柔嫩艾美耳球虫含HD结构域蛋白特性和功能初步研究

doi:10.11843/j.issn.0366-6964.2022.08.019

摘要/Abstract

摘要： 鸡球虫病是由艾美耳球虫引起的一种危害严重的肠道寄生虫病，每年都会给世界各地的养禽业带来巨大的经济损失。目前，该病主要依靠抗球虫药物进行防治，但由于药物的长期及不合理使用导致鸡球虫几乎对所有使用过的抗球虫药均产生耐药性。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含HD域蛋白（EtHDCP）在耐药株中上调表达。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，成功克隆出EtHDCP基因，构建了原核表达重组质粒pGEX-4T-EtHDCP，并成功诱导表达了重组蛋白rEtHDCP。利用qRT-PCR和Western blot对柔嫩艾美耳球虫敏感株不同发育阶段的转录和翻译水平进行分析，结果显示，EtHDCP在第二代裂殖子的转录和翻译水平高于其他三个阶段（未孢子化卵囊、孢子化卵囊和子孢子）。同时利用Western blot分析了EtHDCP在柔嫩艾美耳球虫敏感株、地克珠利耐药株、马杜拉霉素耐药株中的翻译水平，结果显示，EtHDCP在耐药株中的蛋白翻译水平显著高于敏感株。间接免疫荧光定位结果显示，该蛋白主要定位在子孢子和裂殖子的表面以及裂殖子的胞质内。入侵抑制试验表明，抗rEtHDCP多克隆抗体可有效抑制子孢子对宿主细胞的入侵。这些结果说明该蛋白可能参与了虫体在宿主细胞内的生长发育、耐药性的产生以及子孢子入侵宿主细胞的过程。

关键词: 柔嫩艾美耳球虫, 含HD结构域蛋白, 入侵, 耐药性

Abstract: Coccidiosis is a serious intestinal parasitic disease caused by Eimeria spp., which causes huge economic losses to poultry industry all over the world every year. At present, coccidiosis is mainly controlled by anti-coccidial drugs. Drug resistance of Eimeria to all of used drugs has been developed due to the long-term and unreasonable use of drugs. To study the molecular mechanism of the resistance of Eimeria to drugs, differentially expressed genes between two drug-resistant strains (diclazuril-resistant strains, maduramicin-resistant strains) and drug-sensitive strain of Eimeria tenella were obtained by using RNA sequncing in our lab. We found that E.tenella contained HD domain protein (EtHDCP) up-regulated in two drug-resistant strains. In this paper, EtHDCP gene was successfully cloned from the first cDNA strand of E.tenella drug sensitive strain sporulated oocysts (SO) as template, and the recombinant plasmid pGEX-4T-EtHDCP was constructed, and the recombinant protein rEtHDCP was induced successfully. qRT-PCR and Western blot were used to analyze the transcription and translation levels of EtHDCP in the four different developmental stages of E. tenella drug sensitive strains. At the same time, the translation levels of two drug-resistant strains and drug sensitive of E.tenella were detected. The results showed that the transcription and translation level of EtHDCP in second-generation merozoites stage were the highest, the protein translation level of EtHDCP in drug-resistant strains were significantly higher than those in drug sensitive strains. Indirect immunofluorescence localization showed that the protein was mainly located on the surface of sporozoites and merozoites and in the cytoplasm of merozoites. Invasion inhibition test showed that rabbit anti-rEtHDCP polyclonal antibody could effectively inhibit invasion of sporozoites to host cells. These results showed that the protein may be involved in the growth and development of parasite in host cells, the development of drug resistance and the invasion of sporozoites into host cells.

Key words: Eimeria tenella, HD domain protein, invasion, resistance

中图分类号:

S852.723

肖克, 陈婷, 赵其平, 朱顺海, 董辉, 刘曼玉, 于钰, 黄兵, 韩红玉. 柔嫩艾美耳球虫含HD结构域蛋白特性和功能初步研究[J]. 畜牧兽医学报, 2022, 53(8): 2608-2620.

XIAO Ke, CHEN Ting, ZHAO Qiping, ZHU Shunhai, DONG Hui, LIU Manyu, YU Yu, HUANG Bing, HAN Hongyu. Preliminary Study on the Characteristics and Functions of HD Dome-containing Proteins in Eimeria tenella[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2608-2620.

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