鸡柔嫩艾美耳球虫特定发育阶段共同抗原确认及其免疫学机制研究

Chicken coccidiosis is a parasitic protozoan disease which hazard to chicken industry seriously, and it is particularly serious as damage from Eimeria tenella. Immunoproteomic and bioinformatics assays were used in our study. The common antigens were recognized from soluble proteins which could be identificated by hyper-immune serum of Eimeria tenella of the different developmental stages including sporozoite，the second-generation merozoite, gametocyte and unsporulated oocyst of Eimeria tenella. The encode genes of the common antigens were cloned and expressed by PCR or RACE, then the immunological mechanism would to be studied by flow cytometry and cytokines assay. And immuno-histochemistry (IHC) and indirect immunofluorescent antibody test (IFAT) would to be used for detection of the common antigens in the different developmental stages. Then the expression level of mRNA of the common antigen would to be detected by Real time quantitative PCR (RT-PCR). The findings from the proposed study will provide insights for recognizing new cycle protective antigen and expounding immunological mechanisms of Eimeria tenella, and lay the foundation for more effective security DNA vaccine and new diagnostic reagents.

鸡球虫病是一种严重危害养鸡业的寄生性原虫病，其中柔嫩艾美尔球虫危害尤为严重。本研究拟采用免疫蛋白组学和生物信息学方法，以柔嫩艾美耳球虫南昌株不同发育阶段虫体---子孢子、第二代裂殖子、配子体、未孢子化卵囊为研究对象，系统确认四个阶段性虫体可溶性蛋白中被鸡柔嫩艾美耳球虫高免血清所识别的共同抗原。采用PCR、RACE等方法获得共同抗原基因并进行表达，利用流式细胞术和细胞因子测定等方法探究共同抗原免疫学机制；进而通过免疫组织化学法（IHC）、间接免疫荧光抗体试验（IFAT）等检测共同抗原在各阶段的表达，并采用实时荧光定量PCR法（RT-PCR）对共同抗原基因在各发育阶段mRNA表达水平进行检测。拟研究成果的取得将为确认新的柔嫩艾美耳球虫循环保护性抗原、阐明柔嫩艾美耳球虫免疫机理提供理论依据，为研发更有效、安全的DNA疫苗以及新的诊断试剂奠定基础。

本研究采用免疫蛋白组学和生物信息学方法，开展了柔嫩艾美耳球虫（江西株）不同发育阶段虫体可溶性蛋白中被柔嫩艾美耳球虫高免血清所识别的共同抗原研究。发现柔嫩艾美耳球虫子孢子可溶性蛋白684个，其中128个蛋白能够被高免血清所识别；第二代裂殖子可溶性蛋白987个，其中148个蛋白能够被高免血清所识别；未孢子化卵囊可溶性蛋白768个，其中109个蛋白被高免血清所识别。分别选取其中73、111和86个蛋白进行蛋白质谱和串联质谱鉴定，利用Mascot通过NCBI数据库检索，发现了6个在不同发育阶段均能够被柔嫩艾美耳球虫高免血清所识别的共同免疫原性蛋白。分别为：Leucine rich repeat protein，Heat shock protein 60，Acetyltransferase domain-containing protein，Peptidyl-prolyl cis-trans isomerase d-like protein，Serine hydroxymethyltransferase和Triosephosphate isomerase。共同抗原基因合成后分别与原核表达载体pEt28a连接、转化宿主细胞，经IPTG诱导有4个基因（Acetyltransferase domain-containing protein，Peptidyl-prolyl cis-trans isomerase d-like protein，Serine hydroxymethyltransferase 和 Triosephosphate isomerase）实现重组蛋白表达且均能够被鸡高免血清所识别。将6个共同抗原基因分别与真核表达载体pVAX1.0连接构建DNA疫苗，并通过动物免疫保护实验，检验DNA疫苗免疫保护效果。pVAX1.0-acetyltransferase domain-containing protein、pVAX1.0-peptidyl-prolyl cis-trans isomerase d-like protein、pVAX1.0-serine hydroxymethyltransferase和pVAX1.0-triosephosphate isomerase四种DNA疫苗有一定的保护效果，可作为柔嫩艾美耳球虫的候选抗原，其免疫学机制有待于进一步研究。