柔嫩艾美耳球虫伴侣蛋白TCP-1亚基α的特性和功能初步分析

MOLECULAR CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF TCP-1 SUBUNIT α OF EIMERIA TENELLA

柔嫩艾美耳球虫是一种重要的寄生于鸡盲肠细胞内的寄生原虫,可引起危害严重的鸡球虫病。目前,对鸡球虫病的预防和治疗主要依靠抗球虫药,而药物的广泛使用使球虫产生了严重的耐药性。为研究球虫耐药性产生的机制,本实验室前期对诱导的地克珠利耐药株、马杜拉霉素耐药株和亲本敏感株进行转录组测序,经分析发现TCP-1亚基α为耐药株和敏感株的差异表达基因且在耐药株上调表达。本文以柔嫩艾美耳球虫敏感株为模板,成功克隆出TCP-1亚基α基因,构建了原核表达重组质粒p ET-Et TCP-1α,并成功诱导表达了重组蛋白r Et TCP-1α,用纯化的重组蛋白免疫新西兰大白兔和BALB/C小鼠获得多克隆抗体。用荧光定量PCR和Western blot方法检测柔嫩艾美耳球虫敏感株、地克珠利耐药株和马杜拉霉素耐药株TCP-1亚基α基因的m RNA转录和翻译水平,同时检测了该基因在球虫不同发育阶段的转录水平和翻译水平。结果显示,Et TCP-1α在2个耐药株的m RNA转录和蛋白翻译水平明显高于敏感株,而且该蛋白在第二代裂殖子的表达最高;间接免疫荧光实验显示该蛋白主要位于子孢子和第二代裂殖子表面,当虫体入侵DF-1细胞进行裂殖生殖后,该蛋白分布于整个虫体,且表达量明显升高。因此,推测Et TCP-1α在柔嫩艾美耳球虫细胞内的生长发育和耐药性的产生过程中发挥了一定的作用。

Eimeria tenella is an important intracellular parasite that invades cecal epithelial cells of chickens,causing serious coccidiosis.At present,prevention and treatment mainly rely on anti-coccidiosis drugs.As a result,the widespread use of drug causes severe resistance to E.tenella.In our previous study,we analysized differentially expressed genes between two drug-resistant strains(diclazurilresistant and maduramicin-resistant strains)and drug-sensitive strain of E.tenella through RNA sequencing.TCP-1 subunitαof E.tenella,one of the differentially expressed genes,was up-regulated in diclazuril-resistant and maduramicin-resistant strains.In this study,E.tenella TCP-1 subunitα(Et TCP-1α)gene was cloned in PCR and ligated into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-Et TCP-1α.The recombination protein rEt TCP-1αwas expressed after induction with IPTG and then used to immunize New Zealand rabbits and BALB/C mouse to prepare polyclonal antibodies.Quantitative real time PCR and Western blot were applied to analyze the mRNA transcription and translation levels of TCP-1 subunitαin resistant and sensitive strains and test the expression levels of TCP-1 subunitαin four developmental stages of E.tenella.The results revealed that Et TCP-1αwas expressed at significantly higher levels in the two drug-resistant strains than drug-sensitive strains and the highest expression occurred in the second generation merozoite stage.The protein Et TCP-1αwas mainly located in the surfaces of sporozoites and the second generation merozoites as shown in IFA.After the sporozoites invaded DF-1 cells,Et TCP-1αwas uniformly distributed in the immature and mature schizonts and its expression increased signifi cantly.Therefore,it was presumed that Et TCP-1αplayed a certain role during the growth in the host cells and development of drug resistance of E.tenella.