犬钩虫(Ancylostoma caninum)daf-1基因的克隆和表达鉴定

Abstract

[中文文摘] 目的对犬钩虫(Ancylostoma caninum)TGF-βⅠ型受体daf-1基因进行了克隆和鉴定,并在原核系统中进行表达。方法以犬钩虫总RNA为模板,用RT-PCR对犬钩虫TGF-β受体基因daf-1的cDNA进行扩增和克隆,用PCR筛选阳性克隆,双酶切鉴定并测序。将测序正确的阳性克隆双酶切后构建到表达载体pET-32m中,转化到E.coli DH5α内,提取质粒,酶切鉴定阳性,再转化入表达宿主E.coli BL21(DE3)菌株内,对转化菌株用IPTG进行诱导培养。收集培养液,破菌、离心进行SDS-PAGE检测重组蛋白的表达。蛋白纯化后进行免疫,并通过免疫印迹检测在钩虫各时期的表达。结果电泳发现转化了重组质粒的菌株在IPTG诱导下表达的重组蛋白为58kDa,在钩虫各时期都有AcDAF-1的表达。结论成功进行了犬钩虫TGF-βⅠ型受体daf-1基因的扩增、克隆和表达并在各期检测到该蛋白的表达。[英文文摘]The aim is to clone,express and identify the TGF-βtypeⅠreceptor daf-1fromAncylostoma caninumfor the further study.The gene encoding a TGF-βtypeⅠreceptor daf-1of Ancylostoma caninum was amplified from the total RNA by using RT-PCR.The amplified product was cloned into pMD-18Tvector.After PCR selection,enzyme digestion,and sequencing,the right plasmid was digested by enzyme and ligated with expression vector pET-32aand transformed into E.coli DH5α strain.The plasmid containing the right insert was sequenced to confirm its identity,and then retransformed into E.coli BL21 (DE3) strain.Bacterial lysates from cultures induced with IPTG were analyzed by SDS-PAGE.The expression of AcDAF-1in larval stage,adult male and females were detected by western blot.Results showed that a specific protein band with a molecular mass of 58kDa could be visualized in gel after IPTG induction．The recombinant protein was used to raise the antibody and AcDAF-1was expressed in larvae and adult worm stages by western blot．A TGF-βtypeⅠreceptor was successful cloned from A.caninumand expressing in E.coli,and AcDAF-1was expressed in larvae and adult worm stages of A.caninum.