柔嫩艾美耳球虫微线蛋白1(EtMIC1)基因的原核表达与重组蛋白的免疫原性分析  

Prokaryotic expression of microneme protein 1(EtMIC1)gene of Eimeria tenella and immunogenicity analysis of recombinant protein

作　　者：张圳 王松[1] 孙铭飞 胡建和[1] 王丹妮 朱惠丽[1] ZHANG Zhen;WANG Song;SUN Mingfei;HU Jianhe;WANG Danni;ZHU Huili(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China;Institute of Animal Health,Guangdong Academy of Agriculture Sciences,Guangzhou 510640,China)

机构地区：[1]河南科技学院动物科技学院,河南新乡453003 [2]广东省农业科学院动物卫生研究所,广州510640

出　　处：《黑龙江畜牧兽医》2023年第20期77-82,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：河南省重点研发与推广专项(科技攻关)项目(202102110101)。

摘　　要：为了解原核表达柔嫩艾美耳球虫微线蛋白1(Eimeria tenella microneme 1,EtMIC1)重组蛋白的免疫原性,试验以柔嫩艾美耳球虫广东株孢子化卵囊为材料,首先采用PCR技术扩增去除信号肽的EtMIC1基因,将其插入原核表达载体pET-32a(+)中,构建重组原核表达载体,然后将重组原核表达载体pET32a(+)-EtMIC1转化至大肠杆菌T7pLysY感受态细胞中,用IPTG进行诱导表达,最后对重组蛋白进行SDS-PAGE分析和Western-blot鉴定。结果表明:PCR扩增出大小为2055 bp的去信号肽EtMIC1基因片段,与预期结果一致;构建的重组原核表达载体pET32a(+)-EtMIC1可在大肠杆菌T7pLysY感受态细胞中表达,重组蛋白分子质量大小约为93 ku,主要表达在上清液中,可被自然感染抗柔嫩艾美耳球虫鸡血清和EtMIC1重组蛋白多克隆抗体特异性识别。说明试验成功表达了EtMIC1重组蛋白,原核表达的EtMIC1重组蛋白具有良好的免疫原性。In order to understand the immunogenicity of prokaryotic recombinant protein expressing Eimeria tenella microneme 1(EtMIC1),in this experiment,the sporulated oocysts of Eimeria tenella Guangdong strain were used as materials,and PCR technology was first used to amplify the EtMIC1 gene that removed the signal peptide,which was inserted into the prokaryotic expression vector pET-32a(+)to construct a recombinant prokaryotic expression vector pET32a(+)-EtMIC1.The recombinant prokaryotic expression vector pET32a(+)-EtMIC1 was then transformed into E.coli T7pLysY competent cells;by inducing expression with IPTG,SDS-PAGE analysis and Western-blot identification of the recombinant protein were carried out finally.The results showed that the PCR amplification of the de-signaling peptide EtMIC1 gene fragments with a size of 2055 bp was consistent with the expected results.The constructed recombinant prokaryotic expression vector could be expressed in E.coli T7pLysY competent cells.The molecular mass of the recombinant protein was about 93 ku,which was mainly expressed in the supernatant,and could be specifically recognized by anti-Eimeria tenella sera from naturally infected chicken and polyclonal antibodies from recombinant protein EtMIC1.The results suggested that EtMIC1 recombinant protein was successfully expressed and the prokaryotic expression of EtMIC1 recombinant protein had good immunogenicity.

关 键 词：柔嫩艾美耳球虫 EtMIC1 原核表达 免疫原性 SDS-PAGE分析 Western-blot鉴定 

分 类 号：S855.9[农业科学—临床兽医学]