携带红色荧光蛋白报告基因的重组腺病毒载体的构建  

The construction of recombinant adenovirus vector carrying red fluorescent protein

作　　者：卢建溪[1] 阳莉[1] 钱师宇[2] 王强[1] 

机构地区：[1]中山大学附属第三医院疫苗研究所,广东广州510630 [2]暨南大学医学院,广东广州510632

出　　处：《中国微生态学杂志》2011年第8期677-680,共4页Chinese Journal of Microecology

基　　金：国家高技术研究发展计划(863计划)(2007AA021002);广东省自然科学基金资助项目(7001521);广东省科技计划资助项目(2008B030301101)

摘　　要：目的采用体外连接技术构建含红色荧光蛋白(RFP)报告基因的重组腺病毒载体,为基因治疗与基因疫苗研究提供重要工具。方法将pTurbo RFP-N上的含红色荧光蛋白基因片段,经酶切连接定向克隆至转移载体pShuttle上,构成重组质粒pShuttle-TurboRFP-N。用I-CeuI/PI-SceI双酶切重组质粒pShuttle-TurboRFP-N和骨架质粒pH5′040 pkGFP-II,回收目的片段,连接,获得重组腺病毒载体pH5.′040.CMV.RFP-N。将其线性化后,经脂质体介导转染至AD293细胞内包装出重组腺病毒颗粒。通过荧光显微镜观察其在AD293细胞内的包装效率和RFP表达情况。扩增并再次感染AD293细胞检测病毒颗粒感染能力,并测定其生物活性及滴度。结果经酶切鉴定,证明已正确构建重组腺病毒载体AdH5.′040.CMV.RFP-N;经AD293细胞包装成具有感染性的病毒颗粒,并能在真核细胞中高效表达目的基因;扩增纯化后获得重组腺病毒颗粒数为3.6×1012vp/mL,滴度达1×1010pfu/mL。结论成功构建了携带红色荧光蛋白报告基因的重组腺病毒载体,并能够在AD293细胞中高效地表达,为基因治疗与基因疫苗研究提供重要工具。Objective To construct adenovirus vector containing red fluorescent protein（RFP） by in vitro recombinant ligation approach,and provide important tools for gene therapy and gene vaccine research.Method The recombinant vector pShuttle-TurboRFP-N was constructed;the red fluorescent protein gene fragment of pTurboRFP-N was digested and ligated to pShuttle transfer vector.Then we used I-Ceul/PI-Scel to double digest recombinant vector pShuttle-TurboRFP-N and backbone vector pH5′040pkGFP-II,collected target fragment,ligated and obtained recombinant adeno virus vector pH5′.040.CMV.RFP-N.After linearization,we transfected the vector into AD293 cells mediated by liposome for virus packaging.We also examined the virus packaging efficiency and RFP expression level in AD293 cells using fluorescent microscope.We amplified and re-transfected AD293 cells to examine the transfection ability of the virus.In addition,we tested its biological ability and titer.Result We had constructed recombinant adeno virus vector AdH5′.040.CMV.RFP-N correctly.The vector can be packaged into virus having the ability to transfect and express target gene in eukaryote cells.The number of recombinant adenoviruses and titer after amplification and purification was 3.6×10^12 vp/mL and 1×10^10 pfu/mL respectively.Conclusion We have constructed recombinant adenovirus vector carrying red fluorescent protein reporting gene.The vector can be highly expressed in AD293 cells.It provides an important tool for gene therapy and gene vaccine research.

关 键 词：腺病毒载体 红色荧光蛋白 基因重组 

分 类 号：Q781[生物学—分子生物学]