猪硒蛋白W的原核表达及IgY多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2019.06.028

《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (6): 1801-1807.doi: 10.16431/j.cnki.1671-7236.2019.06.028

猪硒蛋白W的原核表达及IgY多克隆抗体制备

周继萍¹, 曹瑞敏¹, 米克热木·沙依布扎提¹, 蒋志惠², 张小莺^1,2,3

1. 新疆农业大学动物医学学院, 乌鲁木齐 830052;
2. 安阳工学院现代生物技术研究所, 河南省兽用生物制品研发与应用国际联合实验室, 安阳 455000;
3. 西北农林科技大学动物医学院, 杨凌 712100

收稿日期:2018-12-14 出版日期:2019-06-20 发布日期:2019-06-19
通讯作者: 蒋志惠, 张小莺 E-mail:jiangzhihui19870326@126.com;zhang.xy@nwsuaf.edu.cn
作者简介:周继萍(1996-),女,新疆昌吉人,硕士生,研究方向:抗体工程,E-mail:1538694137@qq.com
基金资助:
乌鲁木齐市科技计划项目（P16130001）；陕西省国际科技合作项目（2017KW-ZD-10）；河南省高校科技创新人才支持计划（18HASTIT035）；河南省高等学校重点科研项目应用研究计划（19B180001）；安阳工学院博士启动金（BSJ2017002）

Prokaryotic Expression of Porcine Selenoprotein W and Preparation of IgY Polyclonal Antibody

ZHOU Jiping¹, CAO Ruimin¹, MIKEREMU Shayibuzhati¹, JIANG Zhihui², ZHANG Xiaoying^1,2,3

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
2. Henan International Joint Laboratory for the Development and Application of Veterinary Biological Products, Institute of Modern Biotechnology, Anyang Institute of Technology, Anyang 455000, China;
3. College of Veterinary Medicine, Northwest A & F University, Yangling 712100, China

Received:2018-12-14 Online:2019-06-20 Published:2019-06-19

摘要/Abstract

摘要：

试验旨在优化硒蛋白W （selenoprotein W，SelW）的原核表达系统，并评估SelW的免疫原性和基于IgY抗体检测猪体内SelW的可行性。将获得的猪SelW基因序列密码子进行优化、合成并连接至pET-32a （+）表达载体中，构建原核表达重组质粒pET32a （+）-SelW，转化E.coli BL21（DE3）宿主菌中，进行IPTG诱导表达，SDS-PAGE鉴定重组猪SelW的表达情况，并免疫产蛋鸡制备抗SelW的IgY多克隆抗体，通过间接ELISA检测IgY抗体滴度，采用Western blotting检测IgY识别抗原的特异性。SDS-PAGE结果显示，SelW基因在原核细胞中成功表达，得到了大小约为33 ku的重组蛋白，IPTG最佳诱导浓度及时间分别为0.5 mmol/L和6 h；可溶性分析结果显示，重组蛋白SelW主要以包涵体形式存在。间接ELISA检测结果显示，免疫后45 d的IgY多克隆抗体的效价可达1：51 200。Western blotting检测结果显示，重组蛋白具有良好的免疫原性，所制备的IgY抗体与SelW的亲和力较高。本试验成功构建并优化了SelW蛋白的原核表达系统，提高了SelW蛋白的表达量，获得了具有良好免疫原性的SelW蛋白，制备的IgY抗体能特异性识别猪肌肉组织中的SelW，可用于检测猪组织中SelW的表达、预防硒中毒和缺硒性疾病的监测等，为进一步探究SelW的生物学功能奠定基础。

关键词: 猪; 硒蛋白W(SelW); 原核表达; IgY多克隆抗体

Abstract:

The aim of the experiment was to optimize the prokaryotic expression system of selenoprotein W (SelW),and evaluate the immunogenicity of SelW and the feasibility of detecting SelW in swine based on IgY antibodies.The obtained gene sequence codon of porcine SelW was optimized,synthesized and ligated into pET-32a(+) expression vector,and the prokaryotic expression recombinant plasmid pET32a(+)-SelW was constructed and transformed into E.coli BL21 host strain,which was induced by IPTG.The expression of SelW in recombinant swine was identified by SDS-PAGE,and the IgY polyclonal antibody against SelW was prepared by immunizing laying hens,the IgY antibody titer was detected by indirect ELISA,the specificity of the IgY recognition antigen was examined using Western blotting.The results of SDS-PAGE showed that SelW gene was successfully expressed in prokaryotic cells,and a recombinant protein with a size of about 33 ku was obtained.The optimal expression concentration and time of IPTG were 0.5 mmol/L and 6 h,respectively.Soluble analysis results showed that the recombinant protein existed mainly in the form of inclusion bodies.The results of indirect ELISA showed that the titer of IgY polyclonal antibody at 45 days after immunization could reach 1:51 200.Western blotting results showed that the recombinant protein had good immunogenicity,and the prepared IgY antibody had higher affinity with SelW.This experiment successfully constructed and optimized the prokaryotic expression system of SelW,increased the expression of SelW,and obtained SelW with good immunogenicity,and the prepared IgY antibody specifically recognized SelW in swine muscle tissue,which could be used to detect the expression of SelW in swine tissues.By measuring the expression of SelW to reflect the level of selenium in swine,it could be used to prevent selenium poisoning and selenium-deficient disease monitoring,and laid a foundation for further exploration of SelW biological function.

Key words: swine; selenprotein W(SelW); prokaryotic expression; IgY polyclonal antibody

中图分类号:

S828

周继萍, 曹瑞敏, 米克热木·沙依布扎提, 蒋志惠, 张小莺. 猪硒蛋白W的原核表达及IgY多克隆抗体制备[J]. 《中国畜牧兽医》, 2019, 46(6): 1801-1807.

ZHOU Jiping, CAO Ruimin, MIKEREMU Shayibuzhati, JIANG Zhihui, ZHANG Xiaoying. Prokaryotic Expression of Porcine Selenoprotein W and Preparation of IgY Polyclonal Antibody[J]. , 2019, 46(6): 1801-1807.

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猪硒蛋白W的原核表达及IgY多克隆抗体制备

Prokaryotic Expression of Porcine Selenoprotein W and Preparation of IgY Polyclonal Antibody

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