捻转血矛线虫胰岛素样肽编码基因的鉴定、表达与组织定位

Tissue localization of insulin-like peptide in Haemonchus contortus and identification and expression of its encoding genes

胰岛素样肽(insulin-like peptides,ILPs)在线虫新陈代谢、信号转导、发育进程等生物学过程中发挥重要作用,是控制动物寄生线虫感染的潜在靶标。为探究ILPs在寄生线虫发育与感染中的具体作用,对捻转血矛线虫(Haemonchus contortus,H.contortus)ILPs编码基因进行全基因组鉴定、序列和进化分析;通过实时荧光定量PCR方法检测H.contortus不同发育时期ILPs编码基因的转录水平;经原核表达ILPs重组蛋白用于多克隆抗体制备及Western blot分析;采用间接免疫荧光定位试验明确ILPs在H.contortus虫体不同发育阶段的组织分布情况。结果显示,与秀丽隐杆线虫(Caenorhabditis elegans)相比,H.contortus ILPs编码基因发生显著缩减,仅有3个具备拮抗性分子特性的ILPs编码基因;ILPs编码基因在H.contortus感染性三期幼虫阶段的转录水平最高;ILPs蛋白主要分布于感染性幼虫的肠道与皮下组织。本研究鉴定3个可能参与调控H.contortus幼虫发育进程及感染过程的拮抗性ILPs编码基因,为寄生线虫在宿主体内的滞育现象研究以及潜在干预靶标的筛选奠定了基础。

Insulin-like peptides(ILPs)play a crucial role in the biological processes of nematode metabolism,signal transduction,and developmental processes,representing potential therapeutic targets for nematode infections in animals.In order to explore the specific role of ILPs in nematode development and infection,genome-wide identification,sequence and evolution analysis of the ILPs coding genes were conducted on Haemonchus contortus(H.contortus);transcriptional levels of these ILPs coding genes among developmental stages were determined by a real-time quantitative PCR method;recombinant ILPs were produced in prokaryotic system for polyclonal antibody preparation and Western blot analysis;indirect immunofluorescence localization experiment was used to reveal the tissue distribution of ILPs at different developmental stages.It was showed that the number of ILPs coding genes in H.contortus were significantly reduced compared with that of Caenorhabditis elegans,namely three members of ILPs coding genes exhibiting antagonistic features;the highest transcriptional levels of ILPs coding genes was detected in the infective third stage larvae of H.contortus;ILPs were found dominant in the intestine and hypodermis of the infective larvae of this parasitic nematode.In this study,three genes encoding antagonistic ILPs were identified in H.contortus,they might play a role in regulating the development and infection processes.The finding lay a foundation for the study of nematode hypobiosis(larval diapause)in animals and the screening of potential intervention targets.