捻转血矛线虫疫苗候选抗原H11亚型基因的序列结构及启动子活性分析  

Genomic structure and transcriptional activity of 5′-flanking region of H11 isoforms in Haemonchus contortus

作　　者：杨怡[1] 段丽君 张红丽[3] 周前进[4] 陈学秋[1] 杜爱芳[1] YANG Yi;DUAN Lijun;ZHANG Hongli;ZHOU Qianjin;CHEN Xueqiu;DU Aifang(Zhejiang Province Key Laboratory of Animal Preventive Medicine,College of Animal Sciences,Zhejiang University,Hangzhou 310058,China;Technical Service Station of Animal Husbandry and Veterinary Medicine in Haishu District,Ningbo,Zhejiang 315000,China;Zhejiang Animal Disease Prevention and Control Center,Hangzhou 310058,China;College of Marine Science,Ningbo University,Ningbo,Zhejiang 315211,China)

机构地区：[1]浙江大学动物科学学院,浙江省动物预防医学重点实验室,浙江杭州310058 [2]宁波市海曙区畜牧兽医技术管理服务站,浙江宁波315000 [3]浙江省动物疫病预防控制中心,浙江杭州310058 [4]宁波大学海洋学院,浙江宁波315211

出　　处：《中国兽医学报》2021年第5期929-936,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0501200);浙江省基础公益研究计划资助项目(LGN20C180005);中央高校基本科研业务费专项资金资助项目(2019QNA6025)。

摘　　要：捻转血矛线虫微粒体氨肽酶H11是有效的疫苗候选抗原,其重组蛋白免疫保护效果可能与基因亚型相关。为分析H11亚型基因的序列结构及启动子活性,本研究扩增并鉴定了H11-1、H11-2亚型基因的开放阅读框(2 937,299 bp),发现其与已知H11 (H11-3)、H11-4亚型氨基酸序列高度同源,具有保守的糖基化位点、跨膜区及微粒体氨肽酶活性中心锌指基序。分段扩增获得了H11-1、H11-2、H11-4亚型基因DNA序列(11 698,17 995,19 804 bp),比对分析发现,与H11(H11-3)基因结构相似,H11-1、H11-2亚型基因均含25个外显子和24个内含子,通过典型的GT…AG结构剪接,对应的外显子大小基本一致,内含子大小各异,同源性低;推测各亚型不是由同一基因选择性剪接形成,但剪接模式相同。运用基因步移技术扩增鉴定了H11-1、H11-2、H11-4亚型基因5′侧翼区(4 606,3 465,2 361 bp),利用pPD95.77载体构建了重组表达载体,通过显微注射将其导入秀丽线虫观察其启动转录活性,结果表明H11-4 5′侧翼区可启动GFP在秀丽线虫除胚胎以外的各时期表达,且表达集中于虫体肠道起始和末端;未检测到H11-1或H11-2 5′侧翼区启动的GFP表达,推测其转录活性或GFP表达较弱。本研究为后续探明H11基因各亚型生物学功能、参与免疫保护机制及高效重组疫苗研究奠定了基础。The microsomal aminopeptidase H11 of Haemonchus contortus is an effective vaccine antigen candidate.This research was performed to study the genomic structure and transcriptional activity of 5′-flanking region of H11 isoforms in H.contortus.The ORF of H11-1 and H11-2 gene were amplified and identified, their size was 2 937,2 919 bp, respectively.Sequence analysis showed that H11-1 and H11-2 gene shared high homology with H11-3 and H11-4,which exhibited conserved N-glycosylation sites, a relative conserved transmembrane region and zinc-binding motif characteristic of microsomal aminopeptidase.The full sequences of H11-1,H11-2,and H11-4 were successfully obtained, which were 11 698,16 479 and 19 804 bp in size, respectively.Similar to the genetic structure of H11(H11-3),the three isoforms all contain 25 exons and 24 introns, and the exon and intron were spliced by a typical GT...AG structure.The size of the exons was basically the same,while the size of the introns was different and the homology was low.The results confirmed the hypothesis that each isoform was not formed by alternative splicing of the same gene,but the splicing pattern was the same.The 5′-flanking regions of three H11 isoforms were amplified by genome walking(H11-1,4 606 bp;H11-2,3 465 bp;H11-4,2 361 bp)and cloned to upstream of GFP gene in pPD95.77 vector,respectively.The recombinants were microinjected into the posterior gonad of Caenorhabditis elegans(C.elegans)to verify the transcriptional activity of 5′-flanking sequence.The transformed C.elegans progeny,with H11-4 5′-flanking sequence exhibited fluorescence in the most anterior intestine and distal intestine,occasionally in the middle intestine,during all the life-cycle,except embryo phase,while no apparent fluorescence was observed in the transformed worms with H11-1 5′-flanking sequence or H11-2 5′-flanking sequence.One possible reason was that the transcriptional activity or expression of GFP in transformed worms was too low to be observed.Our results laid the foundation for t

关 键 词：捻转血矛线虫 H11亚型基因 5′侧翼区 启动转录活性 

分 类 号：S852.73[农业科学—基础兽医学]