毒害艾美耳球虫氧化还原酶EnOXIO1的原核表达与定位分析  

Procaryotic Expression and Immunolocalization Analysis of the EnOXIO1 of Eimeria necatrix

作　　者：叶状 王乐乐[1,2] 汪飞燕 刘悦 彭月梅 宿世杰 候照峰[1,2] 许金俊 陶建平 刘丹丹[1,2] YE Zhuang;WANG Lele;WANG Feiyan;LIU Yue;PENG Yuemei;SU Shijie;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》2022年第5期1553-1561,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31972698,31602039);江苏省高等学校自然科学研究面上项目(19KJD230004);江苏高校优势学科建设工程项目。

摘　　要：旨在探析毒害艾美耳球虫氧化还原酶EnOXIO1的功能。提取毒害艾美耳球虫扬州株配子体总RNA,应用RT-PCR获取EnOXIO1编码基因,构建原核表达载体pET-28a(+)-EnOXIO1,转化至大肠杆菌BL21(DE3),体外诱导表达,对重组蛋白进行抗原性分析,应用制备的多抗进行激光共聚焦免疫荧光定位。结果显示,获得的EnOXIO1基因全长为2535 bp,体外重组表达的蛋白大小约100 ku,主要以包涵体的形式存在,表达量约为0.5 mg·mL^(-1)。Western blot分析结果显示,该重组蛋白能被6×HIS标签单克隆抗体识别,同时能被制备的鼠多抗以及毒害艾美耳球虫、堆型艾美耳球虫和柔嫩艾美耳球虫病鸡康复血清所识别,表明该重组蛋白具有较好的反应原性和交叉反应原性。激光共聚焦免疫荧光定位结果显示,EnOXIO1基因表达产物主要存在于配子体内的成壁体上,参与了卵囊壁的形成。本研究成功克隆和表达了毒害艾美耳球虫EnOXIO1蛋白,并将其定位于配子体和卵囊壁上,为进一步解析EnOXIO1蛋白参与卵囊壁形成的分子机制奠定基础,为研制新型免疫阻断型球虫亚单位疫苗提供重要靶抗原。The purpose of this study was to analyze the function of EnOXIO1,an oxidoreductase came from Eimeria necatrix.Total RNA was extracted from the gametophyte of E.necatrix Yangzhou strain to amplify EnOXIO1 gene by using RT-PCR.After cloning and sequence analysis,the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into E.coli BL21(DE3).The recombinant protein was expressed by inducing with IPTG,and its antigenicity and immunofluorescence localization was analyzed.Here we show that the full length of EnOXIO1 gene was 2535 bp.The recombinant protein had a molecular weight of about 100 ku and mainly expressed in inclusion body.The expression level was about 0.5 mg·mL^(-1) bacteria.Western blot analysis showed that the recombinant protein could be specifically recognized by 6×HIS labeled monoclonal antibody,the mouse anti-recombinant proteins polyclonal antibody,the recovery serum from chickens infected with E.necatrix,E.acervulina,and E.tenella sporulated oocysts,respectively.These results indicated that the recombinant protein had good antigenicity and cross-reactivity.The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein mainly existed in the wall forming body in the gametocyte and participated in the formation of the oocyst wall.In this study,EnOXIO1 protein was successfully cloned and expressed,and was located on gametocytes and oocyst wall,which laid a foundation for further analysis of the molecular mechanism of EnOXIO1 protein involved in the formation of oocyst wall,and provided important target antigens for the development of a novel immune-blocking anti-coccidiosis subunit vaccine.

关 键 词：毒害艾美耳球虫 EnOXIO1 克隆表达 免疫荧光定位 功能鉴定 

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