鸡毒害艾美耳球虫小热休克蛋白基因EnsHsp20的克隆表达与蛋白定位  

Cloning,expression and protein localization of small heat shock protein gene EnsHsp20 from Eimeria necatrix

作　　者：张知之 冯茜茜 范雪莲 刘丹丹[1,2] 候照峰 许金俊 陶建平[1,2] ZHANG Zhi-zhi;FENG Qian-qian;FAN Xue-lian;LIU Dan-dan;HOU Zhao-feng;XU Jin-jun;TAO Jian-ping(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2023年第7期893-901,共9页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31972698);江苏省高校优势学科建设四期工程项目(2022-2025);高等学校学科创新引智计划项目(D18007)。

摘　　要：克隆、表达毒害艾美耳球虫小热休克蛋白基因EnsHsp20,检测其天然蛋白及其在虫体内的亚细胞定位。以毒害艾美耳球虫子孢子的总RNA为模板,用RT-PCR扩增EnsHsp20基因后,连接至pGEM-T Easy载体;测序后构建pET28a(+)-EnsHsp20表达载体,转化至大肠杆菌BL21(DE3);经双酶切和测序鉴定后,将重组菌诱导表达;表达产物经Western-blot鉴定、纯化与复性后,免疫BALB/c小鼠,制备小鼠抗rEnsHsp20多克隆抗体;利用多克隆抗体,分别用Western-blot和间接免疫荧光试验检测子孢子和第2代裂殖子中EnsHsp20的天然蛋白及其定位;最后,应用qRT-PCR分析EnsHsp20基因在未孢子化卵囊、子孢子和第2代裂殖子中的转录水平。结果显示,该基因全长549 bp,编码183个氨基酸,预测分子质量为20.3 ku;重组蛋白主要以包涵体形式存在,可以被6×His标签单抗和鸡抗毒害艾美耳球虫、抗柔嫩艾美耳球虫、抗堆型艾美耳球虫和抗巨型艾美耳球虫的阳性血清识别;在第2代裂殖子中检测到EnsHsp20的天然蛋白,分子质量约为36 ku;EnsHsp20蛋白定位在子孢子和第2代裂殖子的细胞膜或细胞质;EnsHsp20基因在子孢子中的转录水平极显著高于第2代裂殖子和未孢子化卵囊(P<0.01)。本研究结果为进一步探析EnsHsp20蛋白在虫体入侵与生长发育中的作用奠定了基础。The principal objective of this study was to clone and express small heat shock protein(EnsHsp20)from Eimeria necatrix,and detect native protein of EnsHsp20 and its subcellular localization in E.necatrix.The gene(EnsHsp20)was amplified from the total RNA of sporozoites(SZ)of E.necatrix by RT-PCR.After being inserted into pGEM-T Easy vector and sequencing analysis,EnsHsp20 was subcloned into pET-28a(+)vector and transformed into Escherichia coli BL21(DE3).Following from the double enzyme digestion identification and sequencing analysis,the recombinant E.coli was induced to express.The recombinant protein was identified by Western-blot,then purified and renatured before being immunized to BALB/c mice to prepare the anti-rEnsHsp20 polyclonal antibodies.The native protein of EnsHsp20 and its localization in SZ and the second generation merozoites(MZ-2)of E.necatrix were detected by Western-blot and indirect immuno-fluorescence assay,respectively.Finally,the transcriptional level of EnsHsp20 in unsporulated oocysts(UO),SZ and MZ-2 of E.necatrix was analyzed by qRT-PCR.The results demonstrated that the target gene was 549 bp in size,encoding 183 amino acids with a predicated molecular weight of 20.3 ku.The recombinant protein mainly expressed in inclusion body,and could be specifically recog-nized by anti-6×His tag mouse monoclonal antibodies and the convalescent sera of chicken infected with E.necatrix,E.tenella,E.acervulina and E.maxima,respectively.Native protein of EnsHsp20 was de-tected in MZ-2 with a molecular weight of 36 ku,and localized in the cytomembrane or cytoplasm of SZ and MZ-2.The transcription level of EnsHsp20 was significantly higher in SZ than that in MZ-2 and in UO(P<0.01).These findings lay the foundation for further study the role of EnsHsp20 protein in invasion and growth of parasites.

关 键 词：毒害艾美耳球虫 热休克蛋白20基因 克隆 表达 定位 

分 类 号：S852.723[农业科学—基础兽医学]