锡兰钩虫HPI基因的原核表达与生物信息学分析  

Prokaryotic expression and bioinformatics analysis of HPI gene of Ancylostoma ceylanicum

作　　者：黄岳 刘云秋 颜欣欣 杭建雄 冉荣坤 孙永祥 李国清[1] HUANG Yue;LIU Yun-qiu;YAN Xin-xin;HANG Jian-xiong;RAN Rong-kun;SUN Yong-xiang;LI Guo-qing(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学兽医学院

出　　处：《中国人兽共患病学报》2019年第8期706-710,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(No.31672541);广东省科技计划项目(No.2014A020214005)联合资助~~

摘　　要：目的克隆表达锡兰钩虫血小板抑制剂(hookworm platelet inhibitor, HPI)基因,并研究其编码蛋白的生物学特性。方法 以锡兰钩虫cDNA 为模板,参考已公布的犬钩虫血小板抑制剂(AcaHPI)基因序列设计特异性引物,运用RT-PCR 扩增其AceHPI基因序列,并将其连接至pET-28a表达载体,诱导其表达并纯化。利用在线软件对其编码蛋白的氨基酸序列进行同源性比对和生物信息学分析。结果 锡兰钩虫AceHPI基因的ORF序列全长603 bp,编码200个氨基酸,与犬钩虫AcaHPI氨基酸序列同源性为91%。构建的重组表达质粒pET28a-AceHPI经诱导表达与纯化,获得了分子质量约为26 kDa的可溶性融合蛋白。预测分析表明该蛋白为疏水蛋白,前17个氨基酸为信号肽序列且无跨膜结构域。结论 成功克隆表达了AceHPI基因并对其编码氨基酸进行了生物信息学分析,为进一步研究AceHPI在感染宿主体内的作用机制奠定了基础。In order to clone and express the gene of hookworm platelet inhibitor from Ancylostoma ceylanicum (AceHPI) and analyze its biological characteristics, the specific primers were designed referring to the published sequence of AcaHPI from Ancylostoma caninum . The gene was amplified by RT-RCR, and the PCR product was cloned into pET-28a vector for expression by induction of IPTG. Meanwhile, the homology and bioinformatics analysis of amino acid sequences encoded by the gene was conducted by online software. The results showed that the open reading frame of AceHPI gene was 603 bp in length, encoding a protein of 200 amino acids, and its sequence homology with AcaHPI was 91%. The recombinant expression plasmid pET28a-AceHPI was constructed and expressed, and a soluble fusion protein with a molecular mass of about 26 kDa was obtained. Predictive analysis indicated that the protein was hydrophobin and in the first 17 amino acids was signal peptide sequences, without transmembrane domain. In this study, AceHPI gene was successfully cloned and expressed, and its encoded amino acids were analyzed by bioinformatics, laying a foundation for the further study on the mechanism of AceHPI’s role in the infected host.

关 键 词：锡兰钩虫 钩虫血小板抑制剂 原核表达 序列分析 

分 类 号：R383.1[医药卫生—医学寄生虫学]