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
发布时间：2018-08-26 14:57
【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。
[Abstract]:[objective] to clone and recombine the human respiratory syncytial virus (human respiratory syncytial virus,RSV) microgenomic cDNA expressing the enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP) reporter gene, and to rescue the recombinant plasmid. In order to study and verify the role of RSV polymerase protein or auxiliary protein in RSV reverse genetic manipulation. [methods] using the method of whole gene synthesis and molecular biology combination, On the basis of obtaining pUC57-RSV-EGFP and pUC57-RSV-ORF1, which can express EGFP and non-bioactive proteins respectively, the microgenome plasmids encoding EGFP and non-bioactive proteins were further cloned into pBR322B vector, and the double cis microgenome plasmids encoding EGFP and non-bioactive proteins were obtained. After the analysis of restriction endonuclease and nucleic acid sequence, co-transfection was carried out into BHK-T7 cells with coplasmids expressing four kinds of coproteins. The expression of EGFP was observed by fluorescence microscope and the transcription level of EGFP mRNA was quantitatively analyzed by RT-qPCR. [results] the microgenomic plasmid pBR322B-RSV 鈪，
本文编号：2205222
[Abstract]:[objective] to clone and recombine the human respiratory syncytial virus (human respiratory syncytial virus,RSV) microgenomic cDNA expressing the enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP) reporter gene, and to rescue the recombinant plasmid. In order to study and verify the role of RSV polymerase protein or auxiliary protein in RSV reverse genetic manipulation. [methods] using the method of whole gene synthesis and molecular biology combination, On the basis of obtaining pUC57-RSV-EGFP and pUC57-RSV-ORF1, which can express EGFP and non-bioactive proteins respectively, the microgenome plasmids encoding EGFP and non-bioactive proteins were further cloned into pBR322B vector, and the double cis microgenome plasmids encoding EGFP and non-bioactive proteins were obtained. After the analysis of restriction endonuclease and nucleic acid sequence, co-transfection was carried out into BHK-T7 cells with coplasmids expressing four kinds of coproteins. The expression of EGFP was observed by fluorescence microscope and the transcription level of EGFP mRNA was quantitatively analyzed by RT-qPCR. [results] the microgenomic plasmid pBR322B-RSV 鈪，
本文编号：2205222
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