可用于辅助蛋白功能研究的人呼吸道合胞病毒双顺反子微型基因组的构建及拯救  

Construction and application of dicistronic minigenome of human respiratory syncytial virus

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作  者:郑元博[1] 张秀娟[1] 许敏[1] 姜男[1] 彭向雷[1] 付远辉[1] 郑妍鹏[1] 洪涛[1] 何金生[1] 

机构地区:[1]北京交通大学生命科学与生物工程研究院,北京100044

出  处:《微生物学报》2017年第7期1004-1013,共10页Acta Microbiologica Sinica

基  金:"十二五"国家重大新药创制专项课题(2013ZX09103-003-011)~~

摘  要:【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 clarify the role of nucleocapsid/polymerase proteins or helper proteins of human respiratory syncytial virus (RSV) in rescuing recombinant RSV, plasmids of RSV dicistronic minigenome encoding enhanced green fluorescent protein (EGFP) gene was constructed and rescued. [Methods] Based on the methods of gene synthesis and molecular cloning, RSV monocistronic minigenome plasmids, pUC57-RSV-EGFP encoding EGFP and pUC57-RSV-ORF1 encoding pseudo-virus protein, were constructed. Then, RSV dicistronic minigenome was further cloned through these two monocistronic minigenome plasmids and pBR322B vector, and identified by the analyses of restriction endonuclease and nucleotide acid sequence. Following co-transfecting helper plasmids to BHK-T7 cells together with RSV dicistronic minigenome plasmid, the function of helper proteins was evaluated based on the transcribed EGFP mRNA by real-time quantitative polymerase chain reaction (RT-qPCR) and the expressed EGFP by fluorescent microscope. [Results] The recombinant plasmid of RSV dicistronic minigenome encoding EGFP gene and driven by T7 promoter, pBR322B-RSVII-EGFP, was constructed successfully. The differential expressions of EGFP both transcriptionally and translationally occurred following the dicistronic minigenome rescued by different combinations of the helper plasmids, which showed the helper proteins functioned differently in the replication of RSV. [Conclusion] The constructed RSV dicistronic minigenome containing EGFP gene is a useful tool for function analysis of the helper proteins, and the M2-1 protein, capable of elongating the transcription, is confirmed.

关 键 词:人呼吸道合胞病毒 双顺反子 微型基因组 辅助蛋白 

分 类 号:R373[医药卫生—病原生物学]

 

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