸿飞, 贾红. 非洲猪瘟病毒B438L蛋白的原核表达及其多克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2021, 48(3): 991-1000.

LIU Xueting, WANG Zhaoyang, XIN Ting, GUO Xiaoyu, JIANG Yitong, CUI Shuai, YU Hainan, ZHU Hongfei, JIA Hong. Prokaryotic Expression and Polyclonal Antibody Preparation and Identification of African Swine Fever Virus B438L Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 991-1000.

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