CYP2B1新型自杀基因的真核表达载体的构建和瞬时表达

目的构建一种新型的CYP2B1自杀基因表达载体并检测其在肿瘤细胞中的表达。方法根据gene bank中鼠CYP2B1基因的核苷酸序列设计一对引物，以pc3／2B1为模板进行PCR扩增，将PCR CYP2B1基因产物定向插入到pcDNA3．0中．构建CYP2B1基因表达载体pcDNA3．0／CYP2B1；通过酶切、PCR及测序鉴定重组体。采用脂质体介导法将pcDNA3．0／CYP2B1转染三种肿瘤细胞株，RT—PCR检测CYP2B1基因在各细胞株中的表达。结果目的片段均成功插入相应的载体中，CMV启动子调控下的CYP2B1基因在不同的肿瘤细胞株中实现了表达。结论CMV启动子调控的CYP2B1真核基因表达栽体是肿瘤基因治疗中一种新型、高效的治疗载体、

The Construction of Eukaryotic Expression Vector Containing CYP2B1 Suicide Gene

Objective To construct an expression vector harboring CYP2B1 suicide gene, and detect its expressions in tumor cell lines. Methods PCR amplification was performed using primers based on murine CYP2B1 gene sequence from gene bank and pc3/2B1 as template. PCR product was directly inserted an eukaryotic expression plasmid pcDNA3.0. The recombinants were analyzed and identified by restriction enzyme analysis, PCR and sequencing. Then the recombinant vector pcDNA3.0/CYP2B1 was transfected into three tumor cell lines by liposome-mediated method. The expressions of CYP2B1 gene in all the cell lines were detected by RT-PCR method. Results pCDNA3.0/CYP2B1 vector was successfully constructed, and could express CYP2B1 mRNA in the three tumor cell lines. Conclusion Eukaryotic expression vector pcDNA3.0/CYP2B1 containing CYP2B1 gene under the control of a CMV promoter is an novel effective expression vector for tumor gene therapy.