新型鸭呼肠孤病毒DH13株σC蛋白的原核表达及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2020.09.030

摘要/Abstract

摘要： 试验旨在建立新型鸭呼肠孤病毒（NDRV）σC蛋白的原核表达系统，制备σC蛋白的多克隆抗体，并评估所制备抗体的效价。通过RT-PCR方法扩增获得NDRV DH13株σC基因，连接至pET-30a（+）及pET-32a（+）载体中，构建原核表达质粒，并将其转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导获得两种σC重组蛋白，经SDS-PAGE分析蛋白表达形式。利用切胶方法纯化不含His标签σC重组蛋白，利用Ni-NTA柱纯化含His标签σC重组蛋白。以纯化的无His标签蛋白为免疫原免疫兔子获得兔抗σC多克隆抗体，Western blotting分别使用抗His标签的单克隆抗体和NDRV-σC蛋白阳性血清两种一抗评估抗体的特异性，间接ELISA（iELISA）检测抗体滴度。SDS-PAGE结果显示获得34和37 ku两种重组蛋白，且均得到高效表达。Western blotting结果显示制备的多克隆抗体具有良好的特异性，所制备的抗体与σC蛋白亲和力较好。间接ELISA检测结果显示其效价达1：25 600。本试验成功构建σC蛋白的原核表达系统，制备的σC蛋白多克隆抗体为深入研究σC蛋白的生物学功能及开展NDRV基因工程疫苗的研发奠定了基础。

关键词: 新型鸭呼肠孤病毒; σC蛋白; 原核表达; 多克隆抗体

Abstract: The aims of the experiment was to optimize the prokaryotic expression system of σC protein,prepare polyclonal antibody against σC protein of novel duck reovirus (NDRV),and evaluate the titer of the antibody.The σC gene of NDRV-DH13 strain was amplified by RT-PCR,ligated into pET-30a(+) and pET-32a(+) expression vector,constructed prokaryotic expression plasmid,which were transformed into E.coli BL21(DE3) and the expression of the σC protein were induced by IPTG.The proteins expression were analyzed by SDS-PAGE.The recombinant protein without His tag was purified by digestion,and the recombinant protein with His-tagged was purified by Ni-NTA column.Then the polyclonal antibody was obtained from rabbits which had been immunized by the purified protein without His tag.Anti-His-labeled mAb and NDRV-σC positive serum were used as primary antibodies to evaluate antibodies specificity,the antibodies titer was detected by indirect ELISA (iELISA).SDS-PAGE results showed that the molecular weight of the expression on recombinant proteins were 34 and 37 ku respectively,the proteins were highly expression.Western blotting showed that they had the specific reaction and the prepared antibodies had higher affinity with σC protein,the titer were about 1:25 600 by iELISA detection.This study successfully constructed and optimized the prokaryotic expression system of the polyclonal antibody against σC protein,laid a foundation for the further study of σC protein and the research of genetically engineered vaccine.

Key words: novel duck reovirus; σC protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺9.4

李文俊, 黄惠兰, 杨惠湖, 谢梓民, 崔惠仪, 黄淑坚, 张雪莲. 新型鸭呼肠孤病毒DH13株σC蛋白的原核表达及其多克隆抗体制备[J]. 中国畜牧兽医, 2020, 47(9): 2953-2959.

LI Wenjun, HUANG Huilan, YANG Huihu, XIE Zimin, CUI Huiyi, HUANG Shujian, ZHANG Xuelian. Prokaryotic Expression and Polyclonal Antibody Preparation of σC Protein of Novel Duck Reovirus DH13 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2953-2959.

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