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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 Der f1 eukaryotic expression vector of dust mite allergen, transfect eukaryotic cells and express protein. Methods: According to the nucleic acid sequence of Der f1 gene in Genebank (AB034946), primers were designed and the Der f1 coding gene was amplified by PCR from the deposited JM109 engineering bacteria and cloned into the eukaryotic expression plasmid pcDNA3.1 / myc-his A CHO cells were transfected by liposome method and screened by G418 to screen and identify stable expression cell lines. Results: The target gene Der f1 was successfully cloned into pcDNA3.1 / myc-hisA-Derf1 and transfected into CHO cells to obtain a stable CHO cell line. Conclusion: The Der f1 eukaryotic expression vector of dust mite allergen was successfully constructed and transfected into CHO cells to express the protein.