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目的 构建以甲胎蛋白增强子(AFP E)和白蛋白启动子(ALB P)联合转录调控序列(AFPenh+ALBprom, EP)为启动子的重组腺相关病毒基因治疗载体,用于肝细胞肝癌(HCC)的靶向基因治疗研究。方法 用PCR方法扩增EP基因片段,将EP基因片段顺义克隆至重组腺相关病毒载体系统(AAV Helper Free System)中表达质粒 pAAV IRES hrGFP的启动子位点,并替换其原有的巨细胞病毒(CMV)启动子,构建出以 EP为启动子的表达质粒 pAAV IRES hrGFP EP; 再将 pAAV IRES hrGFP EP与 AAV Helper Free System中的控制质粒pAAV RC和辅助质粒 pHelper用脂质体转染法共转染人胚肾细胞(293 细胞),生成以 EP 为启动子的、可用于HCC靶向基因治疗的重组腺相关病毒载体(rAAV EP)。结果 成功构建并包装出以 EP为启动子的重组腺相关病毒载体 rAAV2 EP,病毒滴度达1.2×105/ml。结论 构建的以腺相关病毒为载体、受 AFP E和 ALB P联合转录调控序列驱动的重组腺相关病毒载体 rAAV2 EP,可望能用于HCC靶向基因治疗研究,并为肝脏疾病的靶向基因治疗提供先进载体系统。
Objective To construct recombinant adeno-associated virus (AAV) gene therapy vector with AFP E and ALB P combined with transcriptional regulatory sequence (AFPenh + ALBprom, EP) as promoter for hepatocellular carcinoma HCC) targeted gene therapy research. Methods The EP gene fragment was amplified by PCR. The EP gene fragment was cloned into the AAV Helper Free System to express the promoter region of plasmid pAAV IRES hrGFP, and replaced with its original cytomegalovirus (CMV) promoter to construct the expression plasmid pAAV IRES hrGFP EP using EP as promoter. The pAAV IRES hrGFP EP and the control plasmid pAAV RC in the AAV Helper Free System and the helper plasmid pHelper were further transfected with the plasmid Human embryonic kidney cells (293 cells) were transfected to generate a recombinant adeno-associated virus vector (rAAV EP) with EP as promoter for HCC targeted gene therapy. Results The recombinant adeno-associated virus vector rAAV2 EP with EP as the promoter was successfully constructed and packaged. The virus titer reached 1.2 × 10 5 / ml. CONCLUSION: The recombinant adeno-associated virus vector rAAV2 EP driven by AFP E and ALB P combined with adeno-associated virus vector is expected to be able to be used in targeted gene therapy of HCC and is a target gene of liver disease Treatment provides advanced carrier system.