调控鸡球虫性别分化与发育相关蛋白基因的克隆与鉴定

Avian coccidiosis is a disease of major economic importance to the poultry industry, which is caused by protozoan parasites of the genus Eimeria. Unlike mammalian gender types, micro- and macrogametes of any clonal strain of an apicomplexan species are genetically identical, yet still develop into morphologically distinct cells. Thus, apicomplexan sexual differentiation is a purely phenotypic phenomenon. This indicates that sexual differentiation and development is dependent on the differential expression of sexual stage-specific genes, rather than presence or absence of sex chromosomes. The morphologically distinct micro- and macrogametocytes are generally regarded as the sexual stages in Eimeria but it can be inferred that sex determination occurs in the generation of merozoites immediately preceding gametocytogenesis. The life cycles of poultry Eimeria are, arguably, the most straight forward of any apicomplexan parasites. In Eimeria, a set number of rounds of asexual reproduction (merogony) precedes gametocytogenesis. The exact number of rounds of merogony that takes place before gametocytogenesis varies for different species but the norm may be three, at least in poultry Eimeria. In the life cycle of E. necatrix, the first and second generation meronts are primarily located within the mid-intestinal epithelium cells of chickens, while the third generation meront and gametocytes development occurs only in the caecum epithelium cells, and therefore the highly pure gametocytes and meronts of E. necatrix may be easily separated from the intestinal tract tissues of chickens. In this project E. necatrix of the second and third meronts and gametocytes are separated and purifed respectively,and are used to clone and characterize the differential expressed genes at the second and third generation meronts and macrogametes using the technique of the suppression subtractive hybridization (SSH), cDNA microarray, qRT-PCR anslysis and the rapid amplification of cDNA ends (RACE) technique. Furthermore,the differential expressed genes are ligated to prokaryotic expression vectors and expressed,and the functions of the recombinant proteins are analyzed by tissue section technique, immunocytochemistry, immunoelectron microscope, ELISA and flow cytometry. Our results will help to understand the molecule basis of the sexual differentiation and development in Eimeria, which maybe provided important rationales for studying new vaccines and drugs against coccidiosis.

鸡球虫病是由艾美耳球虫引起的一种严重危害养鸡业的重大疾病。艾美耳球虫的生活史在顶复门原虫中最简单直接，由卵囊经粪-口在宿主间传播。认为艾美耳球虫的性别分化和发育取决于有性阶段特异基因的差异表达，有性阶段基因在最后一代裂殖子中可能就有表达，但对这些差异表达的基因及其功能知之甚少。鸡球虫的裂殖生殖一般是3代。毒害艾美耳球虫的第一、二代裂殖生殖发生在小肠黏膜上皮细胞内，第三代裂殖生殖和配子生殖发生在盲肠黏膜上皮细胞内，易于鉴别分离。因此，本项目采用SSH、cDNA芯片、实时定量PCR、SMART-RACE等技术，克隆与鉴定毒害艾美耳球虫第二代裂殖子、第三代裂殖子、大配子体阶段的特异表达基因，并进行原核表达；采用组织切片、免疫细胞化学、免疫电镜、ELISA、流式细胞术等方法或技术，分析与鉴定这些发育阶段特异性表达基因的功能，解析艾美耳球虫性别分化和发育的分子基础，为寻找防控鸡球虫病新方法奠定基础。

艾美耳球虫的生长发育和性别分化取决于基因在虫体不同发育阶段的差异表达，性别分化相关基因可能在最后一代裂殖子中就有表达，但对这些差异表达的基因及其功能知之甚少。本项目在建立了分离与纯化毒害艾美耳球虫子孢子、第2代裂殖子、第3代裂殖子和配子体方法的基础上，通过对第2、3代裂殖子间消减cDNA文库的构建与分析，第2代裂殖子、第3代裂殖子、配子体的转录组比较与分析，子孢子、第2代裂殖子、第3代裂殖子、配子体cDNA噬菌体表达文库的构建与分析，获得球虫不同发育的差异表达基因以及球虫生长发育相关基因序列。差异表达基因经GO功能富集分析和KEGG通路富集分析后，筛选出与球虫生长发育、性别分化和发育的相关基因，并通过qRT-PCR验证。选择差异表达基因进行克隆、表达与功能研究。共构建了4个cDNA噬菌体表达文库，筛选后获得29个基因序列。构建了2个消减cDNA文库，获得了11个差异表达基因。完成了第2、3代裂殖子和配子体的转录组测序与分析。在第2、3代裂殖子间鉴定出2053个差异表达基因，其中第2代裂殖子显著上调表达基因1216个，特异性表达基因95个，第3代裂殖子显著上调表达基因837个，特异性表达基因48个，主要包括ApiAP2 DNA结合蛋白、艾美耳球虫特异性表面抗原、CMGC激酶、DEAD/DEAH盒解旋酶结构域蛋白和增殖相关蛋白2G4基因等。在第3代裂殖子和配子体间鉴定出4267个差异表达基因，其中第3代裂殖子显著上调表达基因1478个，特异性表达基因 329个，第3代裂殖子显著上调表达基因2789个，特异性表达基因1289个，有51个与卵囊壁生物合成相关，22个与卵囊壁抗酸脂层的合成与重塑相关，33个与小配子体发育相关。克隆、表达了EnPIPK、EnCK2、EnAGC、EnHAP2、EnDlc、EnArm、EnMAPRE3、EnSNF2基因，免疫荧光定位研究显示这些基因在球虫不同发育阶段以及雌雄配子体上差异表达或特异性表达，预示参与调控球虫生长发育、性别分化和发育。重组蛋白EnHAP2和EnSNF2免疫雏鸡能产生一定的免疫保护力。本项目研究结果为进一步解析艾美耳球虫性别分化和发育奠定了重要的基础，也为研制鸡球虫病亚单位疫苗提供了候选基因。