真核表达质粒pVAX1-Der p1的构建及在真核细胞中的表达  

Construction of Eukaryotic Expression Plasmid pVAX1-Der p 1

作　　者：曹炜[1] 许昱[1] 曹瑞娟[1] 

机构地区：[1]武汉大学人民医院耳鼻咽喉-头颈外科,湖北武汉430060

出　　处：《武汉大学学报（医学版）》2011年第6期713-716,722,F0002,共6页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:30973280/H1301)

摘　　要：目的:构建真核表达质粒pVAX1-Der p 1,检测质粒编码的重组蛋白在真核细胞293T中的表达,为进一步的动物实验打下基础。方法:分离屋尘螨总RNA,根据GenBank已公布的Der p 1核酸序列设计引物,PCR扩增Der p 1编码基因,以其全长为目的基因,定向克隆至真核表达载体pVAX1,将其转化大肠杆菌DH5α,筛选含有重组质粒pVAX1-Der p 1的阳性克隆,培养后提取质粒并对其进行双酶切鉴定和序列测定及分析;应用Lipo-fectamine 2000脂质体转染剂,重组基因pVAX1-Der p 1和空质粒pVAX1分别转染真核细胞293T,采用细胞免疫荧光方法,以抗Der p 1特异性抗体检测质粒编码的重组蛋白在293T细胞中的表达情况。结果:获取的Der p 1基因与Genbank上Der p 1cDNA全长序列完全一致,且构建的表达载体实现了目的蛋白的表达。结论:我国本土屋尘螨Der p 1序列与GenBank已公布的核酸序列完全一致;成功构建含Der p 1全长序列的真核表达质粒pVAX1-Der p 1,其能在真核细胞成功表达重组蛋白。Objective： To construct the eukaryotic expression plasmid pVAX1-Der p 1 and lay the foundation for further animal experiments.Methods： The total RNA of house dust mite were seperated,and based on the GenBank published sequences of Der p 1 DNA primers,the full length Der p 1 gene was amplified by PCR,and then was cloned into the eukaryotic expression vector pVAX1 and transformed into E.coli DH5α.Recombinant plasmids containing pVAX1-Der p 1 positive cloning were screened,extracted,cultured,and identified by double restriction enzyme digestion and sequencing analysis.With Lipofectamine 2000 agent,recombinant pVAX1-Der p 1 and empty vectors pVAX1 were transfected into eukaryotic cells 293T,and then the recombinant protein was identified by immunofluorescence method and with anti-Der p 1 specific antibodies.Results： This Der p 1 gene acquired has the same sequence to the Der p 1 cDNA sequence published on the Genbank,and the expression vector was successfully constructed.Conclusion： China＇s domestic house dust mite Der p 1 sequence is the same as GenBank nucleotide sequence.The eukaryotic expression plasmid pVAX1-Der p 1 containing full-length sequence of Der p 1 gene be successfully constructed,and the recombinant protein can be successfully expressed in eukaryotic cells.

关 键 词：屋尘螨 Derp1 基因克隆 蛋白表达 

分 类 号：R762[医药卫生—耳鼻咽喉科]