真核表达质粒pVAX1-Der p 1的构建及在真核细胞中的表达

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DH...

Construction of Eukaryotic Expression Plasmid pVAX1-Der p 1

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.