可用于輔助蛋白功能研究的人呼吸道合胞病毒雙順反子微型基因組的構(gòu)建及拯救
發(fā)布時(shí)間：2018-08-26 14:57
【摘要】：【目的】構(gòu)建可表達(dá)增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)報(bào)告基因的人呼吸道合胞病毒(human respiratory syncytial virus,RSV)雙順反子微型基因組cDNA克隆及重組質(zhì)粒,并進(jìn)行拯救,以探討并驗(yàn)證RSV的聚合酶蛋白或輔助蛋白在RSV反向遺傳學(xué)操作中的作用�！痉椒ā坷萌蚝铣珊头肿由飳W(xué)相結(jié)合的方法,在獲得可分別表達(dá)EGFP和無生物活性蛋白的單順反子微型基因組質(zhì)粒pUC57-RSV-EGFP和pUC57-RSV-ORF1的基礎(chǔ)上,進(jìn)一步克隆至pBR322B載體,獲得編碼EGFP及無生物活性蛋白的雙順反子微型基因組質(zhì)粒,經(jīng)限制性內(nèi)切酶和核酸序列分析正確后,與可表達(dá)4種輔助蛋白的輔助質(zhì)粒共轉(zhuǎn)染至BHK-T7細(xì)胞,通過熒光顯微鏡觀察EGFP的表達(dá)以及RT-qPCR對(duì)EGFP mRNA的轉(zhuǎn)錄水平進(jìn)行定量分析�！窘Y(jié)果】成功構(gòu)建了編碼EGFP和無生物活性蛋白的RSV雙順反子微型基因組質(zhì)粒pBR322B-RSVⅡ-EGFP,經(jīng)與編碼4種輔助蛋白的輔助質(zhì)粒共轉(zhuǎn)染至BHK-T7細(xì)胞,發(fā)現(xiàn)4種輔助蛋白對(duì)EGFP的表達(dá)具有不同的功能活性。【結(jié)論】以可表達(dá)EGFP報(bào)告基因的RSV雙順反子微型基因組重組質(zhì)粒,實(shí)現(xiàn)了對(duì)4種輔助蛋白的功能驗(yàn)證,其中M2-1蛋白在雙順反子微型基因組拯救過程中具有轉(zhuǎn)錄延長(zhǎng)的生物學(xué)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，
本文編號(hào)：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，
本文編號(hào)：2205222
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