导　　师： 李祥瑞

学科专业： 090602

授予学位： 硕士

作　　者： ;

机构地区： 南京农业大学

摘　　要： 鸡球虫病是严重危害养禽业发展的重要疾病之一，目前对该病的防治还存在许多问题。pEtK2基因编码的钙调蛋白激酶/(EtCDPK/)存在于柔嫩艾美球虫子孢子/(E. tenella/)顶器中，能刺激感染球虫鸡的T淋巴细胞增殖。鸡白细胞介素2是机体免疫调节网络中具有重要免疫调节作用的生物活性物质，能刺激T细胞的生长和增殖，在机体免疫调节中起重要作用。本研究应用DNA重组技术将柔嫩艾美球虫pEtK2基因和鸡白细胞介素2/(IL-2/)基因分别克隆入原核和DNA疫苗载体，制备免疫调节型DNA疫苗，并用构建的DNA疫苗分别免疫动物，观察其诱导产生的特异性免疫应答，以及对E. tenella攻虫的保护作用。本研究主要分以下几个部分： 1．E. tenella pEtK2基因的克隆、鉴定及序列分析 根据国外报道的pEtK2基因序列，利用计算机设计一对引物，从纯化的E. tenella孢子化卵囊子孢子中提取总RNA，然后应用RT—PCR技术扩增出E. tenella pEtK2基因并对其进行测序。测序结果表明，pEtK2基因全长1464bp，具有一个完整的开放阅读框，编码487个氨基酸，与国外发表的序列比较，两序列问有10个碱基不同，其中3个无义突变，7个有义突变，同源性为99％。pEtK2基因编码55KD的钙结合域蛋白酶/(EtCDPK/)，推测可为子孢子的入侵提供重要的离子通道。 2．E. tenella pEtK2基因的原核表达及表达产物的纯化 应用DNA重组技术，将pEtK2基因克隆到原核表达载体pET-28a/(+/)中，并转化大肠杆菌E. coli BL21，通过IPTG诱导表达重组pEtK2蛋白。SDS-PAGE电泳显示，表达的重组蛋白分子量在55KD左右，且在诱导表达5小时后表达量最多，约占总菌体蛋白的32％。细菌经超声波裂解破碎，TritonX-100洗涤，得到的包涵体被纯化，SDS-PAGE电泳显示，pET-28a/(+/)-pEtK2的表达产物绝大部分存在于包涵体� Avian coccidiosis is an important parasitosis, which led to serious economic losses. Eimeria tenella calmodulin-domain protein kinase /(EtCDPK/), converged at the apical end of the sporozoite was encoded by pEtK2 gene. It had been shown that this protein could stimulate T-lymphocyte proliferation of chicken infected with coccidia. Chicken interleukin-2 /(IL-2/) plays an important role in immune regulatory network. In this study, pEtK2 gene of Eimeria tenella and chicken IL-2 gene were cloned into pET28a/(+/), pVAXl.O and pcDNA4.0/(c/)vectors respectively. The DNA vaccine were then used to immunize chicken in order to evaluate their immuno-protective effect.1. Cloning and sequence analysis of pEtK2 gene of Eimeria tenellaBased on pEtK2 cDNA sequence published by GenBank, a pair of special primers were designed by computer software. A 1464bp DNA fragment was amplified by RT-PCR using total RNA template extracted from Eimeria tenella sporozoites. In order to identify this gene, the nucleotide was sequenced and compared with existing data in the GenBank published by Dunn. The result indicated this pEtK2 gene included an open reading frame /(ORF/) which encoded 487 amino acids residues, and had a 99/% homology with pEtK2 in GenBank, in which 10 nucleotides were different from those reported by Dunn. Seven nucleotides produced sense mutation, while the other three nucleotides resulted in non-sense mutation. The pEtK2 gene encoded a 55kDa calmodulin-domain protein kinases /(EtCDPK /) which was thought to play an important role in the sporozoite invasion.2. Expression of pEtK2 cDNA and purification of the recombinant proteinpEtK2 gene was subcloned into pET28a/(+/), and then transformed into E. coli /(BL21/). After induced by IPTG, 55kDa recombinant protein was expressed and could be detected by SDS-PAGE. Analysied by TCL-scanning, it was found that the target protein reach its peak at about 5 hour after induction, which covered more than 32 percent of the total proteins. To purify the recombinant proteins, the inclusion

关 键 词： 疫苗

领　　域： [农业科学] [农业科学] [农业科学]