细粒棘球绦虫3-羟基酰基-CoA脱氢酶基因的克隆及在其不同阶段的差异表达和生物信息学分析  

Cloning of the 3-hydroxyacyl-CoA dehydrogenase gene from Echinococcus granulosus,its differential expression,and analysis of its biological-information at different stages

作　　者：县锦雯 王炜烨 王芸菲 陆宝燕 张艳艳[2] 孟季蒙[2] 王正荣[2] 薄新文[1,2] XIAN Jin-wen;WANG Wei-ye;WANG Yun-fei;LU Bao-yan;ZHANG Yan-yan;MENG Ji-meng;WANG Zheng-rong;BO Xin-wen(College of Animal Science and Technology,Shihezi University,Xinjiang China 832000;State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding/Animal Husbandry and Veterinary Institute,Xinjiang Academy of Agricultural and Reclamation Sciences)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆农垦科学院畜牧兽医研究所

出　　处：《中国病原生物学杂志》2020年第11期1289-1294,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31860701,31360608);国家重点基础研究发展计划(973计划)项目(No.2015CB150300);新疆生产建设兵团国际科技合作项目(No.2017BC003,2020BC007)。

摘　　要：目的研究细粒棘球绦虫3-羟基酰基-CoA脱氢酶分子特性及其在细粒棘球绦虫不同发育阶段的表达差异。方法通过分析细粒棘球绦虫原头蚴高通量转录组测序文库对Unigene进行KEGG通路分析,筛选出一个脂肪酸代谢酶通路及该通路的一个主要调控基因3-羟基酰基-CoA脱氢酶基因。PCR扩增3-羟基酰基-CoA脱氢酶基因,分析其核苷酸序列及其编码蛋白的结构和功能;通过实时荧光定量PCR分析虫体的原头蚴、成虫发育阶段的mRNA的转录情况。结果细粒棘球绦虫3-羟基酰基-CoA脱氢酶基因具有完整的开放阅读框,cDNA全长为822bp,编码274个氨基酸。生物信息学预测3-羟基酰基-CoA脱氢酶蛋白质的相对分子质量及等电点分别为32.98×10^5和8.87。该蛋白无跨膜螺旋区,具有亲水性,且具有多个潜在的优势抗原表位。氨基酸序列多重比对分析显示其与多房棘球绦虫亲缘关系较近。实时荧光定量PCR检测对该基因在原头蚴和成虫期均有表达,以成虫阶段表达量更高。结论细粒棘球绦虫3-羟基酰基-CoA脱氢酶基因在原头蚴和成虫期高表达,其编码蛋白具有潜在的优势抗原表位该基因可作为细粒棘球绦虫终末宿主抗原候选基因。Objective In order to investigate the molecular characteristics of Echinococcus granulosus 3-hydroxyacyl-CoA dehydrogenase and differences in its expression in E.granulosus protoscoleces and adult worms.Methods KEGG pathway analysis of unigenes was performed by analyzing the high-throughput transcriptome sequencing library of E.granulosus and screening a fatty acid metabolism enzyme pathway and a major regulatory gene 3-hydroxyacyl-CoA dehydrogenase gene.The 3-hydroxyacylCoA dehydrogenase gene was amplified using PCR,and the structure and function of its encoded protein were analyzed.mRNA transcription in the protoscolex and adult stages of E.granulosus was analyzed using real-time fluorescent quantitative PCR.Results Results indicated that the 3-hydroxyacyl-CoA dehydrogenase gene of E.granulosus had a complete open reading frame.cDNA was 822 bp in length,and the gene,encoded 274 amino acids.Bioinformatics predicted that the protein’s relative molecular mass was 32.98×10^5 and its isoelectric point was 8.87.The protein had no transmembrane helix regions,it was hydrophilic,and it may have many dominant epitopes.Multiple alignment analysis of the amino acid sequence indicated that it was closely related to E.multilocularis.Expression of the gene in different stages was analyzed using real-time fluorescence quantitative PCR.Real-time PCR indicated that the gene was expressed in both the protoscolex and adult stages,though the level was higher in the adult stage.Conclusion The 3-hydroxyacyl-CoA dehydrogenase gene of E.granulosus is highly expressed in the protoscolex and adult stages.Its encoded protein may have many dominant epitopes.Results indicated that this gene could potentially be used as an E.granulosus antigen in definitive hosts.

关 键 词：细粒棘球绦虫 3-羟基酰基-CoA脱氢酶 基因克隆 生物信息学分析 差异表达分析 

分 类 号：R383.33[医药卫生—医学寄生虫学]