携带红色荧光蛋白报告基因的重组腺病毒载体的构建及其体外试验研究

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

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pTurboRFP-N上的含RFP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-Ceu I/PI-Sce I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AdH5'.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。将所构建的重组腺病毒载体AdH5'.040.CMV.RFP-N与对照腺病毒载体AdH5.CMV.EGFP在体外感染人肺癌细胞系A549和乳腺癌细胞系MDA-MB-231。通过荧光蛋白RFP和EGFP的表达,比较它们对以上肿瘤细胞的感染效率。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3.6×1015vp/L,滴度达1.8×1013pfu/L。重组腺病毒载体AdH5'.040.CMV.RFP-N感染人肺癌细胞系A549和乳腺癌细胞系MDA-MB-231的感染效率,高于对照腺病毒载体AdH5.CMV.EGFP(P<0.05)。结论:成功构建了携带RFP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,对肿瘤细胞的感染效率高。RFP对GFP是一种很好的代替和补充,为基因治疗与基因疫苗研究提供更多的荧光标签。

AIM：To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein（RFP） reporter gene with the approach of in vitro recombinant ligation.METHODS： The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N.I-Ceu I/PI-Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5＇040.pkGFP-II.The target fragment was collected and ligated,and recombinant adenovirus vector AdH5＇.040.CMV.RFP-N was obtained.After linearization,the vector was transfected into AD293 cells by liposome for virus packaging.The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope.In addition,the biological activity and titer of the virus were tested.Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5＇.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively.The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein（EGFP）.RESULTS： The recombinant adenovirus vector AdH5＇.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion.The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells.The target gene in eukaryotic cells was also expressed.The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×1015 vp/L and 1×1013 pfu/L,respectively.The infection efficiencies of recombinant adenovirus vector Ad5＇.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP（P〈0.05）.CONCLUSION： We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research.The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells.RFP is a good substitution and supplement to green fluorescent protein.