人乙酰肝素酶基因真核表达载体的构建与表达

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基冈,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lipefectmi...

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

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.