人呼吸道合胞病毒微型基因組的研究
發(fā)布時間：2018-08-27 20:35
【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)是導(dǎo)致嬰幼兒嚴(yán)重下呼吸道感染的最重要的病毒病原,目前尚無有效的防治方法。利用RNA病毒反向遺傳學(xué)操作獲得的減毒RSV活病毒,具有穩(wěn)定的安全性和良好的免疫原性,用于制備RSV疫苗有較好的前景。該技術(shù)的關(guān)鍵在于首先得到含有整個病毒基因組的感染性cDNA克隆,然后在培養(yǎng)細胞中重新拯救出活病毒。RSV基因組為單股負(fù)鏈RNA,為加深對其基因組復(fù)制特性的認(rèn)識,積累RSV拯救的必要知識,擬先嘗試構(gòu)建微型基因組(minigenome),即在RSV轉(zhuǎn)錄起始和終止信號之間插入報告基因或較短的病毒基因組片段,兩端為病毒RNA復(fù)制轉(zhuǎn)錄所必需的病毒前導(dǎo)序列和尾隨序列等調(diào)控序列,構(gòu)成cDNA來源的微型基因組,與表達輔助蛋白(N、P、L、M2-1)的重組質(zhì)粒共轉(zhuǎn)染或以RSV病毒作為輔助病毒在細胞內(nèi)進行復(fù)制和轉(zhuǎn)錄,通過鑒定報告基因的表達即可初步鑒定所建立的微型基因組的功能。目前常用T7 RNA聚合酶(T7 RNA Polymerase,T7 RNP)將含有病毒微型基因組的重組質(zhì)粒在細胞內(nèi)轉(zhuǎn)錄合成病毒的基因組RNA。本研究旨在探討T7轉(zhuǎn)錄系統(tǒng)及功能,并進一步構(gòu)建攜帶增強型綠色熒光蛋白(Enhanced Green Fluorescent Protein, EGFP)基因的RSV微型基因組重組質(zhì)粒,通過轉(zhuǎn)染可表達T7 RNP的BSR T7/5細胞系,并以RSV作為輔助病毒觀察EGFP的表達情況,明確RSV微型基因組的功能,為研究RSV感染性cDNA奠定基礎(chǔ)。 方法:根據(jù)編碼T7 RNP的基因序列,設(shè)計一對引入EcoR V和Xho I酶切位點的引物。提取含有T7 RNP的E.coli BL21(DE3)基因組DNA,以該DNA為模板,應(yīng)用PCR技術(shù),擴增T7 RNP全長基因,經(jīng)過中間載體將其克隆至真核表達載體pcDNA3.1(+)。與此同時,將px8δt載體切下的T7 RNP轉(zhuǎn)錄識別的終止信號(Terminator,TER)和從pGEM-T easy/EGFP上切下的EGFP基因克隆至pcDNAII,且保證EGFP位于T7啟動子與TER之間。將獲得的兩個重組質(zhì)粒共轉(zhuǎn)染BHK細胞。48 h后,通過熒光顯微鏡觀察EGFP在真核細胞內(nèi)的表達情況。同時,根據(jù)文獻獲得RSV轉(zhuǎn)錄復(fù)制時所需的最小順式作用元件,即轉(zhuǎn)錄起始(Gene Start, GS)信號和轉(zhuǎn)錄終止(Gene End, GE)信號以及基因組啟動子(Leader)與反基因組的啟動子(Trailer)序列。設(shè)計好信號序列的順序并在其中引入多克隆酶切位點,命名為GSGE,基因合成得到GSGE的cDNA,以該段序列為模板合成兩對分別在上下游帶有T7啟動子的引物,同時在T7啟動子端引入Hind III酶切位點,行PCR,得到的產(chǎn)物分別命名為GSGE1和GSGE2,之后將這兩段序列克隆入px8δT載體,并進一步將EGFP基因定向克隆至其中得到兩個含有RSV微型基因組的重組載體,分別命名為px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP。為驗證這兩個重組載體中RSV微型基因組的功能,通過脂質(zhì)體法轉(zhuǎn)染BSR T7/5細胞系,進一步利用RSV作為輔助病毒提供RSV轉(zhuǎn)錄和復(fù)制必須的所有功能蛋白,72h后在倒置熒光顯微鏡下觀察報告基因EGFP的表達情況。 結(jié)果:成功構(gòu)建了真核表達載體pcDNA3.1(+)/T7 RNP和重組載體pcDNAII/EGFP/TER。在共轉(zhuǎn)染BHK細胞后,熒光顯微鏡下可觀察到EGFP表達的綠色熒光。成功構(gòu)建含有RSV微型基因組的px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP載體,轉(zhuǎn)染BSR T7/5細胞,以RSV作為輔助病毒,倒置熒光顯微鏡下觀察到BSR T7/5細胞表達綠色熒光。 結(jié)論:pcDNA3.1(+)/T7 RNP可在真核細胞BHK內(nèi)表達T7 RNP,通過與T7啟動子和TER的相互作用,實現(xiàn)了pcDNA II/EGFP/TER中EGFP基因的轉(zhuǎn)錄及表達。構(gòu)建的帶有RSV微型基因組的重組載體,在RSV的輔助下可實現(xiàn)EGFP的成功表達,微型基因組具有RSV病毒轉(zhuǎn)錄和復(fù)制的功能,為進一步的RSV反向遺傳學(xué)操作提供了堅實保障。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373.1
本文編號：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373.1
【共引文獻】
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