人呼吸道合胞病毒微型基因组的研究
发布时间：2018-08-27 20:35
【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)是导致婴幼儿严重下呼吸道感染的最重要的病毒病原,目前尚无有效的防治方法。利用RNA病毒反向遗传学操作获得的减毒RSV活病毒,具有稳定的安全性和良好的免疫原性,用于制备RSV疫苗有较好的前景。该技术的关键在于首先得到含有整个病毒基因组的感染性cDNA克隆,然后在培养细胞中重新拯救出活病毒。RSV基因组为单股负链RNA,为加深对其基因组复制特性的认识,积累RSV拯救的必要知识,拟先尝试构建微型基因组(minigenome),即在RSV转录起始和终止信号之间插入报告基因或较短的病毒基因组片段,两端为病毒RNA复制转录所必需的病毒前导序列和尾随序列等调控序列,构成cDNA来源的微型基因组,与表达辅助蛋白(N、P、L、M2-1)的重组质粒共转染或以RSV病毒作为辅助病毒在细胞内进行复制和转录,通过鉴定报告基因的表达即可初步鉴定所建立的微型基因组的功能。目前常用T7 RNA聚合酶(T7 RNA Polymerase,T7 RNP)将含有病毒微型基因组的重组质粒在细胞内转录合成病毒的基因组RNA。本研究旨在探讨T7转录系统及功能,并进一步构建携带增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein, EGFP)基因的RSV微型基因组重组质粒,通过转染可表达T7 RNP的BSR T7/5细胞系,并以RSV作为辅助病毒观察EGFP的表达情况,明确RSV微型基因组的功能,为研究RSV感染性cDNA奠定基础。 方法:根据编码T7 RNP的基因序列,设计一对引入EcoR V和Xho I酶切位点的引物。提取含有T7 RNP的E.coli BL21(DE3)基因组DNA,以该DNA为模板,应用PCR技术,扩增T7 RNP全长基因,经过中间载体将其克隆至真核表达载体pcDNA3.1(+)。与此同时,将px8δt载体切下的T7 RNP转录识别的终止信号(Terminator,TER)和从pGEM-T easy/EGFP上切下的EGFP基因克隆至pcDNAII,且保证EGFP位于T7启动子与TER之间。将获得的两个重组质粒共转染BHK细胞。48 h后,通过荧光显微镜观察EGFP在真核细胞内的表达情况。同时,根据文献获得RSV转录复制时所需的最小顺式作用元件,即转录起始(Gene Start, GS)信号和转录终止(Gene End, GE)信号以及基因组启动子(Leader)与反基因组的启动子(Trailer)序列。设计好信号序列的顺序并在其中引入多克隆酶切位点,命名为GSGE,基因合成得到GSGE的cDNA,以该段序列为模板合成两对分别在上下游带有T7启动子的引物,同时在T7启动子端引入Hind III酶切位点,行PCR,得到的产物分别命名为GSGE1和GSGE2,之后将这两段序列克隆入px8δT载体,并进一步将EGFP基因定向克隆至其中得到两个含有RSV微型基因组的重组载体,分别命名为px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP。为验证这两个重组载体中RSV微型基因组的功能,通过脂质体法转染BSR T7/5细胞系,进一步利用RSV作为辅助病毒提供RSV转录和复制必须的所有功能蛋白,72h后在倒置荧光显微镜下观察报告基因EGFP的表达情况。 结果:成功构建了真核表达载体pcDNA3.1(+)/T7 RNP和重组载体pcDNAII/EGFP/TER。在共转染BHK细胞后,荧光显微镜下可观察到EGFP表达的绿色荧光。成功构建含有RSV微型基因组的px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP载体,转染BSR T7/5细胞,以RSV作为辅助病毒,倒置荧光显微镜下观察到BSR T7/5细胞表达绿色荧光。 结论:pcDNA3.1(+)/T7 RNP可在真核细胞BHK内表达T7 RNP,通过与T7启动子和TER的相互作用,实现了pcDNA II/EGFP/TER中EGFP基因的转录及表达。构建的带有RSV微型基因组的重组载体,在RSV的辅助下可实现EGFP的成功表达,微型基因组具有RSV病毒转录和复制的功能,为进一步的RSV反向遗传学操作提供了坚实保障。
[Abstract]:Objective: Human Respiratory Syncytial Virus (RSV) is one of the most important viral pathogens causing severe lower respiratory tract infections in infants and young children, and there is no effective method to prevent and cure it. RSV vaccines have a promising future. The key to this technique is to obtain infectious cDNA clones containing the entire viral genome first, and then re-rescue the live virus in cultured cells. Minigenome, in which a reporter gene or a shorter viral genome segment is inserted between the initiation and termination signals of RSV transcription, and the viral precursor and trailing sequences at both ends are necessary for viral RNA replication and transcription, constitute a microgenome derived from a cDNA and are co-transfected or co-transfected with recombinant plasmids expressing helper proteins (N, P, L, M2-1). RSV is used as a helper virus to replicate and transcribe in cells, and the function of the microgenome can be preliminarily