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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 eukaryotic expression plasmid pVAX1-Der p 1 to detect the expression of plasmid-encoded recombinant protein in eukaryotic 293T cells, which laid the foundation for further animal experiments. Methods: The total RNA of house dust mite was isolated. The primers of Der p 1 were designed according to the published Der p 1 nucleic acid sequence in GenBank. The gene encoding Der p 1 was amplified by PCR. The full-length gene was cloned into eukaryotic expression vector pVAX1. Escherichia coli DH5α was transformed into E.coli DH5α, and the positive clones containing the recombinant plasmid pVAX1-Der p 1 were screened. After cultivation, the plasmids were extracted and identified by double restriction enzyme digestion and sequence analysis. Lipo-fectamine 2000 lipofectamine transfection reagent, Eukaryotic cells 293T were transfected with pVAX1-Der p 1 and empty plasmid pVAX1, respectively, and the expression of the recombinant protein encoded by the plasmid was detected in 293T cells by anti-Der p 1-specific antibody by immunofluorescence staining. Results: The Der p 1 gene was identical to the full-length Der p 1 cDNA in Genbank. The constructed expression vector achieved the expression of the target protein. CONCLUSION: The Der p 1 sequence of house dust mite in China is exactly the same as the published nucleotide sequence in GenBank. The eukaryotic expression plasmid pVAX1-Der p 1 containing the full-length sequence of Der p 1 was successfully constructed and successfully expressed in eukaryotic cells protein.