摘要
为表达锡兰钩虫金属蛋白酶组织抑制剂(Ace-TIMP)重组蛋白,本实验根据GenBank中锡兰钩虫TIMP (ANCCEY-04405)基因序列设计引物扩增犬源锡兰钩虫Ace-TIMP基因,利用生物信息学软件对其序列鉴定;根据编码的氨基酸序列进行遗传进化关系分析;构建其重组表达质粒p ET-32a-Ace-TIMP-MP,转化大肠杆菌,并经诱导表达。结果显示,Ace-TIMP基因ORF为648 bp,编码215个氨基酸,包含16个氨基酸的信号肽,具有TIMP家族Cys-X-Cys特征性序列。进化树分析显示Ace-TIMP与犬钩虫TMP-2位于同一分支。经诱导表达与纯化,获得了分子质量约为42 ku的可溶性融合蛋白。本实验克隆了Ace-TIMP基因并利用原核表达系统表达了可溶性的重组蛋白,为Ace-TIMP生物学活性的研究和免疫潜能的评价奠定了基础。
To prepare recombinant Ancylostoma ceylanicum tissue inhibitor of metalloproteinase(Ace-TIMP), primers were designed according to the sequence in GenBank(ANNECY-04405) to amplify the Ace-TIMP gene from dog-derived parasites.Bioinformatic softwares were used to identify the amplified sequence and phylogenetic analysis was conducted based on the deduced amino acid sequences. A plasmid for expressing Ace-TIMP(pET-32a-Ace-TIMP-MP) in E.coli was constructed to express recombinant protein. Results showed that the ORF of Ace-TIMP is 648 bp and encodes 215 amino acids. Ace-TIMP contains a signal peptide with 16 amino acids and the characteristic sequence of TIMP family(Cys-X-Cys). Ace-TIMP is in the same branch with A.caninum TMP-2 in the phylogenetic tree. Finally, soluble fusion protein with molecular weight of about 42 ku was expressed and purified from the induced E.coli. In this study, the coding sequence of Ace-TIMP was cloned and expressed through a prokaryotic expression system. The data laid a foundation for further study on biological activities of the protein and evaluation of its immunogenic potential.
作者
颜欣欣
杭建雄
刘云秋
黄岳
冉荣坤
赵琪
何龙
李秀
刘菊玫
李国清
YAN Xin-xin;HANG Jian-xiong;LIU Yun-qiu;HUANG Yue;RAN Rong-kun;ZHAO Qi;HE Long;LI xiu;LIU Ju-mei;LI Guo-qing(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2019年第9期969-972,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(31672541)
广东省科技计划项目(2014A020214005)
关键词
锡兰钩虫
金属蛋白酶组织抑制剂
基因克隆
原核表达
序列分析
Ancylostoma ceylanicum
tissue inhibitor of metalloproteinase(TIMP)
cloning
prokaryotic expression
phylogenetic analysis
