柔嫩艾美耳球虫保守蛋白基因EtCHP40的分子特性研究

Molercular Characteristics of a Conserved Hypothetical Protein Gene EtCHP40 in Eimeria tenella

鸡球虫病是由几种艾美耳球虫寄生鸡肠道引起的一种危害严重的寄生虫病.目前,球虫病的防治主要依赖于药物,但由于药物长期广泛使用使鸡球虫对药物产生了严重的耐药性.为研究鸡球虫耐药性产生的分子机制,本实验室前期利用RNA-sequence对柔嫩艾美耳球虫地克珠利耐药株、马杜拉霉素耐药株与敏感株进行了转录组分析,获得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40(EtCHP40)在马杜拉霉素耐药株表达显著上调.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40(EtCHP40)基因,并在原核表达系统中成功表达了重组蛋白(rEtCHP40).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(耐药株和敏感株)的mRNA转录水平.结果显示EtCHP40基因在第二代裂殖子的mRNA转录水平最高;与敏感株相比,EtCHP40基因的mRNA转录水平在耐药株上调,且在马杜拉霉素耐药株的转录水平显著高于其他耐药株,另外发现在不同浓度的盐霉素作用下,随着药物浓度的升高,EtCHP40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的表面和顶端.因此,推测该蛋白可能参与了虫体在宿主细胞内的生长发育和对药物产生耐药性的过程.

Chicken coccidiosis is a serious parasitic disease caused by several Eimeria species parasitizing chicken intestinal tract.At present,the prevention and treatment of coccidiosis mainly depends on drugs.However,due to the extensive use of drugs for a long time,Eimeria spp have developed serious drug resistance.In order to study the molecular mechanism of drug resistance of Eimeria tenella,the transcriptomes of diclazuril-resistant strain,maduramycin-resistant strain and drug-sensitive strain of E.tenella were analyzed by RNA-sequence in our previous study.The differentially expressed genes between the sensitive and resistant strains were obtained.It was found that the conserved protein 40(EtCHP40)of E.tenella was up-regulated expression in maduramycin-resistant strain.In this study,the ORF of the conserved protein gene 40 of E.tenella(EtCHP40)was successfully cloned using the first strand of sporulated oocyst cDNA of E.tenella drug-sensitive strain as template and ligated to prokaryotic expression vector pET28a(+)to construct the recombinant expression plasmid pET-EtCHP40.rEtCHP40 was expressed as a His6-tagged fusion protein.Real time quantitative PCR(qPCR)was used to analyze the mRNA transcription levels of EtCHP40 in different developmental stages and different strains(drug-resistant strains and drug-sensitive strain)of E.tenella.The results showed that the mRNA transcription level of EtCHP40 gene in the second generation merozoite was the highest;compared with the drug-sensitive strain,the mRNA transcription level of EtCHP40 gene was up-regulated in the drug-resistant strains,and the transcription level was significantly higher in maduramycin-resistant strain than that in other drug-resistant strains.In addition,it was found that the mRNA transcription level of EtCHP40 increased with the increase of salinomycin concentration.Indirect immunofluorescence showed that the conserved protein was mainly distributed on the surface and the anterior of sporozoites.Therefore,it is speculated that the protein may be involved in growth and development of parasite in the host cells and the drug-resistant development of E.tenella.