目的:构建尘螨变应原Der f1真核表达载体,转染真核细胞并进行蛋白表达。方法:根据Genebank中Der f1基因的核酸序列(AB034946),设计引物,采用PCR法,从保存的JM109工程菌中扩增Der f1编码基因,克隆到真核表达质粒pcDNA3.1/myc-his A上,以脂质体法转染CHO细胞,经G418筛选,进行稳定表达细胞株的筛选和鉴定。结果:将目的基因Der f1成功连接到pcDNA3.1/myc-hisA-Derf1并转染CHO细胞,获得稳定表达的CHO细胞株。结论:成功构建了尘螨变应原Der f1真核表达载体,并转染CHO细胞表达蛋白质。
Objective: To construct eukaryotic expressing vector of derf1 gene of dermatophagoides farinae,and transfect CHO cells for protein expression.Methods: According to the Genebank nucleic acid sequences of Derf1(No.AB034946),designed primers,amplified the Der f 1 gene from the preservative engineering bacteria JM109 by PCR,then cloned it into plasmid pcDNA3.1/myc-his A;and then transfected the plasmid into CHO cells by liposomes and screening the positive cell clone use G418 to express the protein of Derf1 gene.Results: The Derf1 gene was connected to plasmid pcDNA3.1/myc-hisA and screening into CHO eukaryotic expression cells successfully.Conclusions: The eukaryotic expression vector of Derf1 was constructed well and got its protein.