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巨型艾美耳球虫子孢子表面蛋白IMP1的特性分析

Characterization of the Eimeria maxima sporozoite surface protein IMP1.

作者信息

Jenkins M C, Fetterer R, Miska K, Tuo W, Kwok O, Dubey J P

机构信息

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD 20705, USA.

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD 20705, USA.

出版信息

Vet Parasitol. 2015 Jul 30;211(3-4):146-52. doi: 10.1016/j.vetpar.2015.05.009. Epub 2015 May 14.

Abstract

The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection.

摘要

本研究的目的是对巨型艾美耳球虫免疫图谱蛋白1(IMP1)进行表征,据推测该蛋白在引发鸡对巨型艾美耳球虫感染的保护性免疫中发挥作用。对未孢子化和正在孢子化的巨型艾美耳球虫卵囊的RNA进行RT-PCR分析,结果显示在孢子化6 - 12小时时转录水平最高,此后显著下调。对巨型艾美耳球虫霍顿株、韦布里奇株和APU - 1株的IMP1编码序列进行比对,发现单核苷酸多态性,在某些情况下导致编码的蛋白质序列中的氨基酸变化。克隆了巨型艾美耳球虫(APU - 1)IMP1 cDNA序列,并在2种不同的多聚组氨酸大肠杆菌表达载体中进行表达。无论使用何种表达载体,重组巨型艾美耳球虫IMP1(rEmaxIMP1)在非变性缓冲液中相当不稳定,这与一级氨基酸序列的稳定性分析一致。针对rEmaxIMP1的抗血清通过非还原或还原SDS - PAGE/免疫印迹鉴定出一种72 kDa的单一蛋白质或一种61 kDa的蛋白质。用抗rEmaxIMP1进行免疫荧光染色,显示巨型艾美耳球虫子孢子表面有强烈染色,而裂殖子阶段染色可忽略不计。对感染巨型艾美耳球虫的鸡肠道组织进行免疫组织化学染色,发现在感染后24小时和48小时,固有层和隐窝中的巨型艾美耳球虫发育阶段均有染色,此后染色可忽略不计。IMP1在体内发育早期的表达及其在子孢子表面的定位可能部分解释了该蛋白对巨型艾美耳球虫感染的免疫保护作用。

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