不同启动子调控的PNP基因载体的构建和表达差异性分析

Construction of Two Vectors Containing PNP Gene under Control of Two Different Promoters and Analysis of the Expression Difference between Them

目的 分别构建巨细胞病毒 (cytomegalovirus, CMV) 通用启动子和甲胎蛋白 (α fetoprotein, AFP) 组织特异性启动子调控的PNP基因表达载体并分析二者表达的差异性。方法 将 AF0. 3 启动子插入载体 pcDNA3. 0,构建肝癌细胞特异表达载体 pAF0 .3; 将 PNP基因分别插入到 pcDNA3 .0 和 pAF0. 3 中, 构建不同启动子调控下的PNP基因表达载体 pcDNA3 .0/PNP和 pAF0. 3/PNP; 通过酶切、PCR及测序鉴定各重组体。采用脂质体介导法将两种PNP基因表达载体转染不同的细胞株, RT-PCR检测PNP基因在各细胞株中的表达, 分析二者表达的不同。结果各目的片段均成功插入相应的载体中, CMV启动子调控下的 PNP基因在各株细胞中均实现了表达, 而 AF0 3 启动子调控下的PNP基因则在AFP阳性肝癌细胞中实现了组织特异性表达。结论 两 PNP基因表达载体是肝癌基因治疗中新型、高效的治疗载体, 且 pAF0. 3/PNP还实现了在AFP阳性肝癌细胞中的靶向性表达。

Objective To construct two expression vectors harboring PNP gene under the control of cytomegalovirus (CMV) promoter and the α-fetoprotein (AFP) tissue-specific promoter and detect their expression in different cell lines. Methods AF0.3 promoter was inserted into pcDNA3.0 vector,and an recombinant vector controlled by AFP promoter,pAF0.3,was constructed. After PNP gene was inserted into pcDNA3.0 and pAF0.3 vectors separately,two expression vectors containing PNP gene driven by two different promoters,pcDNA3.0/PNP and pAF0.3/PNP,were constructed by using recombinant DNA techniques.The recombinants were analyzed and identified by restriction enzyme,PCR and sequencing. pcDNA3.0/PNP and pAF0.3/PNP were transfected into human hepatocellular carcinoma cell lines by liposome-mediated method. The expression of PNP gene in 4 different cell lines was detected by RT-PCR method. Results All target fragments were separately cloned into corresponding vectors. The expression of PNP gene under the control of CMV promoter in 4 cell lines was detectable,and the tissue-specific expression of PNP gene under the control of AF0.3 promoter in AFP (+) hepatocellular carcinoma cell line HepG2 was positive. Conclusion Two expression vectors containing PNP gene are novel and effective vectors for human hepatocellular carcinoma gene therapy,and pAF0.3/PNP is a target-expressing vector in AFP positive hepatocellular carcinoma cells.