马泰勒虫棒状体颈部蛋白4基因的克隆与原核表达分析  

Cloning and prokaryotic expression analysis of Theileria equi rhoptry neck protein 4 gene

作　　者：芦星 范士龙 王水怡 王金明 李思媛 刘明明 刘雨桐 巴音查汗[1] 刘丹丹 张伟[1] LU Xing;FAN Shilong;WANG Shuiyi;WANG Jinming;LI Siyuan;LIU Mingming;LIU Yutong;BAYIN Chahan;LIU Dandan;ZHANG Wei(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi,Xinjiang 830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《西北农林科技大学学报（自然科学版）》2023年第9期1-10,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：新疆维吾尔自治区天山青年计划-优秀青年科技人才项目(2020Q014);新疆农业大学博士后科研流动站资助项目。

摘　　要：【目的】克隆马泰勒虫新疆株棒状体颈部蛋白4基因(RON4)并原核表达RON4蛋白。【方法】参考美国马泰勒虫WA株基因组数据及其他顶复门原虫RON4基因信息,设计特异性引物,以马泰勒虫新疆株DNA为模板克隆RON4基因片段,构建pET-32a-RON4原核表达载体后进行原核表达与鉴定,并对RON4基因编码蛋白进行生物信息学分析。【结果】获得马泰勒虫新疆株RON4基因(NCBI登录号为:ON254212)。马泰勒虫新疆株与马泰勒虫WA株亲缘关系最近,RON4基因的核苷酸和氨基酸相似性分别为99.4%和99.0%;RON4基因在种间高度保守,其氨基酸序列与其他顶复门原虫具有高度相似的保守区域。成功构建了RON4蛋白原核表达载体,诱导表达获得分子质量为50 ku的重组包涵体蛋白;重组蛋白可与马泰勒虫阳性马血清特异性反应。生物信息学分析表明,马泰勒虫新疆株RON4蛋白的亲水性、柔韧性以及表面可及性较弱,B细胞表面抗原表位区域较少,形成表面抗原表位的结构基础条件不足。【结论】克隆获得了马泰勒虫新疆株RON4基因,并完成了原核表达及鉴定,该基因高度保守,在不同物种间可能具有相似的功能。【Objective】This study aimed to clone the rhoptry neck protein 4(RON4)gene of Theileria equi Xinjiang strain and to express RON4 protein in prokaryotes.【Method】Specific primers were designed by referring to the genome data of T.equi WA strain from the United States as well as the RON4 gene from other apicomplexa parasites.The target gene fragment of RON4 was cloned from T.equi Xinjiang strain and used as DNA template,and the pET-32a-RON4 expression vector was constructed.The protein was expressed with prokaryotic expression system and identified by SDS-PAGE and Western Blot.Finally,the amino acids of RON4 gene were analyzed by bioinformatics informatics.【Result】The RON4 gene of T.equi Xinjiang strain was successfully obtained with NCBI accession number of ON254212.Genetic evolution analysis showed that the T . equi Xinjiang strain and the WA strain were located in the same branch.The nucleotide and amino acid similarities of RON4 gene between the two strains were 99.4% and 99.0%,respectively.The RON4 gene was highly conserved,and its amino acid sequence was highly similar to that of other apicomplexa parasites.The RON4 prokaryotic expression vector was successfully construc-ted, and the recombinant inclusion body protein with molecular weight of 50 ku was obtained.The recombi-nant protein was specifically reacted with the positive horse serum.Bioinform-atics analysis showed that the hydrophilicity,flexibility and surface accessibility of the TeRON4 protein were weak,the epitope re-gions formed of B cell epitopes were few,the structural basis conditions for the formation of epitopes were insufficient,and the antigenicity was poor.【Conclusion】 The RON4 gene of T . equi Xinjiang strain was successfully obtained and its prokaryotic expression and identification were constructed.The gene was highly conserved and might have similar functions among different species.

关 键 词：马泰勒虫 RON4基因 原核表达 B细胞抗原表位 

分 类 号：S858.21[农业科学—临床兽医学] S852.7[农业科学—兽医学]