携带人二氢叶酸还原酶基因慢病毒表达载体构建及鉴定

Construction of a Lentiviral Expression Vector Harboring Human Dihydrofolate Reductase Gene

目的:构建携带人二氢叶酸还原酶(DHFR)基因的慢病毒表达载体pWPI。方法:采用PCR方法扩增二氢叶酸还原酶cDNA全长,与EZ-T克隆载体连接,HindIII及BamHI-HF限制性内切酶双酶切回收的PCR片段并补平其缺口。慢病毒系统载体使用pWPI系统,采用PmeI酶切载体后回收片段,将其磷酸化,T4酶连接载体与目的基因。表达载体鉴定均采用核苷酸序列测定,重组质粒采用脂质体转染293T包装细胞后获得包装的病毒颗粒。结果:成功扩增二氢叶酸还原酶全长并连接入pWPI载体构建成重组表达载体DHFR-pWPI,重组质粒测序结果显与DHFR基因的同源性达100%,按标准生产程序转染293T后有DHFR基因的表达。结论:成功采用慢病毒载体系统构建了二氢叶酸还原酶重组慢病毒转基因,为探讨DHFR在肿瘤多药耐药过程中的分子机理奠定基础。

Objective： To construct a lentiviral expressing vector harboring human Dihydrofolate reductase（DHFR）gene.Methods： The cDNA length of DHFR gene was amplified by PCR and was connected to cloned vector EZ-T,then the recovered PCR fragment was obtained by digesting with restriction enzymes named HindIII and BamHI-HF,and then blunted at the gap.Lentiviral vector system adopted pWPI system.The vector pWPI was digested with PmeI enzyme,and then it was recovered fragment and phosphorylated.The phosphorylated vector was connected with DHFR gene by T4 enzyme.Recombinant plasmid was confirmed by PCR and sequencing nucleotide.The recombinant retroviral vector pWPI was selected to transfect packaging cell 293T to gain virus particles with infection ability.Results： The recombinant plasmid,named DHFR-pWPI,which was constructed and identified Packaged by packaging cell 293T.The homology between sequencing results and DHFR gene sequence was up to 100%,and the expression of DHFR in 293T cells was determined by RT-PCR.Conclusion： The lentiviral expressing vector harboring human DHFR has been constructed successfully,which made the foundation to explore the molecular mechanism of multidrug-resistance in tumor.