中文摘要：

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pAd—BM5-GFP-mAFP，并测定其滴度。方法从Hepal-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TCBn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3．2×10^8PFU／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。

英文摘要：

Objective To construct a novel recombinant adenovirus vector pAdBM5-GFP-mAFP and detect virus titer. Methods Total RNA were extracted from hepal-6 murine liver cancer cell line, mAFP（mu- fine alpha fetoprotein,mAFP） gene was cloned by RT-PCR method. The gene was identified by sequencing, then it was inserted into pAdBMS-GFP shuttle vector by Bgl Ⅱ and Pme Ⅰ restrict enzyme digestion to construct pAdBM.5-GFP-mAFP recombinant vector, mAFP gene was expressed in adenovirus by homologous recombinant in HEK293A cells after pAdBMS-GFP-mAFP vector and virus skeleton plasmid cotransfected HEK293A cells by standard calcium phosphate coprecipitation method. Virus titer was detected by TCID50 method. Results pAdBMS-GFP-mAFP recombinant vector was successfully constructed and high titer recombinant adenovirus carrying mAFP gene was harvested. Virus titer reached to 3. 2 10^8 PFU/ml. Conclusion pAdBM.5-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.