牛源副结核分枝杆菌MAP3732c基因的原核表达与多克隆抗体的制备
Prokaryotic expression of Mycobacterium avium subspecies paratu-berculosis MAP3732c of cow origin and preparation of polyclonal antibodies
马聿田 1李阳 1李晓宇 1其勒木格 1吉林台 1张文彦 2希尼尼根1
作者信息
- 1. 内蒙古农业大学兽医学院,呼和浩特 010018
- 2. 鄂尔多斯市动物疫病预防控制中心,内蒙古 鄂尔多斯 017010
- 折叠
摘要
为表达副结核分枝杆菌MAP3732c蛋白,研究构建原核表达质粒pET28a-MAP3732c,诱导表达鉴定后采用His标签亲和层析纯化获得MAP3732c重组蛋白,多次免疫Balb/c小鼠制备多克隆抗体,采用间接ELISA方法检测其抗体效价.结果表明,成功构建克隆重组质粒T1-MAP3732c与表达重组质粒pET28a-MAP3732c;在IPTG终浓度为0.5 mmol·L-1、37℃条件下成功诱导MAP3732c重组蛋白表达;SDS-PAGE结果表明,MAP3732c重组蛋白大小约为28 ku,且以包涵体形式存在;Western blot鉴定结果显示,重组蛋白与His抗体和牛源副结核病阳性血清均有良好免疫反应、阴性血清无非特异性结合;ELISA方法检测结果显示,抗MAP3732c多克隆抗体效价为1·204 800.研究为副结核分枝杆菌MAP3732c蛋白免疫原性分析及副结核病检测方法建立提供理论参考.
Abstract
In order to exogenously express the MAP3732c protein of Mycobacterium avium subspecies paratuberculosis,this study constructed the prokaryotic expression plasmid pET28a-MAP3732c,purified the recombinant protein of MAP3732c by His-tag affinity chromatography after the induced expression and identification,and then immunised Balb/c mice several times to prepare the polyclonal antibody,detected the antibody potency by indirect ELISA method.The results showed that the recombinant plasmid T1-MAP3732c was successfully constructed and the recombinant plasmid pET28a-MAP3732c was successfully induced at a final concentration of IPTG of 0.5 mmol·L-1 at 37℃;SDS-PAGE results showed that MAP3732c recombinant protein was about 28 ku in size and existed in the form of inclusion bodies;Western blot results showed that the recombinant protein had good immunoreactivity with His antibody and bovine paratuberculosis positive serum,while negative serum did not bind non-specifically;the ELISA results showed that the anti-MAP3732c polyclonal antibody had a potency of 1:204 800.This study provided a reference for the immunogenicity analysis of MAP3732c protein of Mycobacterium avium subspecies paratuberculosis and detection methodology of paratuberculosis.
关键词
副结核分枝杆菌/MAP3732c/原核表达/蛋白纯化/多克隆抗体Key words
Mycobacterium avium subspecies paratuberculosis/MAP3732c/prokaryotic expression/protein purification/polyclonal antibody引用本文复制引用
基金项目
内蒙古自治区高等学校科学研究项目(NJZZ22490)
鄂尔多斯市科技重大专项项目(2022EEDSKJZDZX025)
出版年
2024
NETL
NSTL
万方数据