【作者】 刘义伟；

【导师】 秦建华；

【作者基本信息】 河北农业大学 ， 预防兽医学， 2012， 硕士

【摘要】 鸡球虫病(Coccidiosis)是由艾美耳属(Eimeria)的多种球虫寄生于鸡消化道引起感染的一种严重危害养鸡业发展的原虫病,目前该病的防治主要依靠化学药物,长期应用药物易使球虫产生耐药性,以及在肉蛋中药物残留危害人体健康,因此研究人员期望用更好的手段来控制鸡球虫病。近年来随着分子生物学和基因工程学的发展,亚单位疫苗作为一种安全、稳定、高效和易于制备的疫苗而得到广泛研究。本试验对鸡堆型艾美耳球虫(Eimeria acervulina)相关抗原基因ADF和3-1E进行了重组克隆共表达,并用重组蛋白进行了免疫保护试验,为研制高效、安全的抗球虫多价疫苗奠定基础。参考GenBank中收录的E.acervulina肌动蛋白解聚因子(actin-depolymerizing factor,ADF)基因序列,设计特异性引物,以保定株堆型艾美耳球虫卵囊子孢子DNA为模板,用聚合酶链式反应(PCR)法扩增ADF基因,经检测扩增产物为729bp。将扩增的ADF基因克隆至pMD-18T载体,转化于感受态细胞DH5α,蓝白斑法筛选出阳性重组子,提取阳性质粒DNA并测序。序列分析结果表明,该株的ADF基因序列与参考序列同源性达99.6%。将扩增产物与原核表达载体pET32a(+)连接,构建重组质粒pET32a-ADF,转化入大肠杆菌BL21(DE3)中诱导表达,表达产物经SDS-PAGE鉴定。结果表明表达出的ADF基因重组蛋白分子量大小约为46kDa。应用重叠PCR技术(SOE-PCR)将ADF基因和3-1E基因融合得到ADF-3-1E重组基因,并将融合基因插入到pET-32a(+)载体中,构建成了pET-32a-ADF-3-1E共表达载体,转化BL21宿主菌中进行诱导表达及表达产物的鉴定。SDS-PAGE检测结果表明,表达的融合蛋白分子量约为68 kDa,与预测的蛋白大小相符。经Western blot鉴定表明,表达的融合蛋白具有很好的反应原性。将100只雏鸡随机分为5组,分别设ADF重组蛋白免疫组、3-1E重组蛋白免疫组、ADF-3-1E重组蛋白免疫组、非免疫感染组和健康对照组。在7日龄时首免,14日龄加强免疫,二免一周后用4×106个E.acervulina BD株球虫孢子化卵囊进行攻毒试验,观察重组ADF-3-1E蛋白对鸡堆型艾美耳球虫的免疫保护效果。结果表明, ADF-3-1E重组蛋白免组可产生较强的抗虫效果,卵囊产量下降67.88%,肠道病变记分降低67.21%,鸡的相对增重率达88.36%,ACI值为166.76。更多还原

【Abstract】 Avian coccidiosis is a serve problem for the poultry industry, caused by intracellular protozoa including several species of the coccidia. Although coccidiosis is mainly controlled by the use of chemotherapeutic agents, alternative control strategies are needed due to the increasing emergence of drug-resistant parasite strains in commercial settings. The reaserch constructed a recombinant vector to co-express ADF and 3-1E genes of Eimeria acervulina. It built solid foundation for the development of effective and safe vaccines of anticoccodial as well as new drugs.Based on the sequence alignment of the actin-depolymerizing factor(ADF) gene of E.acervulina,two primers were designed and synthesized. Using the total DNA of sporozoites of E.acervulina Baoding strain isolated from Hebei province of China as template, a partial segment of ADF gene was amplified by PCR.The gene fragment 729bp in length was cloned into pMD-18T vector, and the recombinant plasmid was identified by PCR,restriction enzyme analysis and sequencing. The homology analysis revealed that the nucleotide sequences similar of the ADF gene of the E.acervulina Baodong strain with reference sequence were 99.6%. The plasmid pMD-18T-ADF was cloned into pET32a(+) vector,constructed recombinant plasmid pet32a-ADF,then transformed into E.coil BL21 strain to Expression. The ADF fusion protein band of about 46kDa was identificated by SDS-PAGE. Western blot analysis indicated that the recombinant protein could react specifically with Eimeria acervulina polyclonal antibody. The gene could synthesis a fusion protein at high levels.The fusion gene of ADF and3-1E was cloned by SOE-PCR, inserting into pET-32a(+) vector, the recombinant vector of pET-32a-ADF-3-1E was constructed correctly. Then it was transformedinto E.coli BL21 (DE3) and induced with IPTG. The expressed protein was detected bySDS-PAGE, with the expected molecular sizes of 68 kDa. The expressed protein was consistent with the prediction.The immuoprotection induced by recombinant ADF-3-1E antigen in chicken.The chicken were inoculated with r ADF-3-1E at 7 days and 14 days, then were challenged with Eimeria acervulina oocysts. The results showed that the pET-32a-ADF-3-1E test chickens can produce stronger resistance effect: Insects attack the egg sac of chicken production fell 67.88%, lower intestinal lesion score 67.21%, and the relative weight of chicken rate of 88.36%, ACI value of 166.76.更多还原