捻转血矛线虫ZJ株疫苗候选抗原H11亚型基因H11-4的克隆及序列分析  

Cloning and sequence analysis of the isoforms H11-4 of the vaccine candidate antigen H11 from Haemonchus contortus

作　　者：段丽君[1] 周前进[1] 张红丽[1] 杨怡[1] 闫宝龙[1] 杜爱芳[1] 

机构地区：[1]浙江大学动物科学学院,浙江杭州310058

出　　处：《中国兽医学报》2013年第3期381-385,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31101807;30972174);中国博士后科学基金资助项目(84951)

摘　　要：微粒体氨肽酶H11天然提取物是目前捻转血矛线虫(Haemonchus contortus)防治研究中最好的疫苗候选抗原之一,但其重组形式均不能提供有效的免疫保护效果;同时报道H11蛋白存在多种亚型,推测其某种亚型或亚型组合可能在天然提取物参与免疫保护中起了关键作用。本试验参考NCBI公布的H.contortus H11-4基因序列,设计2对特异性引物,以H.contortus ZJ株总RNA为模板,利用RT-PCR技术分段扩增出该基因的部分片段,并进行T-A克隆。测序正确后,利用含有不同片段的阳性质粒经BamHⅠ/NcoⅠ消化,连接后获得H11-4基因的全长cDNA序列。测序结果显示成功获得H11-4基因,开放阅读框大小为2 916bp,与NCBI中公布的核苷酸序列同源性为97.8%,氨基酸序列同源性为97.6%。生物信息学分析,已获得的H11-4与H11(H11-3)亚型氨基酸序列高度同源,且具有保守糖基化位点、相对保守的跨膜区与微粒体氨肽酶活性中心锌指基序。为进一步分析H11天然提取物各亚型在参与免疫保护的机制和角色分工奠定了基础。The recombinant microsomal aminopeptidase Hll antigen has not shown highly protec- tive efficacy against Haemonchus contortus compared to its native extract which is considered to be the most efficient vaccine candidate antigen. It is reported that there exist several isoforms of native H11 and it was supposed that one or combined isoforms of native Hll play a key role in immune protection. Fragments of H11-4 gene were amplified by reverse transcription polymerase chain reaction （RT-PCR） with two pairs of primers designed according to the published gene se- quence from NCBI. Then the fragments of Hll-4 gene were ligated to the T-A cloning vector pMD18-T and sequenced. Positive clones representative the different two fragments were digested with BamH Ⅰ / Nco Ⅰ and then ligated to obtain the full-length cDNA. Sequence analysis shows that with 2 916 bp,H11-4 gene shared 97.8% nucleotide identity and 97.3% protein identity to the sequence on NCBI. It infers that these two isoforms H11-4 and Hll （H11-3） of Hll share high homology after bioinformatics analysis and have few N-glycosylation sites, a relative con- served transmembrane region and zinc-binding motif characteristic of microsomal aminopeptidase. These data help to study the mechanism and interaction of Hll isoforms as a target for immune intervention against H. contortus.

关 键 词：捻转血矛线虫 疫苗候选抗原基因 H11-4 

分 类 号：S852.731[农业科学—基础兽医学]