鸡柔嫩艾美耳球虫MIC2转基因玉米表达体系构建及其免疫效果研究

With the development of gene engineering, transgenic plant express exogenous protein is a healthy and economic express system for vaccine relative to the traditional way. Corn is the main feed for poultry industry. If genes of protective antigen of coccidium were transferred to corn and expressed, chicken can have immunity to prevent the coccidium from invading by feeding with the transgenic maize. Coccidiosis is one of the main disease for poultry industry with high mortality. For a long time, anticoccidial drugs of many different chemical types have been the dominant means to prevent and control chicken coccidiosis.Drug residue and drug-resistant are big problems in animal husbandry right now. Vaccine is a good way to control the disease. Etmic2 is a kind of protein secreted by microneme of Eimeria tenella. It is reported that Etmic2 protein can prevent Eimeria tenella from invading into the intestinal epithelial cell of chicken. But how to prevent Eimeria tenella from invading into the intestinal epithelial cell with the genetically modified maize is seldom reported. We will synthetize EtMIC2 gene according to the plant preference codon in this project. In order to promote the target gene expression in seeds of maize, specific expression promoter of endosperm will be selected which can regulate and control the expression of EtMIC2 gene. Agrobacterium will be used to transfer EtMIC2 gene to the embryo of maize. After we get the seeds of maize which contain EtMIC2 gene, we will vaccinate healthy chicken by feeding with the seeds. Then the immune effect will be tested by cell mediated immunity and humoral immunity. And we will evaluate the security of the chicken fed with the transgenic maize . This project will afford a new way to control Eimeria tenella.

随着植物基因工程的发展，用转基因植物表达外源蛋白相对于传统疫苗而言是一种更安全、更经济的表达系统。玉米是养鸡业的主体饲料，如果将鸡球虫保护性抗原基因整合到玉米基因组中并表达，再用转基因玉米饲喂鸡，便可预防鸡球虫病。鸡球虫病是养鸡业多发病，死亡率高，对该病通常以药物防控为主,但由此带来的诸如药物残留以及抗药性问题是显而易见的。疫苗是预防鸡球虫病较为理想的方法。Etmic2是柔嫩艾美耳球虫微线分泌的蛋白，研究表明EtMIC2基因表达产物能抵御柔嫩艾美耳球虫入侵，但有关艾美耳球虫转基因植物疫苗的研究却鲜有报道。本项目拟根据植物偏爱密码子重新合成EtMIC2基因，并构建由玉米胚乳特异表达启动子驱动EtMIC2基因的植物表达载体，用农杆菌法转化玉米，使EtMIC2基因在种子中表达，然后用转EtMIC2玉米种子免疫健康鸡,检测免疫效果,并对免疫后的鸡进行安全性评价,从而为控制鸡球虫病提供新途径。

鸡球虫病是养鸡业多发病，死亡率高，对该病通常以药物防控为主,但由此带来的诸如药物残留以及抗药性问题是显而易见的。疫苗是预防鸡球虫病较为理想的方法。Etmic2是柔嫩艾美耳球虫微线分泌的蛋白，研究表明EtMIC2基因表达产物能抵御柔嫩艾美耳球虫入侵，本项目拟将柔嫩艾美耳球虫EtMIC2基因转入玉米，并构建EtMIC2基因的植物表达载体，用农杆菌法转化玉米，使EtMIC2基因在玉米中表达，本研究为利用转基因玉米控制鸡球虫病提供新途径。.首先，我们从沈阳地区患球虫病的鸡盲肠中分离纯化柔嫩艾美耳球虫，用PCR扩增EtMIC2基因，测序后进行同源性分析，表明沈阳分离的EtMIC2基因与Houghton株EtMIC2基因核苷酸序列同源性达98%以上。然后，我们成功地构建了由CaMV 35s启动子驱动MIC2基因表达的载体pCAMBIA1301-MIC2，该载体经过PCR、双酶切、测序鉴定完全正确。再用农杆菌法转化玉米自交系SN3和杂交种KN1。经农杆菌转化后的愈伤通过潮霉素抗性筛选、PCR检测、Southern blot检测，证实MIC2基因能够转入玉米幼胚中并呈双拷贝，并获得转基因苗。同时将植物表达载体pCAMBIA1301-MIC2转化玉米幼胚，以转空载体pCAMBIA1301为对照，并对共培养3天后的幼胚进行GUS染色检测，发现有呈阳性的幼胚，说明MIC2基因可能转入玉米幼胚细胞内并进行瞬时表达。此外，我们还做了玉米自交系和杂交种阳性率和转化率的比较。发现杂交种KN1相对自交系SN3无论GUS阳性率还是转化率都略高。但无论自交系还是杂交种获得抗性愈伤都很少。最后，我们对EtMIC2原核表达的蛋白进行了口服和肌注免疫，并用ELISA方法和淋巴细胞转化方法(MTT法)以及鸡只抗球虫感染动物试验，对实验鸡只分别检测和统计，证明了重组蛋白口服和肌肉注射鸡只免疫后均能在机体内产生一定的抗体水平和抗球虫感染保护力。