绵羊肺炎支原体核糖体蛋白30S rpsE基因的原核表达及多克隆抗体的制备  

Prokaryotic expression of Mycoplasma ovipneumoniae ribosomal protein 30S rpsE gene and preparation of its polyclonal antibody

作　　者：刘晓艳 王永飞 邓博文 哈尔勒哈·阿曼太 蔡江 李有文[1,2] LIU Xiaoyan;WANG Yongfei;DENG Bowen;HAERLEHA·Amantai;CAI Jiang;LI Youwen(Key Laboratory of Tarim Animal Husbandry Science and Technology Corps of Xinjiang Production and Construction Corps,College of Animal Science and Technology,Tarim University,Alaer 843300,China;Engineering Laboratory for Diagnosis and Prevention and Control of Animal Diseases in South Xinjiang,Xinjiang Production and Construction Corps,Alaer 843300,China)

机构地区：[1]塔里木大学动物科学与技术学院,新疆生产建设兵团塔里木畜牧科技兵团重点实验室,新疆阿拉尔843300 [2]新疆生产建设兵团南疆动物疫病诊断与防控工程实验室,新疆阿拉尔843300

出　　处：《畜牧与兽医》2024年第7期78-84,共7页Animal Husbandry & Veterinary Medicine

基　　金：新疆生产建设兵团区域创新基金项目(2021BB014);塔里木大学校长基金项目(TDZKCX202305)。

摘　　要：旨在了解绵羊肺炎支原体(Mycoplasma ovipneumoniae)30S rpsE蛋白的生物学特性及其蛋白免疫原性。用在线分析软件预测30S rpsE蛋白的理化性质、B细胞抗原表位、糖基化位点、二级结构及三级结构等;采用PCR方法从病羊鼻拭子样品中扩增得到30S rpsE基因片段,经NdeⅠ、XhoⅠ酶切纯化后,将其连接到pET-42b载体上,构建原核表达载体pET-42b-30S rpsE,使用不同浓度IPTG诱导蛋白在大肠杆菌BL21中表达;重组蛋白通过SDS-PAGE和Western blot进行鉴定,用镍层析法纯化蛋白后,与弗氏佐剂1∶1混合注射免疫家兔,然后采取全血,检测血清抗体效价及特异性。结果:经预测30S rpsE蛋白是亲水无信号肽的膜外蛋白,无跨膜结构域,有一个糖基化位点;成功构建了原核表达载体pET-42b-30S rpsE,表达的重组蛋白分子量约为25.9 ku,以包涵体和可溶性的形式存在;免疫家兔后血清抗体效价为1∶25 600,证实30S rpsE蛋白具有较好的免疫原性。The study aimed to understand the biological characteristics of Mycoplasma ovipneumoniae ribosomal protein 30S rpsE and its protein immunogenicity.The physicochemical properties of the recombinant protein,its B-cell antigenic epitopes,glycosylation sites,secondary and tertiary structures were predicted based on an online analysis software.The gene fragment of 30S rpsE was obtained by PCR amplification,and after NdeⅠand XhoⅠdigestion,it was ligated to pET-42b,and the prokaryotic expression vector pET-42b-30S rpsE was prepared,and the protein was induced to be expressed in BL21 bacterium using different concentrations of gradient IPTG.Recombinant proteins were identified by SDS-PAGE and Western blot,and the adjuvants were mixed with the recombinant proteins and were immunized,while their sera were collected later and the antiserum potency and specificity were measured.The results showed that the 30S rpsE protein was predicted to be a hydrophilic,signal peptide-free,extramembrane protein with no transmembrane structural domains but one glycosylation site.The prokaryotic expression vector pET-42b-30S rpsE was successfully prepared,and the recombinant protein expressed had a relative molecular weight of about 25.9 ku,which could exist in BL21 bacteria as inclusion bodies and soluble forms.The antiserum titer was 1∶25600 after immunization of rabbits,confirming the good immunogenicity of 30S rpsE.

关 键 词：肺炎支原体 30S rpsE基因 原核表达 多克隆抗体 

分 类 号：S852.62[农业科学—基础兽医学]