identified by identifying the expression of the reporter gene. The purpose of this study was to explore the transcriptional system and function of T7, and to construct a recombinant RSV mini-genome plasmid carrying Enhanced Green Fluorescent Protein (EGFP) gene. The BSR T7/5 cell line expressing T7 RNP was transfected with the recombinant plasmid. The expression of EGFP was observed by using RSV as an auxiliary virus. The function of the group laid the foundation for studying the infectious cDNA of RSV.
METHODS: A pair of primers were designed for introducing EcoR V and Xho I digestion sites according to the gene sequence encoding T7 RNP. The genomic DNA of E.coli BL21 (DE3) containing T7 RNP was extracted. Using this DNA as template, the full-length gene of T7 RNP was amplified by PCR and cloned into eukaryotic expression vector pcDNA3.1 (+) via an intermediate vector. The T7 RNP transcriptional recognition termination signal (TER) and the EGFP gene cut from pGEM-T easy/EGFP were cloned into pcDNAII and the EGFP was located between T7 promoter and TER. After co-transfection of the two recombinant plasmids into BHK cells for 48 hours, the expression of EGFP in eukaryotic cells was observed by fluorescence microscopy. The minimal cis-acting elements required for transcriptional replication of RSV, i.e. Gene Start (GS) signal and Gene End (GE) signal, genome promoter (Leader) and anti-genome promoter (Trailer) sequence, were presented. The sequence of the signal sequence was designed and polyclonal enzyme digestion site was introduced into the sequence, named GSGE. Two pairs of primers with T7 promoter were synthesized. Hind III cleavage site was introduced into T7 promoter and PCR was performed. The products were named GSGE1 and GSGE2, respectively. The two sequences were cloned into px8delta T vector and EGFP gene was cloned into the vector. Two recombinant vectors containing RSV microgenome were obtained, named px8delta T/GSGE1/EGFP and px8delta T/GSGE2/EGFP. To verify the function of the RSV microgenome in these two recombinant vectors, BSR T7/5 cell lines were transfected by liposome method, and RSV was used as a helper virus to provide all the functional proteins necessary for RSV transcription and replication. The expression of reporter gene EGFP was observed under inverted fluorescence microscope.
Results: Eukaryotic expression vector pcDNA3.1 (+) / T7 RNP and recombinant vector pcDNAII / EGFP / TER were successfully constructed. After co-transfection with BHK cells, the green fluorescence of EGFP expression was observed under fluorescence microscope. The px8 Delta T / GSGE1 / EGFP and px8 Delta T / GSGE2 / EGFP vectors containing RSV Minigenome were successfully constructed and transfected into BSR T7 / 5 cells with RSV as accessory virus. BSR T7/5 cells expressed green fluorescence under inverted fluorescence microscope.
