犬钩虫C型凝集素AcaCTL-1基因的克隆及表达  

Cloning and expression of a C-type lectin(AcaCTL-1) gene from Ancylostoma caninum

作　　者：邓莉[1] 金娴[1] 何庆丰[1] 许琴英[1] 吴亚敏[1] 彭礼飞[1] 

机构地区：[1]广东医学院寄生虫学暨l临床寄生虫检验学教研室,广东湛江524023

出　　处：《中国病原生物学杂志》2010年第12期918-921,共4页Journal of Pathogen Biology

基　　金：广东省自然科学基金项目(No.04011381)

摘　　要：目的分离鉴定犬钩虫C型凝集素AcaCTL-1基因,并在大肠埃希菌中表达。方法根据EST序列设计引物,用RT-PCR及3′(RACE技术扩增获得AcaCTL-1全长cDNA序列,并对其进行初步生物信息学分析;将AcaCTL-1成熟肽编码序列克隆、连接到表达载体pET32a,构建重组质粒pET32a/AcaCTL-1;在大肠埃希菌BL21(DE3)中诱导表达重组蛋白,SDS-PAGE分析表达情况,Ni亲和层析纯化可溶性蛋白。结果成功克隆了AcaCTL-1全长cDNA序列,其最大开放阅读框由525 bp组成,预测编码174个氨基酸残基组成的蛋白,含17个氨基酸组成的信号肽,为C型凝集素家族蛋白成员;构建pET32a/AcaCTL-1重组质粒,并在大肠埃希菌中高效表达。表达产物多以包涵体形式存在,小部分为可溶性蛋白。结论成功克隆并表达了犬钩虫C型凝集素AcaCTL-1基因,为进一步了解AcaCTL-1的功能奠定了基础。Objective To isolate,identify,and overexpress AcaCTL-1,a novel C-type lectin gene,from Ancylostoma caninum.Methods The full length cDNA of AcaCTL-1 from A.caninum was amplified by 3′RACE and RT-PCR.The nucleotide sequence encoding mature AcaCTL-1 was inserted into pET32a to construct a recombinant pET32a/AcaCTL-1 plasmid.pET32a/AcaCTL-1 was expressed in E.coli BL21（DE3） by induction with IPTG and was analyzed using SDS-PAGE.The recombinant protein was affinity-purified with Protino Ni-IDA Resin.Results The AcaCTL-1 full-length cDNA in GenBank（accession no.HM535634） contained an open reading frame of 525 bp,which encodes a protein consisting of 174 amino acids with a signal peptide of 17 amino acid residues.The deduced AcaCTL-1 sequence contained a C-type lectin domain and belonged to the C-type lectin superfamily.The mature AcaCTL-1 was successfully inserted into a pET32a plasmid and expressed in E.coli BL21（DE3） after induction with IPTG.Conclusion AcaCTL-1 gene,a novel C-type lectin,was cloned from A.caninum and successfully overexpressed in E.coli.

关 键 词：犬钩虫 C型凝集素 克隆 表达 

分 类 号：R383.13[医药卫生—医学寄生虫学]