重组腺病毒载体pAdBM5-GFP-hIL-2的构建及病毒滴度测定  

Construction of recombinant adenovirus vector pAdBM5-GFP-hIL-2 and the detection of viral titer

作　　者：匡志鹏[1] 李安娜[1] 罗小玲[1] 谢裕安[1] 梁安民[1] 尚俊英[1] 吴继宁[1] 

机构地区：[1]广西肿瘤防治研究所,广西南宁530021

出　　处：《现代肿瘤医学》2009年第5期814-817,共4页Journal of Modern Oncology

基　　金：广西自然科学基金项目(编号:桂科自0640158)

摘　　要：目的:构建重组腺病毒载体pAdBM5-GFP-hIL-2,并测定病毒滴度。方法:从Jurkat细胞中提取mRNA,设计特异性引物,通过RT-PCR法扩增hIL-2基因全编码区序列。将测序正确的片段用BglII和PmeI双酶切定向插入到pAdBM5-GFP腺病毒载体中。通过磷酸钙共沉淀法将线性化重组质粒和腺病毒骨架共转染HEK293A细胞,扩增纯化重组腺病毒,TCID50法测定重组腺病毒滴度。结果:成功构建出表达hIL-2基因的重组腺病毒载体(pAdBM5-GFP-hIL-2),获得了高滴度表达hIL-2基因的重组腺病毒。结论:重组腺病毒表达载体(pAdBM5-GFP-hIL-2)的成功构建及重组腺病毒的获得有利于进一步开展肝癌的转基因治疗研究。Objective ：To construct recombinant adenovirus vector phdBM5 - GFP - hIL - 2 and detect the viral titer. Methods： Total mRNA was extracted from Jurkat tumor cells, and specific primer pairs were designed in order to clone human interleukin - 2 （ hIL - 2） gene cDNA by RT - PCR method, hIL - 2 gene was directional constructed in- to adenovirus vector（ pAdBM5 -GFP） after digestion by restrictive endoenzyme Bgl II,and Pine I. The constructed recombinant adenovirus vector was named pAdBM5 - GFP - hIL - 2. The lined recombinant adenorirus vector and vi- ral skeleton were cotransfected into HEK293A cells by coprecipitate of calcium phosphate. Recombinant adenovirus was amplificated and purificated in HEK293A cells. Results：Recombinant adenovirus vector pAdBM5 -GFP- hIL - 2 was constructed successfully and high titer recombinant adenovirus was obtained. Conclusion：The construction of recombinant adenovirus vector pAdBM5 - GFP - hIL - 2 and the obtain of recombinant adenovirus will lay down the basis for developing gene therapy of primary liver cancer.

关 键 词：HIL-2 重组腺病毒载体 病毒滴度 pAdBM5-GFP 

分 类 号：R73-362[医药卫生—肿瘤]