医学业论文--一种新的重组腺病毒载体pAdBM5GFPmAFP的构建.doc

一种新的重组腺病毒载体pAdBM5GFPmAFP的构建 作者：匡志鹏，谢裕安，梁安民，罗小玲，吴继宁 【摘要】 目的 构建表达小鼠甲胎蛋白（murine alpha fetoprotein，mAFP）基因的重组腺病毒载体pAdBM5GFPmAFP，并测定其滴度。方法 从Hepa16小鼠肝癌细胞中提取总RNA，设计特异性引物，通过RTPCR法扩增mAFP基因，测序确认后,插入到pAdBM5GFP穿梭载体中，双酶切鉴定重组腺病毒载体。线性化重组腺病毒载体与病毒骨架DNA质粒用磷酸钙共沉淀法共转染HEK293A细胞，通过同源重组使腺病毒表达mAFP基因，并在HEK293A细胞中大量扩增含目的基因的重组腺病毒，用TCID50法测定重组腺病毒滴度。结果 成功克隆了小鼠mAFP基因，构建出表达mAFP基因的重组腺病毒载体pAdBM5GFPmAFP，获得了表达mAFP基因的重组腺病毒，病毒滴度达3.2×108PFU／ml。结论 本研究成功构建了pAdBM5GFPmAFP载体，获得了高滴度表达mAFP基因的重组腺病毒，为进一步开展肝癌的免疫基因治疗奠定了基础。 【关键词】 pAdBM5GFP；小鼠甲胎蛋白；重组腺病毒载体 　　Abstract：Objective To construct a novel recombinant adenovirus vector pAdBM5GFPmAFP and detect virus titer. Methods Total RNA were extracted from hepa16 murine liver cancer cell line, mAFP(murine alpha fetoprotein，mAFP) gene was cloned by RTPCR method. The gene was identified by sequencing, then it was inserted into pAdBM5GFP shuttle vector by Bgl II and Pme I restrict enzyme digestion to construct pAdBM5GFPmAFP recombinant vector. mAFP gene was expressed in adenovirus by homologous recombinant in HEK293A cells after pAdBM5GFPmAFP vector and virus skeleton plasmid cotransfected HEK293A cells by standard calcium phosphate coprecipitation method. Virus titer was detected by TCID50 method. Results pAdBM5GFPmAFP recombinant vector was successfully constructed and high titer recombinant adenovirus carrying mAFP gene was harvested. Virus titer reached to 3.2 108 PFU/ml. Conclusion pAdBM5GFPmAFP recombinant adenoviral vector carrying mAFP gene was successfully constructed and high titer recombinant adenovirus was harvested. it will lay down the basis for further developing gene therapy of primary liver cancer. 　　Key words：pAdBM5；mAFP；Recombinant adenovirus vector 　　0 引言 　　大多数肝癌细胞过表达AFP抗原。Meng等［1,2］认为人甲胎蛋白是最重要的一种肝癌治疗性靶抗原。Vollmer 等［3,4］也发现尽管小鼠的免疫系统长期暴露于AFP环境中，当被合适地或特异性地激发时，仍能对这种胚胎性抗原产生较强的T细胞免疫反应。由此说明，AFP能够作为一种潜在的诱导特异性CTL反应的肿瘤抗原用于AFP分泌性肝癌的靶向性免疫治疗。因此，本研究将肝癌相对较为特异的甲胎蛋白（AFP）基因构建到含有强力启动子的pAdBM5腺病毒载体中，从而扩增出大量高效表达目的基因的重组腺病毒，为开展肝癌的靶向性免疫基因治疗完成关键的一步。