青海血蜱Haemalin基因的克隆、生物信息学及转录水平分析  

Cloning,bioinformatics and transcriptional analysis of Haemalin gene in Haemaphysalis qinghaiensis

作　　者：何永彩 李增魁[1] 陈炳华 张连生 李中余 何顺福 陈继勇 耿鹏程 胡晓宇 蒋硕 陈强 徐璐瑶 康明[1] 李英[1,8,9] HE Yong-cai;LI Zeng-kui;CHEN Bing-hua;ZHANG Lian-sheng;LI Zhong-yu;HE Shun-fu;CHEN Ji-yong;GENG Peng-cheng;HU Xiao-yu;JIANG Shuo;CHEN Qiang;XU Lu-yao;KANG Ming;LI Ying(College of Agriculture and Animal Science,Qinghai University,Xining 810016,China;Qinghai Province Ledu County Luhua Township Animal Husbandry and Veterinarian Station,Haidong 810799,China;Qinghai Province Ledu County Lijia Township Animal Husbandry and Veterinarian Station,Haidong 810799,China;Qinghai Xunhua Salar Autonomous County Animal Husbandry and Veterinary Station,Haidong 811100,China;Xining Wildlife Park,Xining 810001,China;Animal Disease Prevention and Control Center of Yushu Prefecture,Yushu 815099,China;Animal Disease Prevention and Control Center of Guoluo Prefecture,Golog 814000,China;Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Diseases and Green Technical Research for Prevention and Control,Xining 810016,China;Key Laboratory of Plateau Cold-water Fish Culture and Eco-environmental Conservation(Co-construction by Ministry and Province),Ministry of Agriculture and Rural Affairs,Qinghai University,Xining 810016,China)

机构地区：[1]青海大学农牧学院,青海西宁810016 [2]海东市乐都区芦花乡畜牧兽医站,青海海东810799 [3]海东市乐都区李家乡畜牧兽医站,青海海东810799 [4]青海省循化撒拉族自治县畜牧兽医站,青海海东811100 [5]西宁野生动物园,青海西宁810001 [6]玉树州动物疫病预防控制中心,青海玉树815099 [7]果洛州动物疫病预防控制中心,青海果洛814000 [8]青海省动物疾病病原诊断与绿色防控技术研究重点实验室,青海西宁810016 [9]青海大学农业农村部高原冷水鱼养殖与生态环境保护重点实验室(部省共建),青海西宁810016

出　　处：《中国预防兽医学报》2024年第7期700-708,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：青海省科学技术厅科技国际合作专项(2021-HZ-801)。

摘　　要：为构建青海血蜱Haemalin基因的cDNA全长序列,并分析该基因的生物信息学特征及其在青海血蜱不同发育阶段和雌蜱不同组织中的转录水平,本研究基于青海血蜱唾液腺转录组文库中检索到的一个功能注释为Haemalin的基因片段,采用cDNA末端快速扩增技术(RACE)引物设计软件设计Haemalin基因3'RACE和5'RACE特异性引物,分别经RACE扩增青海血蜱Haemalin基因的3'端和5'端序列,测序后与其中间序列拼接获得该基因的cDNA全长序列。利用生物信息学软件分析该基因编码蛋白的信号肽、理化性质、跨膜结构域、结构域、蛋白质三级结构、B细胞抗原表位以及氨基酸序列比对,采用NCBI中的BLAST及MEGA 7.0软件分别分析Haemalin基因编码氨基酸序列的相似性及构建其系统进化树,采用qPCR检测Haemalin基因在不同发育阶段青海血蜱和雌蜱不同组织中的转录水平。结果显示,青海血蜱Haemalin基因cDNA全长583 bp,开放阅读框(ORF)423 bp,该基因编码蛋白的信号肽为N端的15个氨基酸,理论等电点4.71,属于亲水性蛋白,无跨膜结构域。该基因编码蛋白的三级结构分析结果显示,其存在两个典型的Kunitz/牛胰蛋白酶抑制剂(Kunitz/BPTI)结构域,两个结构域之间由单链连接,共包括3个α螺旋,2对反向平行的β折叠;在aa35~aa45、aa54~aa60、aa75~aa80、aa91~aa93、aa125~aa131等处可能存在潜在的B细胞抗原表位。相似性与进化树结果显示,青海血蜱Haemalin氨基酸序列与褐黄血蜱凝血酶抑制剂Haemalin氨基酸序列的相似最高达92%,并在进化树中二者处于同一进化分支。表明,青海血蜱Haemalin可能也为一种凝血酶抑制剂。qPCR结果显示,Haemalin基因在不同发育阶段青海血蜱中的转录水平为:卵>半饱血蜱>若蜱>饱血蜱>幼蜱>吸血96 h的雌成蜱>游离雌成蜱,且其在青海血蜱雌蜱中肠中的转录水平极显著高于其他组织(P<0.0001)。表明,Haemalin基因在青海血蜱成蜱�To analyse the bioinformatics characteristics of the Haemalin gene of Haemaphysalis qinghaiensis and the transcriptional levels of the gene in different developmental stages and different tissues of female ticks,the full-length c DNA of the Haemalin gene was constructed.A gene fragment with a functional annotation of Haemalin was retrieved from the transcriptome library of the salivary gland of Haemaphysalis qinghaiensis.The 3'RACE and 5'RACE specific primers of Haemalin gene were designed using c DNA end rapid amplification(RACE) primer design software.The 3' and 5' end sequences of Haemalin gene amplified by RACE,respectively,and the full-length c DNA sequence of the gene was obtained by sequencing and splicing with its intermediate sequence.Bioinformatics software was used to analyze the signal peptide,physicochemical properties,transmembrane domains,structural domains,protein tertiary structure,B cell antigen epitope and amino acid sequence alignment of the protein coded by this gene.NCBI of BLAST and MEGA 7.0software were respectively used to analyze the similarity of amino acid sequences encoded by the Haemalin gene and construct the phylogenetic tree of amino acid sequences encoded by the Haemalin gene,and fluorescence quantitative PCR(q PCR) was used to detect the transcription levels of the Haemalin gene at different developmental stages and different tissues of female ticks.The results showed that the c DNA of the Haemalin gene of Haemaphysalis qinghaiensis was 583bp in length with an open reading frame(ORF) of 423bp.The signal peptide of the protein encoded by the gene was 15 amino acids at the N-terminus.The theoretical isoelectric point of this protein was 4.71.It was a hydrophilic protein without a transmembrane domain.The tertiary structure of the protein showed the presence of two typical Kunitz/Bovine trypsin inhibitor(Kunitz/BPTI) domains,which were connected by a single chain and consisted of three alpha helices and two pairs of antiparallel beta folds.Potential B cell antigen epitopes may be l

关 键 词：青海血蜱 Haemalin基因 克隆 生物信息学 转录水平 

分 类 号：S855.9[农业科学—临床兽医学]