绵羊肺炎支原体EF-Tu蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.02.025

中国畜牧兽医 ›› 2024, Vol. 51 ›› Issue (2): 689-699.doi: 10.16431/j.cnki.1671-7236.2024.02.025

绵羊肺炎支原体EF-Tu蛋白的原核表达及多克隆抗体制备

王永飞^1,2, 邓博文^1,2, 刘晓艳^1,2, 哈尔勒哈·阿曼太^1,2, 郭嘉栋^1,2, 周正国³, 蔡江^1,2, 李有文^1,2

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, 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, 阿拉尔 843300;
3. 阿克苏职业技术学院, 阿克苏 843000

收稿日期:2023-07-26 出版日期:2024-02-05 发布日期:2024-01-29
作者简介:王永飞,E-mail:907586453@qq.com。

Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody

WANG Yongfei^1,2, DENG Bowen^1,2, LIU Xiaoyan^1,2, HAERLEHA·Amantai^1,2, GUO Jiadong^1,2, ZHOU Zhengguo³, CAI Jiang^1,2, LI Youwen^1,2

1. 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;
2. Engineering Laboratory for Diagnosis and Prevention and Control of Animal Diseases in South Xinjiang of Xinjiang Production and Construction Corps, Alaer 843300, China;
3. Aksu Vocational and Technical College, Aksu 843000, China

Received:2023-07-26 Online:2024-02-05 Published:2024-01-29
Contact: 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) E-mail:lyw＿lk@163.com
Supported by:
The project was supported by the National Key Research and Development Program of China (2019YFC1905301);National Natural Science Foundation of China (22078115,21776108,21690083,22008078).

摘要/Abstract

摘要： 【目的】克隆绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)EF-Tu基因,原核表达获得EF-Tu蛋白,制备抗EF-Tu蛋白的兔源多克隆抗体,为研究肺炎支原体EF-Tu蛋白的结构和功能奠定基础。【方法】采用重叠延伸PCR方法将pET-28a-EF-Tu质粒中EF-Tu基因中间的TGA密码子突变为TGG,并对测序结果与其他支原体参考株进行相似性比对和遗传进化分析,利用在线软件对其推测的蛋白序列进行生物信息学分析。将突变后的重组质粒转化大肠杆菌BL21(DE3)感受态细胞进行原核表达,经SDS-PAGE和Western blotting鉴定,利用镍柱亲和层析法纯化,以纯化的EF-Tu融合蛋白免疫家兔制备多克隆抗体,采用间接ELISA和Western blotting检测多克隆抗体效价及免疫反应性。【结果】试验成功突变了EF-Tu基因中TGA位点,并构建了融合表达His标签pET-28a-EF-Tu'原核表达载体。生物信息学分析表明,克隆的EF-Tu基因与绵羊肺炎支原体MoGH3-3菌株相似性最高,亲缘关系最近;编码387个氨基酸,无N-糖基化位点和跨膜区域,存在10个丝氨酸、20个苏氨酸、4个酪氨酸磷酸化位点,二级结构由无规则卷曲(35.14%)、α-螺旋(26.87%)、延伸链(26.87%)及β-转角(11.11%)构成。SDS-PAGE和Western blotting结果显示,目的蛋白大小约为43 ku,蛋白纯化浓度为0.615 g/L。ELISA和Western blotting结果显示,制备的多克隆抗体效价可达1∶128 000,能够特异性识别EF-Tu融合蛋白,具有良好的免疫反应性。【结论】本研究成功突变了EF-Tu基因的TGA密码子,原核表达并纯化获得EF-Tu融合蛋白,制备其多克隆抗体效价为1∶128 000,为后续研究肺炎支原体EF-Tu蛋白结构和生物学功能及其疫苗研发提供了试验基础。

关键词: 绵羊肺炎支原体; 重叠延伸PCR; EF-Tu基因; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to clone EF-Tu gene in Mycoplasma ovipneumoniae (Mo), obtain EF-Tu protein by prokaryotic expression, prepare rabbit-derived polyclonal antibody against EF-Tu protein, laying the foundation for the study of the structure and function of Mo EF-Tu protein.【Method】 TGA codon in the middle of EF-Tu gene in pET-28a-EF-Tu plasmid was mutated to TGG by overlapping extension PCR, and the similarity alignment and genetic evolution analysis were conducted between the sequencing results and other Mycoplasma reference strains, and the putative protein sequences were analyzed for bioinformatics using online software.The mutated recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression, identified by SDS-PAGE and Western blotting, and purified using Ni-chelating affinity chromatography.The polyclonal antibodies were prepared by immunizing rabbits with the purified EF-Tu fusion protein.The polyclonal antibody potency and immunological reactivity were detected by indirect ELISA and Western blotting.【Result】 The TGA site in EF-Tu gene was successfully mutated, and a prokaryotic expression vector expressing the His tag pET-28a-EF-Tu' was constructed.Bioinformatics analysis results showed that the cloned EF-Tu gene had the highest similarity and the closest genetic relationship to Mycoplasma ovipneumoniae strain MoGH3-3, and encoded 387 amino acids, without N-glycosylation site and transmembrane region.There were 10 serine, 20 threonine and 4 tyrosine phosphorylation sites of EF-Tu protein, and the secondary structure consists of random coil (35.14%), alpha helix (26.87%), extended chain (26.87%) and beta turn (11.11%).SDS-PAGE and Western blotting results showed that the size of the target protein was about 43 ku, and the protein purification concentration was 0.615 g/L.ELISA and Western blotting results showed that the prepared polyclonal antibody had a potency of up to 1∶128 000, and could specifically recognize the EF-Tu fusion protein, which had good immunological reactivity.【Conclusion】 The TGA codon of EF-Tu gene was successfully mutated in this study, the EF-Tu fusion protein was obtained by prokaryotic expression and purification, and its polyclonal antibody was prepared with a potency of 1∶128 000.This result provided an experimental basis for the subsequent study of the structure and biological function of Mo EF-Tu protein and its vaccine development.

Key words: Mycoplasma ovipneumoniae; overlapping extension PCR; EF-Tu gene; polyclonal antibody

中图分类号:

S852.62

王永飞, 邓博文, 刘晓艳, 哈尔勒哈·阿曼太, 郭嘉栋, 周正国, 蔡江, 李有文. 绵羊肺炎支原体EF-Tu蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(2): 689-699.

WANG Yongfei, DENG Bowen, LIU Xiaoyan, HAERLEHA·Amantai, GUO Jiadong, ZHOU Zhengguo, CAI Jiang, LI Youwen. Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 689-699.

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绵羊肺炎支原体EF-Tu蛋白的原核表达及多克隆抗体制备

Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody

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