十二指肠钩虫金属蛋白酶组织抑制剂同源物Ad-TIMPL-1基因克隆及其原核表达  

Cloning and prokaryotic expression of Ad-TIMPL-1,a tissue inhibitor of metalloproteinase homologue from Ancylostoma duodenale

作　　者：邓莉[1] 邵正[1] 许琴英[1] 胡晶晶[1] 彭礼飞[1] 

机构地区：[1]广东医学院寄生虫学教研室,广东湛江524023

出　　处：《热带医学杂志》2011年第8期866-869,874,共5页Journal of Tropical Medicine

基　　金：广东省自然科学基金(04011381)

摘　　要：目的克隆十二指肠钩虫金属蛋白酶组织抑制剂同源物(TIMPL)Ad-TIMPL-1基因并进行原核表达。方法分别用3’RACE及RT-PCR技术从十二指肠钩虫成虫cDNA中扩增编码Ad-TIMPL-1的cDNA3’末端及5’末端序列;序列经拼接后进行初步生物信息学分析;将Ad-TIMPL-1成熟肽编码序列克隆至原核表达载体pET-32a,构建重组表达质粒;重组质粒转化至大肠杆菌BL21(DE3)后,用IPTG诱导表达,SDS-PAGE分析表达情况。结果成功克隆获得了编码Ad-TIMPL-1的全长cDNA序列并登记到GenBank(accession no.EF495071);Ad-TIMPL-1完整阅读框为396bp,编码132个氨基酸残基组成的蛋白,含16个氨基酸残基组成的信号肽;成功构建了原核表达重组质粒pET-32a/Ad-TIMPL-1,并在大肠杆菌中得到了表达。结论本研究首先从十二指肠钩虫中克隆到了Ad-TIMPL-1基因并进行了原核表达,为进一步研究其生物学功能奠定了基础。Objective To clone and express the tissue inhibitor of metalloproteinase homologue（TIMP like,TIMPL）,Ad-TIMPL-1,from the hookworm Ancylostoma duodenale.Methods The 5＇ and 3＇ end of cDNA encoding Ad-TIMPL-1 were amplified by 3＇RACE and RT-PCR from the cDNA of adult A.duodenale,respectively.The nucleotide sequence encoding mature Ad-TIMPL-1 was ligated into pET32a to construct the recombinant plasmid pET32a/Ad-TIMPL-1.The recombinant plasmid was transformed into E.coli BL21（DE3）,and then expressed by inducing with IPTG and analysed by SDS-PAGE.Results The full-length cDNA of Ad-TIMPL-1 was obtained from A.duodenale and deposited in GenBank（accession no.EF495071）.The full-length cDNA contains an open reading frame of 396 bp,which encoding the Ad-TIMPL-1 precursor of 132 amino acids with a signal peptide of 16 amino-acid-residue.The mature Ad-TIMPL-1 was expressed in E.coli BL21（DE3） after inducing by IPTG.Conclusion This study is the first report on the isolation,expression of a TIMP homologue from the human hookworm A.duodenale.Our results laid a foundation for futher research the biological function of Ad-TIMPL-1.

关 键 词：十二指肠钩虫 金属蛋白酶组织抑制剂 克隆 原核表达 

分 类 号：R383.13[医药卫生—医学寄生虫学]