捻转血矛线虫ES24抗原基因的克隆与表达特性分析  

Cloning and expression characteristics of ES24 gene in Haemonchus contortus

作　　者：牛延萍[1] 高丽丽[1] 宋小凯[1] 徐立新[1] 李祥瑞[1] 严若峰[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2012年第11期17-21,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家自然基金(31001059);江苏高校优势学科建设工程资助项目

摘　　要：根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。A gene fragment at a length of 670 bp was produced by RT-PCR, using a pair of primers based on the ES24 sequence （No. U64793.1 ） of Haemonchus contortus. Then this fragment was cloned into pMD18-T vector, sequenced and analysed. It was founded that the gene was 96% - 98% similar with the gene of H. contortus ES24 available in the GenBank. The prokaryotic expression plasmid pET28/ES24 was constructed by cloning the ORF of ES24 into the vector pET28a （＋）. Then the recombinants were transformed into Esche- richia coli BL21 and induced by isopropyl-B-D-thiogalactopyranoside （IPTG）. The SDS-PAGE analysis showed that the recombinant protein was expressed with the molecular weight of 25 ku. The ES24 RNA levels in egg, the third stage larva （ L3 ） , adult male and female worms were detected by Real-time PCR. The result showed that ES24 was expressed at the highest level in the adult male worms and lowest in the L3.

关 键 词：捻转血矛线虫 排泄分泌抗原 ES24 阶段性表达 实时荧光定量PCR 

分 类 号：S852.73[农业科学—基础兽医学]