捻转血矛线虫blister-5基因序列特征及表达特性分析  

Analysis of sequence characteristics and expression properties of blister-5 in Haemonchus contortus

作　　者：汤洪宁 吴飞 项继忠 童丹妮 杜振东 陈学秋[1] 马光旭 杨怡[1] TANG Hongning;WU Fei;XIANG Jizhong;TONG Danni;DU Zhendong;CHEN Xueqiu;MA Guangxu;YANG Yi(Zhejiang Province Key Laboratory of Animal Preventive Medicine/College of Animal Sciences,Zhejiang University,Hangzhou 310058,China;Huzhou Miemieyang Animal Husbandry Co.,Ltd,Huzhou 313023,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,杭州310058 [2]湖州咩咩羊牧业有限公司,浙江湖州313023

出　　处：《中国农业大学学报》2024年第10期199-207,共9页Journal of China Agricultural University

基　　金：国家自然科学基金项目(32202829,32172877);浙江省自然科学基金项目(LZ22C180003,LQ23C180006)。

摘　　要：为明确捻转血矛线虫(Haemonchus contortus)中Kunitz型蛋白BLI-5的表达特性,利用生物信息学软件对其基因结构、保守结构域、进化关系和蛋白质三维结构进行分析;基于WormBase数据库,分析捻转血矛线虫发育过程各个阶段Hc-bli-5与Hc-bli-4的相对转录丰度;通过构建原核表达载体诱导表达重组蛋白,并制备多克隆抗体,采用虫体组织免疫荧光试验分析目标蛋白(Hc-BLI-5)在捻转血矛线虫中的组织定位情况。结果显示:1)Hc-bli-5基因全长2088 bp,由4个外显子和3个内含子组成,Hc-BLI-5共有195个氨基酸,132~192位氨基酸为Kunitztype super family结构域,是一种丝氨酸蛋白酶抑制因子。2)进化树分析发现,丝氨酸枯草杆菌蛋白酶超家族蛋白BLI-5在寄生线虫和自由生活线虫中均有较为密切的进化关系,捻转血矛线虫与柏氏血矛线虫(Haemonchus placei)在该基因的进化关系上最为接近,聚集在进化树的同一小分支上。3)蛋白质结构对比分析显示,捻转血矛线虫和秀丽隐杆线虫(Caenorhabditis elegans)BLI-5具有一定的相似性,尤其是Kunitz结构域。4)各时期转录丰度检测显示,Hc-bli-5与Hc-bli-4的表达趋势有一定的重叠,提示二者可能互相作用、参与共同的生物学过程。5)组织定位研究表明,Hc-BLI-5蛋白主要表达于捻转血矛线虫的表皮和肌肉组织。综上,Hc-bli-5可能与Hc-bli-4相互作用参与捻转血矛线虫蜕皮过程。本研究明确了Hc-bli-5的序列特征及其蛋白的表达特性,为探索其是否参与捻转血矛线虫蜕皮过程及其作用机制提供了理论依据。The study was to identify the expression properties of the Kunitz-type protein BLI-5 in Haemonchus contortus.Firstly,analysis of its gene structure,conserved domain,evolutionary relationships and protein three-dimensional structure using bioinformatics software were conducted;Secondly,the relative transcript abundance of Hc-bli-5 and Hc-bli-4 was analyzed based on WormBase database at each developmental stage of H.contortus;Subsequently,a prokaryotic expression vector to obtain recombinant protein and prepared polyclonal antibodies were constructed,while tissue distribution of the protein candidates in the worm was analyzed by the immunolocalization assay.The results revealed that:1)The Hc-bli-5 gene has a total of 2088 bp,and consisted of 4 exons and 3 introns.Hc-BLI-5 has a total of 195 amino acids,with 132-192 aa forming a Kunitz-type super family domain,which is a serine proteinase inhibitor.2)Phylogenetic tree showed that BLI-5 has a close evolutionary relationship in both parasitic and free-living nematodes,and the evolutionary relationship between H.contortus and Haemonchus placei is most closely aligned,clustering together on the same minor branch of the phylogenetic tree;3)Comparative analysis of protein structures indicated that Hc-BLI-5 and Ce-BLI-5 shared certain similarities,especially in the Kunitz domain.4)Transcript abundance analysis at each developmental stage showed that the expression trend of Hc-bli-5 and Hc-bli-4 overlapped,suggesting that they may interact with each other and participate in common biological process.5)Immunolocalization showed that BLI-5 was distinctly expressed at the cuticle and muscle tissue of worm.In summary,Hc-bli-5 may interact with Hc-bli-4 and participate in the molting process of H.contortus.This study clarified the sequence characteristics and expression properties of Hc-bli-5,providing a theoretical basis for exploring its involvement in the molting process and mechanism in H.contortus.

关 键 词：捻转血矛线虫 蜕皮 Hc-bli-5 序列特征 表达特性 

分 类 号：S855.9[农业科学—临床兽医学]