人乙酰肝素酶基因真核表达载体的构建与表达

Construction and expression of the eukaryotic expressing vector of human heparanase gene

目的构建人乙酰肝素酶基因(HPA)的真核表达载体pEGFP-N1/HPA,初步探讨其在人胚肾细胞293T中的表达情况。方法以pOTB7/HPA质粒为模板,PCR扩增HPA基因,经限制性内切酶酶切后,连接到pEGFP-N1载体上,并对重组表达载体pEGFP-N1/HPA进行酶切与测序鉴定。用脂质体lipefectmine2000为介导转染至293T细胞中,观察该基因在293T细胞中的表达情况。结果经限制性内切酶酶切鉴定及测序分析证实pEGFP-N1/HPA基因在771 bp处碱基c突变成t;在919 bp处碱基a突变成g,两处突变均为同义突变。该基因转染293T细胞48 h后,在荧光显微镜下可见转染的293T细胞有绿色荧光蛋白表达。结论成功构建人乙酰肝素酶基因的真核表达载体pEGFP-N1/HPA,并在人293T细胞中成功表达。

Objective To construct eukaryotic expressing vector of human heparanase（HPA） gene,pEGFP-N1/HPA and test its expression in 293T cells.Methods pOTP7/HPA plasmid used as a template,HPA cDNA was amplified by PCR and digested by restriction enzyme.Then the gene segment was inserted into pEGFP-N1 vector and identified by restriction enzyme digestion as well as by DNA sequencing.Subsequently,the recombinant pEGFP-N1/HPA plasmid was transfected into 293T cells with lipofectamine2000.Results The sequence of pEGFP-N1/HPA vector was confirmed by restriction enzyme digestion and DNA sequencing.There were two genetic mutations at 771 bp and 919 bp.But both of mutations belonged to samesense mutations.After transfection at 48 h,the expression of green fluorescent protein was present.Conclusion The recombinant vector pEGFP-N1/HPA has been successfully constructed and expressed in 293T cells.