毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX的原核表达与分析  

Procaryotic Expression and Analysis of the EnGPX of Eimeria necatrix

作　　者：彭月梅 叶状 汪飞燕 王礼跃 冯永翠 王乐乐[1,2] 候照峰 许金俊 陶建平[1,2] 刘丹丹 PENG Yuemei;YE Zhuang;WANG Feiyan;WANG Liyue;FENG Yongcui;WANG Lele;HOU Zhaofeng;XU Jinjun;TAO Jianping;LIU Dandan(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,扬州225009 [2]扬州大学江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《畜牧兽医学报》2024年第2期846-853,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31972698,31602039);江苏高校优势学科建设四期工程项目(2022-2025);高等学校学科创新引智计划资助(D18007)。

摘　　要：旨在研究毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX的反应原性及其在虫体内的亚细胞定位。提取毒害艾美耳球虫(扬州株)配子体总RNA,RT-PCR扩增En GPX的ORF编码序列,构建原核表达质粒pET-28a(+)-En GPX,转化至BL21(DE3)进行体外诱导表达,同时制备鼠抗rEnGPX多克隆抗体,对重组蛋白进行Western blot反应原性分析和激光共聚焦免疫荧光定位分析。结果表明,En GPX ORF序列全长753 bp,编码250个氨基酸,体外重组表达蛋白大小约30 ku,主要以包涵体形式存在。该重组蛋白能被6×HIS标签单克隆抗体,鼠抗rEnGPX多克隆抗体,毒害艾美耳球虫、巨型艾美耳球虫和柔嫩艾美耳球虫病鸡康复血清所识别,表明其具有较好的反应原性和交叉反应原性。在天然配子体蛋白中检测出EnGPX,其编码蛋白主要分布于配子体内的Ⅱ型成壁体(WFBII)及卵囊壁上。本研究成功克隆表达了毒害艾美耳球虫谷胱甘肽过氧化物酶EnGPX,验证其具有良好的反应原性,并定位于配子体及卵囊壁上。以期为研究En GPX参与卵囊壁形成的分子机制提供新的线索,也为研制新型球虫亚单位疫苗提供新的靶标。The aim of this paper was to study the antigenicity of glutathione peroxidase EnGPX and its subcellular localization in Eimeria necatrix.Total RNA was extracted from the gametophyte of E.necatrix(Yangzhou strain),and the ORF coding sequence of En GPX was amplified using RT-PCR.The prokaryotic expression plasmid pET-28a(+)-En GPX was constructed and transformed into BL21(DE3)for in vitro expression,additionally,a mouse anti-rEnGPX polyclonal antibody was prepared and used to analyze the recombinant protein through Western blotting,and laser confocal immunofluorescence localization analysis.The study found that the coding region of the En GPX gene was 753 base pairs in length and encoded a protein consisting of 250 amino acids.The resulting recombinant protein had a molecular weight of around 30 ku and was predominantly present in the form of inclusion bodies.The recombinant protein exhibited favorable reactivity and cross-reactivity,as it was recognized by a 6×HIS-tagged monoclonal antibody,a mouse anti-rEnGPX polyclonal antibody,and convalescent serum from E.necatrix,E.maxima,and E.tenella.En GPX was detected in natural gametophyte proteins,with the encoded protein primarily localized to the type II wall-forming body(WFBII)of the gametophyte and oocyst wall.This study successfully cloned and expressed the glutathione peroxidase(EnGPX)of E.necatrix.The recombinant protein exhibited good reactivity,and natural EnGPX protein was found to be localized on the gametophyte and oocyst wall.The results shed light on the molecular mechanism of EnGPX involvement in oocyst wall formation and identify potential targets for the development of novel subunit vaccines against coccidia.

关 键 词：毒害艾美耳球虫 EnGPX 克隆表达 反应原性 免疫荧光定位 

分 类 号：S855.9[农业科学—临床兽医学] S852.723[农业科学—兽医学]