柔嫩艾美耳球虫保守蛋白EtCHP35特性和功能初步研究  

Molecular Characterization and Relationship to Drug Resistance of the Conserved Hypothetical Protein Gene Et CHP35 of Eimeria tenella

作　　者：张思诗 姚亚文 朱顺海[1] 赵其平[1] 董辉[1] 于钰 黄兵[1] 韩红玉[1] ZHANG Sishi;YAO Yawen;ZHU Shunhai;ZHAO Qiping;DONG Hui;YU Yu;HUANG Bing;HAN Hongyu(Key Laboratory of Animal Parasitology of Ministry of Agriculture and Rural Affairs,Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,农业农村部动物寄生虫学重点实验室,上海200241

出　　处：《中国动物传染病学报》2024年第5期11-21,共11页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31970420);国家寄生虫种质资源共享服务平台(平台-TDRC-22)。

摘　　要：鸡球虫病是由几种艾美耳属寄生虫寄生引起的一种家禽肠道疾病。目前,对于该病控制仍然主要依赖于抗球虫药物,但长期广泛地使用药物造成了球虫耐药性的产生。为研究球虫耐药性产生的分子机制,本实验室前期对柔嫩艾美耳球虫盐霉素耐药株、地克珠利耐药株、马杜拉霉素耐药株以及敏感株进行了转录组测序并获得了敏感株与耐药株的差异表达基因,发现柔嫩艾美耳球虫保守蛋白35(EtCHP35)在盐霉素耐药株和马杜拉霉素耐药株mRNA转录水平显著上调。本研究以柔嫩艾美耳球虫敏感株孢子化卵囊cDNA第一链为模板,成功克隆了柔嫩艾美耳球虫保守蛋白35(EtCHP35)基因,并在原核表达系统中成功表达了重组蛋白(rEtCHP3)。利用实时荧光定量PCR分析了该基因在柔嫩艾美耳球虫不同发育阶段以及不同虫株(敏感株和耐药株)的mRNA转录水平,结果显示EtCHP35基因在敏感株孢子化卵囊的mRNA转录水平最高;与敏感株相比,EtCHP35的mRNA转录水平在马杜拉霉素耐药株和盐霉素耐药株上调,但在地克珠利耐药株mRNA转录水平无差异,且随着盐霉素耐药株浓度的升高,EtCHP35的mRNA转录水平也升高。间接免疫荧光定位结果表明该蛋白主要分布在子孢子和第二代裂殖子的表面和顶部。因此,推测该蛋白可能参与了虫体在宿主体内的生长发育、对宿主细胞的识别附着、逃避宿主免疫应答以及球虫对离子载体类药耐药性产生的过程。Coccidiosis is an intestinal disease of poultry caused by several Eimeria spp..Currently,the disease is primarily controlled by use of anticoccidial drugs.However,the widespread use of drugs has caused the emergence of drug resistant strains.In order to study the molecular mechanism of the resistance of Eimeria to drugs,transcriptome sequencing was performed for salinomycin-resistant strain(SMR),diclazuril-resistant strain(DZR),maduramycin-resistant strain(MRR)and drug-sensitive strain(DS)of Eimeria tenella in our previous study.Differentially expressed genes(DEGs)were revealed between drug-sensitive and drug-resistant strains.Among these DEGs,one conserved hypothetical protein gene(GenBank number:ETH_00025035)was upregulated in the MRR and SMR compared to the DS.In this study,the conserved hypothetical protein gene Et CHP35 of E.tenella was cloned using the fi rst strand of cDNA of sporulated oocyst of E.tenella drug-sensitive strain strain as template and the recombinant protein(rEt CHP35)was expressed in the prokaryotic system.The mRNA transcription levels of E.tenella at diff erent developmental stages and in diff erent strains(drug-sensitive and drug-resistant strains)were analyzed by qRT-PCR.The results showed that the highest mRNA transcription level of Et CHP35 gene was found in sporulated oocysts in E.tenella DS strain.Compared to DS,the mRNA transcription level of EtCHP35 gene was up-regulated in MRR and SMR.In addition,the mRNA transcription level of Et CHP35 increased with the high concentration of salinomycin.Indirect immunofl uorescence localization showed that the protein was mainly distributed on the surface and the top of the sporozoites and the second generation merozoites.Therefore,this protein was speculated to be involved in the survival and development of the parasites in the hosts,recognition and attachment of host cells,escape from the host immune response and emergence of drug resistant E.tenella strains.

关 键 词：柔嫩艾美耳球虫 保守蛋白 免疫逃逸 耐药性 

分 类 号：S852.723[农业科学—基础兽医学]