目的对犬钩虫(Ancylostomacaninum)天冬氨酸蛋白酶Asp基因进行了克隆,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法对犬钩虫天冬氨酸蛋白酶Asp基因进行扩增,获得的AspcDNA产物克隆进pMD-18T载体,用PCR筛选阳性克隆。碱裂解法进行质粒提取,单、双酶切进行鉴定并测序。将测序正确的阳性克隆进行双酶切,构建到表达载体pET-32a中,将此重组质粒先转化到E.coliDH5a内,提取质粒,酶切鉴定阳性,再转化入表达宿主E.coliBL21(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,上清进行SDS-PAGE,通过电转移将胶中蛋白转到硝酸纤维素膜上后进行免疫印迹分析。结果电泳发现转化了重组质粒的菌株有表达蛋白,所表达蛋白相对分子量为40kDa,抗体检测有特异条带大小为40kDa。结论成功进行了犬钩虫天冬氨酸蛋白酶Asp基因的克隆表达。Cloning,expressing and identify the aspartic protease gene from Ancylostoma caninum for the further studying in the application in the diagnosis of Ancylostoma as well as for the development of anti-hookworm vaccine.The gene encoding aspartic protease was amplified from the total RNA by using RT-PCR technique.The amplified product was cloned initially into pMD-18T vector.After PCR selection,enzyme digestion,and sequencing.The positive and right sequence of plasmid was digested by enzyme and ligated with digested expression vector pET-32a by ligase,and then transformed the construct into the competent E.coli DH5а strain.Colonies containing the insert plasmid were selected on LB plus ampicillin(100μg/ml) plate and also by PCR screening,the positive plasmid DNA was extracted and digested with enzymes.Plasmid containing the right insert were sequenced to confirm its identity,and then retransformed the recombinant plasmids into E.coli BL21(DE3)strain.Bacterial lysates from cultures induced with IPTG(1mmol/L) were directly loaded onto SDS-PAGE,and the proteins on the SDS-PAGE gel.Were transferred to nitrocellulose membrane,detected with antiserum against recombinant expressing aspartic protease.Through these procedures,a specific protein with a molecular mass of 40kDa could be visualized on gel.This recombinant expressing protein can be used for further studies in the detection of the effectiveness of immunity and the preparation of antigen and antibodies of aspartic protease in large scale.国家自然科学基金(No30470881);; 福建省自然科学基金重点项目(NoC0320001)联合资
