SP-D基因真核表达质粒的构建及鉴定

Construction and identification of eukaryotic expression plasmid containing SP-D gene

目的构建并鉴定含肺表面活性蛋白D(SP-D)基因的重组真核表达质粒。方法提取A549细胞的总RNA为模板,用RT-PCR方法扩增含SP-D基因的cDNA,将产物克隆进真核载体pGEFP-N1内,构建含SP-D基因的重组真核表达质粒。将pGEFP-N1-SP-D重组质粒和pGEFP-N1空载体分别转染293T细胞,Western blot法检测SP-D的蛋白表达。结果双酶切鉴定及核酸序列分析证实,SP-D已成功插入真核载体pGEFP-N1内。转染pGEFP-N1-SP-D的293T细胞中检测到高表达的SP-D蛋白。结论成功构建含SP-D基因的重组真核表达质粒,并能在293T细胞中高表达。

Objective To construct and identify the recombinant eukaryotic plasmid containing pulmonary surfactant protein D（SP-D）gene.Methods The cDNA of SP-D gene was amplified by RT-PCR with total RNA in A549 cells as a template.The PCR fragments were cloned into pGEFP-N1 vector to construct the recombinant eukaryotic expression plasmid containing SP-D gene.The recombinant plasmid pGEFP-N1-SP-D and empty vector pGEFP-N1 were transfected into 293 Tcells,and the protein expression of SP-D was detected by Western blot.Results Double enzyme digestion and DNA sequence analysis showed that SP-D was successfully inserted into pGEFP-N1 vector.SP-D was highly expressed in 293 T cells transfected with pEGFP-N1-SP-D plasmid.Conclusion The recombinant eukaryotic plasmid containing SP-D gene has been successfully constructed and is highly expressed in 293T cells.