CONCLUSION: pcDNA3.1 (+) / T7 RNP can express T7 RNP in eukaryotic cell BHK. The transcription and expression of EGFP gene in pcDNA II / EGFP / TER can be achieved by interacting with T7 promoter and TER. The constructed recombinant vector with RSV Minigenome can successfully express EGFP with the assistance of RSV, and the Minigenome has RSV transcription and TER. The function of replication provides a solid guarantee for further RSV reverse genetics.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373.1
本文编号：2208380
[Abstract]:Objective: Human Respiratory Syncytial Virus (RSV) is one of the most important viral pathogens causing severe lower respiratory tract infections in infants and young children, and there is no effective method to prevent and cure it. RSV vaccines have a promising future. The key to this technique is to obtain infectious cDNA clones containing the entire viral genome first, and then re-rescue the live virus in cultured cells. Minigenome, in which a reporter gene or a shorter viral genome segment is inserted between the initiation and termination signals of RSV transcription, and the viral precursor and trailing sequences at both ends are necessary for viral RNA replication and transcription, constitute a microgenome derived from a cDNA and are co-transfected or co-transfected with recombinant plasmids expressing helper proteins (N, P, L, M2-1). RSV is used as a helper virus to replicate and transcribe in cells, and the function of the microgenome can be preliminarily identified by identifying the expression of the reporter gene. The purpose of this study was to explore the transcriptional system and function of T7, and to construct a recombinant RSV mini-genome plasmid carrying Enhanced Green Fluorescent Protein (EGFP) gene. The BSR T7/5 cell line expressing T7 RNP was transfected with the recombinant plasmid. The expression of EGFP was observed by using RSV as an auxiliary virus. The function of the group laid the foundation for studying the infectious cDNA of RSV.
METHODS: A pair of primers were designed for introducing EcoR V and Xho I digestion sites according to the gene sequence encoding T7 RNP. The genomic DNA of E.coli BL21 (DE3) containing T7 RNP was extracted. Using this DNA as template, the full-length gene of T7 RNP was amplified by PCR and cloned into eukaryotic expression vector pcDNA3.1 (+) via an intermediate vector. The T7 RNP transcriptional recognition termination signal (TER) and the EGFP gene cut from pGEM-T easy/EGFP were cloned into pcDNAII and the EGFP was located between T7 promoter and TER. After co-transfection of the two recombinant plasmids into BHK cells for 48 hours, the expression of EGFP in eukaryotic cells was observed by fluorescence microscopy. The minimal cis-acting elements required for transcriptional replication of RSV, i.e. Gene Start (GS) signal and Gene End (GE) signal, genome promoter (Leader) and anti-genome promoter (Trailer) sequence, were presented. The sequence of the signal sequence was designed and polyclonal enzyme digestion site was introduced into the sequence, named GSGE. Two pairs of primers with T7 promoter were synthesized. Hind III cleavage site was introduced into T7 promoter and PCR was performed. The products were named GSGE1 and GSGE2, respectively. The two sequences were cloned into px8delta T vector and EGFP gene was cloned into the vector. Two recombinant vectors containing RSV microgenome were obtained, named px8delta T/GSGE1/EGFP and px8delta T/GSGE2/EGFP. To verify the function of the RSV microgenome in these two recombinant vectors, BSR T7/5 cell lines were transfected by liposome method, and RSV was used as a helper virus to provide all the functional proteins necessary for RSV transcription and replication. The expression of reporter gene EGFP was observed under inverted fluorescence microscope.
Results: Eukaryotic expression vector pcDNA3.1 (+) / T7 RNP and recombinant vector pcDNAII / EGFP / TER were successfully constructed. After co-transfection with BHK cells, the green fluorescence of EGFP expression was observed under fluorescence microscope. The px8 Delta T / GSGE1 / EGFP and px8 Delta T / GSGE2 / EGFP vectors containing RSV Minigenome were successfully constructed and transfected into BSR T7 / 5 cells with RSV as accessory virus. BSR T7/5 cells expressed green fluorescence under inverted fluorescence microscope.
CONCLUSION: pcDNA3.1 (+) / T7 RNP can express T7 RNP in eukaryotic cell BHK. The transcription and expression of EGFP gene in pcDNA II / EGFP / TER can be achieved by interacting with T7 promoter and TER. The constructed recombinant vector with RSV Minigenome can successfully express EGFP with the assistance of RSV, and the Minigenome has RSV transcription and TER. The function of replication provides a solid guarantee for further RSV reverse genetics.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373.1
【共引文献】
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2 祁巧芬;稳定表达T7 RNA聚合酶的ST细胞系的建立[D];新疆农业大学;2008年
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