首先根据生物信息学技术拼接得到的柔嫩艾美耳球虫(Eimeria tenella)的顶膜抗原-1的序列，以预测的序列设计特异性引物，以E. tenella总RNA为模板，用RT-PCR的方法扩增出长2000bp左右的E. t AMA1基因的部分cDNA序列，长为1611bp的ORF编码一个包括信号肽的长为536个氨基酸的多肽片段，与其它顶复门原虫的AMA1的同源性在32-40％之间。 选择原核表达载体pET-32a (+)和pMAL-c2x，利用E.coli Rosetta(DE3)重组表达E. t AMA1的序列片段(去除信号肽)，优化表达策略，结果显示仅有pET-32a (+)-E. t AMA1能够进行重组表达；温度对pET-32a (+)载体的表达量有显著的影响，随着温度的降低，表达量显著增加；但低温对该蛋白为包涵体表达没有影响。 通过生物信息学方法，在E. tenella数据库进行搜索比对，确定该蛋白的特异性后；利用上述表达的蛋白，用尿素进行变性和复性后纯化，免疫小鼠制备特异性抗体，利用激光共聚焦(Confocal)研究E. t AMA1在E. tenella子孢子体内的免疫荧光亚细胞定位。结果显示，E. t AMA1在E. tenella子孢子表面尤其是顶部。 选取E. tenella生活史中的未孢子化卵囊、孢子化卵囊、子孢子、入侵1小 时后子孢子、裂殖体和裂殖子，提取上述六个样品的总RNA，反转录为cDNA；选择E. t actin 作为参照基因，E. t AMA1为目的基因，设计符合实时荧光定量PCR的引物，进行实验分析上述个样品中E. t AMA1分泌量的变化。结果显示，E. t AMA1的表达随着卵囊的孢子化而上升，并在子孢子阶段达到最高，然后迅速下降，到裂殖子阶段几乎没有表达。 本研究利用前第三章所制备的抗柔嫩艾美耳球虫(Eimeria tenella)的顶膜抗原-1的多克隆抗体，在体外培养的鸡肾细胞进行抗体阻断实验，发现该抗体对阻止E.tenella 子孢子感染鸡肾细胞能起到一定的抑制作用。 本研究首次克隆了E. tenella的AMA1基因，用原核表达后制备抗体，分析了其亚细胞定位，并利用实时荧光定量PCR技术分析了其分泌量的变化情况。在体外培养的鸡肾细胞进行抗体阻断实验。

In this study, the partial sequence fragment of AMA1 gene was amplified by RT-PCR from Eimeria tenella(Guangdong strain) total RNA template. The ORF of 1611bp encoding a protein of 536 amino acids with a predicted signal peptide. Amino acids sequences alignment of EtAMA1 with other AMA1s from several other apicomplexan parasites by ClustalW showed 32-40％ identity with other AMA1s. The fragment encoding AMA1 was sub cloned into the pET-32a (+) and pMAL-c2x respectively, the recombinant vectors were transformed into E.coli Rosetta(DE3) and induced by IPTG. Comparison of these expressions indicates that only the fragment without signal peptide was expressed in pET-32a (+), the expression was enhanced after induced at the lower temperature, but only insoluble protein was expressed in pET-32a (+) at any temperature. The specify of the fragment encoding E.tAMA1 was used as queries to search the E. tenella EST database at the Sanger institute using tBLASTn algorithm. The BALB/c mice were immunized with purified recombinant protein three times to produce polyclonal antibodies against E.tAMA1. E.tAMA1 biosynthesis protein was demonstrated located in the membrane of the apical party of E. tenella using immunocytochemistry. To study the expression pattern of AMA1 at different life stages, real-time quantitative RT-PCR was carried out, which indicate that AMA1 was expressed in all life stages, including unsporulated oocysts, sporulated oocysts, sporozoites, merozoites. AMA1 mRNA level was significantly up-regualted in unsporulated oocysts, sporulated oocysts and sporozoites down-regualted in sporozoites,, merozoites. We use the antiE.tAMA1 antibody to study the invasion inhibition, we find antiE.tAMA1 antibody can inhibit the invasion of Chicken-kidney Cell Monolayer by E.tenella sporozoite.