Gluc-GFP 双标记系统的构建及验证

Construction and validation of a Gluc-GFP dual-labeled system

目的：构建含Gluc-GFP的双标记系统，为研究病毒的体外标记和活体成像提供技术手段。方法根据文献报道合成split-GFP基因，将GFP拆分为G1-10与G11两部分，分别构建含G1-10与G11的pcDNA3．1表达质粒并通过荧光观察验证其相互作用。然后通过融合PCR技术将Gluc基因与G11基因通过Linker连接并插入到pcDNA3．1上，与G1-10表达质粒共转染293T细胞，测定细胞上清中Gluc荧光素酶的活性，并进行GFP荧光观察。为了更方便对病毒进行细胞水平的观测和筛选，利用慢病毒表达系统构建能稳定表达G1-10的MDCK细胞系，再向细胞系中转染G11质粒，通过红色荧光蛋白（ RFP）标记和嘌呤霉素的抗性对细胞系以及GFP活性进行筛选与验证。结果split-GFP基因共转染细胞后的荧光观察结果证实了split-GFP拆分策略的可行性。 Gluc-G11与G1-10质粒共转染后，细胞能够发出明亮的绿色荧光，上清中荧光素的活性也较高。在此基础上，建立的G1-10 MDCK细胞系能够稳定表达出G1-10，用G11质粒转染该细胞系后，可检测到GFP荧光。结论成功构建了含Gluc-GFP的双标记系统，split-GFP拆分策略可行，Gluc-G11不会影响荧光素酶Gluc的生理活性；G1-10细胞系的稳定表达可简化双标记系统的制备，为在细胞和活体水平研究病毒致病机制提供技术手段。

Objective To construct a Gaussia luciferase-green fluorescent protein （ Gluc-GFP ） dual-labeled system for the investigation of in vitro and in vivo pathogenesis of virus infections .Methods Two DNA fragments encoding G1-10 and G11 were split from the gene encoding GFP and then respectively inserted into pcDNA3.1 vector to construct the recombinant expression plasmids G1-10-pcDNA3.1 and G11-pcDNA3.1.The genes encoding Gluc and G11 were linked by a fusion PCR and then inserted into pcDNA3.1 vector to construct the fusion plasmid.The expression plasmids of Gluc-G11-pcDNA3.1 and G1-10-pcDNA3.1 were transfected into 293T cells.The expression of Gluc in the supernatants was measured and a fluorescence microscopy was used to observe cells with GFP fluorescence.To simplify the construction process, a Madin-Darby canine kidney （ MDCK ） cell line consistently express G1-10 was constructed by Lentivector Expression Systems.The dual-labeled system was constructed by transfecting G11-pcDNA3.1 plasmid into the MDCK cell line.The puromycin and red fluorescent protein （ RFP） were used for screening and validation.Results The feasibility of split-GFP strategy was confirmed by using fluorescence micro-scope.The expression of luciferase in the supernatants and cells with green fluorescence could be detected after co-transfection of G1-10-pcDNA3.1 and Gluc-G11-pcDNA3.1 plasmids into 293T cells.The construc-ted MDCK cell line could stably express G1-10.Conclusion The physiological activity of Gluc was not af-fected by creating Glu-G11 fusion protein.The MDCK cell line stably expressing G1-10 could simplify the construction of the dual-labeled system.The successfully constructed Gluc-GFP dual-labeled system would provide a useful tool for further investigation on the pathogenesis of virus infections.