尘螨变应原Derf1真核表达载体的构建及转染CHO细胞

Construction of Eukaryotic Vector of Derf1 Gene of Dermatophagoides Farinae and Transfection of CHO Cells

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.