柔嫩艾美耳球虫微管蛋白β链基因EtTBC的克隆、表达及特性的初步分析  

Cloning,Expression and Preliminary Analysis of the Characteristics of the Tubulin Beta Chain Gene EtTBC of Eimeria tenella

作　　者：谢馨锐 赵其平[2] 朱顺海[2] 郭慧琳 王丽慧 于钰 董辉[2] 韩红玉[2] 刘伟[1] XIE Xinrui;ZHAO Qiping;ZHU Shunhai;GUO Huilin;WANG Lihui;YU Yu;DONG Hui;HAN Hongyu;LIU Wei(School of Animal Medicine,Hunan Agricultural University,Changsha Hunan 410128,China;Key Laboratory of Animal Parasitology of Ministry of Agriculture,Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200234,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点实验室,上海200234

出　　处：《复旦学报（自然科学版）》2024年第3期405-414,共10页Journal of Fudan University：Natural Science

基　　金：国家重点研发计划揭榜挂帅项目(2023YFD1802400);国家寄生虫种质资源共享服务平台(平台-TDRC-22)。

摘　　要：鸡球虫病是一种由艾美耳属原生动物寄生虫引起的肠道疾病,是影响家禽业的最重要疾病之一。目前,球虫病的控制主要依赖于抗球虫药物的使用,但由于药物的长期广泛使用导致了球虫的严重耐药性。为研究其耐药性机制,本实验室前期对柔嫩艾美耳球虫敏感株、地克珠利耐药株、马杜拉霉素耐药株、盐霉素耐药株进行了转录组测序分析,发现与敏感株相比,柔嫩艾美耳球虫微管蛋白β链(Et TBC)仅在地克珠利耐药株高表达。本研究在此基础上,克隆、表达了EtTBC,并初步分析了其功能及与地克珠利的关系;以柔嫩艾美耳球虫敏感株cDNA为模板成功克隆了EtTBC基因,构建了原核表达重组质粒pET28a-Et TBC,并成功诱导表达了重组蛋白r Et TBC;利用qPCR对柔嫩艾美耳球虫敏感株不同发育阶段以及不同虫株(耐药株、敏感株和田间耐药株)的mRNA转录水平进行了分析,结果显示,该基因的转录水平在敏感株未孢子化卵囊阶段最高,且其mRNA转录水平仅在地克珠利耐药株上调;间接免疫荧光定位结果表明该蛋白主要分布在子孢子除折光体外的细胞质和表面,第二代裂殖子的细胞质和表面。Coccidiosis is an intestinal disease caused by parasites of the genus Eimeria spp.and one of the most important diseases affecting the poultry industry.At present,the control of coccidiosis mainly depends on the use of anticoccidial drugs,but drug resistance of Eimeria develops qucikly due to the long-term and extensive use of drugs.In order to study drug resistant mechanism of Eimeria,we previous study transcriptome sequencing on drug-sensitive strain(DS),diclazuril-resistant strain(DZR),maduramycin-resistant strain(MRR),salinomycin-resistant strain(SMR)of E.tenella.We found that the tubulin bate chain(Et TBC)was highly expressed only in DZR.Based on this,the EtTBC gene was cloned and expressed in this study,and its function was preliminarily analyzed.EtTBC was successfully cloned,the recombinant plasmid pET28a-Et TBC was constructed and the recombinant protein(r Et TBC)was successfully expressed.The mRNA transcription levels of E.tenella at different developmental stages of DS and in different strains(drug-sensitive strain,drug-resistant strains and field isolated diclazuril-resistant strains)were analyzed by qPCR.The results showed that the mRNA transcript level of this gene was highest in the unsporulated oocyst stage,while the other three stages were low in E.tenella DS;Moreover,its mRNA levels was only upregulated in DZR.Indirect immunofluorescence localization showed that the protein was mainly distributed on the cytoplasm and surface of sporozoites and second generation merozoites.Therefore,it is speculated that this protein may be involved in the invasion of the host and the immune evasion and the development of resistance of E.tenella to diclazuril.

关 键 词：柔嫩艾美耳球虫 微管蛋白β链 耐药性 

分 类 号：S858.31[农业科学—临床兽医学]