捻转血矛线虫ES24抗原基因的克隆与表达特性分析

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。

Cloning and expression characteristics of ES24 gene in Haemonchus contortus

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 Escherichia 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.