鸡艾美耳球虫侵入部位特异性关键分子研究

The Eimeria coccidia, Apicomplexa parasites,infected in chicken are E．acervulina,E．praecox,E．maxima,E．necatrix,E．brunetti,E．mitis and E．tenella. They site specifically located at the duodenum,uper, middle , lower of small intestine and caecum.This site specificity of infection was determined by the site specificity for invasion. The previous researches showed that the MIC proteins secreted by the parasites acting as ligands and the receptors on the epithelial cells of intestines might be the key molecules mediating the site specificity.However, the ligands of the coccidia and the receptors of the epithelial cells of chicken were not totally charactered. In this research, the E．acervulina,E．maxima, E．brunetti and E．tenella were used as model to investigate the key molecules of the site specificity invasion. The member proteins or the whole cell proteins of purified sporozoites are used to bind the epithelial cells of the chicken intestine.The binding proteins to the epithelial cells are analyzed by 2-D electrophoresis and probed by the anti-serum against the proteins of the purified sporozoites. The identified binding proteins are further analyzed by MALDI-TOF or MALDI-TOF-TOF to get the ligands genes. Then, the ligands genes are cloned and experssed in E. coli. The recombiant proteins of the ligands are used to test their abilities to block the invasions of the sporozoites and to protect the chicken from the infections of ccocidia and further to identify the ligands. After that, the Yeast two-hybrid will be used to select the receptors on the epithelial cells of intestine. The passively immune protective effects of antiserum to recombiant receptors on the chickens against the coccidia challenges will be carried out to further confirm the receptors.The distrubutions of the receptors on the intestine will also be observed. The results are benificall to the totally understanting of the molecular mechanisms of site specificity of Eimeria.

鸡球虫病由艾美耳属的7种球虫感染引起，分别寄生于十二指肠和小肠上段、中段、下段及盲肠，具有明显的寄生部位特异性。寄生部位特异性取决于侵入部位特异性，但决定部位特异性的虫体配体和细胞表面受体尚不完全明了。本项目拟选取不同部位寄生的代表性虫体，根据虫体配体与细胞表面受体相结合的原理，提取虫体子孢子膜蛋白或全虫可溶性蛋白，与鸡不同肠段上皮细胞结合，提取结合后细胞膜蛋白或总蛋白，进行2-D电泳分析，并用子孢子蛋白高免血清识别，选取识别蛋白点，通过质谱分析，确认虫体配体分子。再观察配体重组蛋白抗血清阻断虫体侵入和配体重组蛋白对攻虫的免疫保护作用，确定虫体配体。在此基础上，用酵母双杂交确认细胞受体分子，用抗血清被动免疫保护试验进一步验证受体功能，并观察受体在整个肠道的分布，从而确定鸡球虫部位特异性的关键分子。对阐明鸡球虫部位特异性的分子机理有重要意义，对研究顶复门其他虫体的部位特异性也有重要借鉴意义。

鸡球虫病由艾美耳属的7种球虫感染引起，鸡球虫感染具有明显的部位特异性。寄生部位特异性取决于侵入部位特异性，但决定部位特异性的虫体配体和细胞表面受体尚不完全明了。本项目选取了不同部位寄生的代表性虫体，根据虫体配体与细胞表面受体相结合的原理，提取子孢子可溶性蛋白，与鸡不同肠段上皮细胞结合，确认虫体配体分子。观察配体重组蛋白抗血清阻断虫体侵入和配体重组蛋白对攻虫的免疫保护作用，确定虫体配体。用酵母双杂交确认细胞受体分子，用抗血清被动免疫保护试验验证受体功能，确定鸡球虫部位特异性关键分子。.鉴定出了鸡球虫特异性寄生肠段上皮细胞结合蛋白，其中85个E. acervulina子孢子蛋白与十二指肠结合，35个E. maxima子孢子蛋白与空肠结合，91个E. mitis子孢子蛋白与小肠后段结合，包含入侵相关分子MIC2、MIC7和MIC3等。免疫保护试验和被动免疫保护试验发现鸡球虫入侵相关分子EaMIC2、EaMIC5、EaMIC3、EmMIC3、EmMIC2、EmMIC7和EmAMA1等可以抵抗球虫感染，可以作为鸡球虫疫苗的候选抗原。观察不同微线蛋白和鸡不同肠段的结合能力，发现EaMIC3只与十二指肠上皮细胞结合，EmMIC3只与空肠上皮细胞结合，EmiMIC3只与小肠后段上皮细胞结合，表明MIC3是E. acervulina，E. maxima和E. mitis寄生部位特异性的关键分子。比较MIC3的不同段MARR与相对应肠上皮细胞的结合能力，发现EaMARR3与十二指肠的结合能力最强，EmiMARR4与小肠后段的结合能力最强。证明决定E. acervulina入侵部位特性的关键分子为位于EaMIC3的MARR3，决定E. mitis入侵部位特性的关键分子为位于EmiMIC3的MARR4。以E. acervulina为模型，鉴定出了入侵部位关键分子EaMIC3在十二指肠上皮细胞上有7种受体分子。通过受体分子抗血清被动免疫试验和重组蛋白的主动免疫试验，发现对入侵部位特异性关键分子MIC受体UBE和VAP的封闭，能够显著阻断球虫的入侵，证实在球虫入侵中发挥重要作用的MIC受体分子是UBE和VAP。.本研究初步鉴定出鸡球虫的侵入和寄生部位特异性关键分子，对深入阐明鸡球虫的部位特异性机制具有重要意义，对于研发新的抗球虫疫苗，减少抗球虫药的使用，提高禽类产品的安全性也具有重要作用。