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 paratuberculosis MAP3732c of cow origin and preparation of polyclonal antibodies

作　　者：马聿田 李阳 李晓宇 其勒木格 吉林台 张文彦 希尼尼根[1] MA Yutian;LI Yang;LI Xiaoyu;Qilemuge;JI Lintai;ZHANG Wenyan;Xininigen(School of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Center for Animal Disease Prevention and Control,Ordos Inner Mongolia 017010,China)

机构地区：[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

出　　处：《东北农业大学学报》2024年第4期42-49,共8页Journal of Northeast Agricultural University

基　　金：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)。

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p ET28a-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1:204800。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。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 1:204800.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 

分 类 号：S852.43[农业科学—基础兽医学]