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【目的】构建可表达增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的人呼吸道合胞病毒(human respiratory syncytial virus,RSV)双顺反子微型基因组cDNA克隆及重组质粒,并进行拯救,以探讨并验证RSV的聚合酶蛋白或辅助蛋白在RSV反向遗传学操作中的作用。【方法】利用全基因合成和分子生物学相结合的方法,在获得可分别表达EGFP和无生物活性蛋白的单顺反子微型基因组质粒pUC57-RSV-EGFP和pUC57-RSV-ORF1的基础上,进一步克隆至pBR322B载体,获得编码EGFP及无生物活性蛋白的双顺反子微型基因组质粒,经限制性内切酶和核酸序列分析正确后,与可表达4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,通过荧光显微镜观察EGFP的表达以及RT-qPCR对EGFP mRNA的转录水平进行定量分析。【结果】成功构建了编码EGFP和无生物活性蛋白的RSV双顺反子微型基因组质粒pBR322B-RSVⅡ-EGFP,经与编码4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,发现4种辅助蛋白对EGFP的表达具有不同的功能活性。【结论】以可表达EGFP报告基因的RSV双顺反子微型基因组重组质粒,实现了对4种辅助蛋白的功能验证,其中M2-1蛋白在双顺反子微型基因组拯救过程中具有转录延长的生物学活性。
【Objective】 To construct cDNA clone and recombinant plasmid of bicistronic microgenomic genome of human respiratory syncytial virus (RSV) that can express enhanced green fluorescent protein (EGFP) reporter gene and to rescue To investigate and validate the role of RSV polymerase or accessory proteins in RSV reverse genetics. 【Method】 Based on the combination of whole gene synthesis and molecular biology, we obtained the monocistronic minigenome plasmid pUC57-RSV-EGFP and pUC57-RSV-ORF1 which can express EGFP and non-bioactive protein respectively, And further cloned into pBR322B vector to obtain bicistronic minigenomes encoding EGFP and biologically inactive proteins. After restriction endonuclease analysis and nucleic acid sequence analysis were correct, co-transfections with helper plasmids expressing four helper proteins BHK-T7 cells, EGFP expression was observed by fluorescence microscopy and RT-qPCR quantitative analysis of EGFP mRNA transcription level. 【Result】 The recombinant plasmid pBR322B-RSVⅡ-EGFP encoding EGFP and biologically inactive protein was successfully constructed and co-transfected into BHK-T7 cells with four helper plasmids encoding four helper proteins. Four Auxiliary proteins have different functional activities on the expression of EGFP. 【Conclusion】 The functional verification of four accessory proteins was achieved by using RSV bicistronic microgenomic recombinant plasmids expressing EGFP reporter gene. Among them, M2-1 protein has a prolonged transcriptional activity in the rescue of bicistronic minigenomes Biological activity.