摘要
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)杂合启动子[HRE] AF调控的PNP基因表达载体并检测、分析二者的差异表达. 方法:将[HRE]AF启动子插入载体pcDNA3.0,构建肝癌细胞特异表达载体p[HRE]AF;将PNP基因分别插入pcDNA3.0 和p[HRE]AF,构建两种启动子调控的PNP基因表达载体pcDNA3.0/PNP和p[HRE]AF/PNP;经酶切、PCR及测序鉴定各重组体.用脂质体介导法将两PNP基因载体转染不同细胞株,RT-PCR检测PNP基因在各细胞株中表达,分析二者表达的特点. 结果:各目的片段均成功插入相应载体中,CMV启动子调控的PNP基因在各株细胞中均实现了表达,而[HRE]AF启动子调控的PNP基因则在AFP阳性和阴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,p[HRE]AF/PNP还实现了在AFP阳性,尤其是AFP阴性肝癌细胞中的靶向性表达.
AIM: To construct two expression vectors carrying PNP gene under a cytomegalovirus (CMV) promoter and a hybrid a-fetoprotein (AFP) tissue-specific promoter, and to detect and analyze their expression in different cell lines. METHODS: [HRE]AF promoter was inserted into pcDNA3.0 vector, and a recombinant vector controlled by the hybrid AFP promoter, p[HRE]AF, was constructed. Inserting PNP gene into pcDNA3.0 and p[HRE]AF vectors separately, two PNP gene expression vectors driven by two different promoters, pcDNA3.0/PNP and p[HRE]AF /PNP, were constructed by using recombinant DNA techniques. The re-combinants were analyzed and identified by restriction enzymes, PCR and sequencing. pcDNA3.0/PNP and p[HRE] AF/PNP were transfected into human hepatocellular carcinoma cell lines by liposome-mediated method. The expression of PNP gene in four different cell lines was detected by RT-PCR method. RESULTS: All target fragments were separately cloned into corresponding vectors. We detected the expression of PNP gene under the control of CMV promoter in all cell lines, and the tissue-specific expression of PNP gene under the control of [HRE]AF promoter in AFP positive and negative hepatocellular carcinoma cell line was positive. CONCLUSION: Two expression vectors harboring PNP gene are novel effective vectors for human hepatocellular car- cinoma gene therapy, and p[HRE]AF/PNP is a target-expressing vector in AFP positive, especially in AFP negative hepatocellular carcinoma cells.
出处
《世界华人消化杂志》
CAS
2004年第9期2036-2040,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金重点项目
No.30330680~~
