反向遺傳構(gòu)建A組輪狀病毒重配毒株的研究
發(fā)布時間：2018-12-11 07:25
【摘要】： 輪狀病毒(Rotavirus,RV)屬于呼腸孤病毒科,輪狀病毒屬;已被確認(rèn)為引起全世界嬰幼兒急性胃腸炎最常見的病原。輪狀病毒感染每年導(dǎo)致全球約1.25億嬰幼兒發(fā)生腹瀉,輪狀病毒腹瀉每年造成全球90萬5歲以下嬰幼兒死亡,嚴(yán)重影響了嬰幼兒的健康并給全球帶來巨大的疾病負(fù)擔(dān)。輪狀病毒引起的腹瀉至今尚無特效藥物進(jìn)行治療,疫苗是目前證實唯一最有效的預(yù)防方法。本論文研究內(nèi)容包括A組輪狀病毒(GARV)武漢地區(qū)G9型流行毒株CC-1的分離及vp7基因的克隆與分析;A組輪狀病毒vp7基因轉(zhuǎn)錄載體的構(gòu)建與檢測;A組輪狀病毒重配株的構(gòu)建。具體內(nèi)容如下: 第一章全面概述了輪狀病毒的形態(tài)結(jié)構(gòu)、理化性質(zhì)、基因組結(jié)構(gòu)及編碼的蛋白質(zhì)、體外培養(yǎng)、以及A組輪狀病毒的流行病學(xué)、輪狀病毒疫苗研究現(xiàn)狀,重點論述了G9型輪狀病毒的流行特征及輪狀病毒的反向遺傳學(xué)研究。 第二章武漢地區(qū)G9型輪狀病毒的分離和vp7基因的克隆與序列分析采用MA104細(xì)胞從糞便樣本中分離CC-1株,RT-PCR獲得vp7基因,并克隆至pGEMT-easy載體,獲得質(zhì)粒pGEMvp7-G9。對質(zhì)粒插入片斷測序并進(jìn)行序列分析。 第三章A組輪狀病毒全長vp7基因轉(zhuǎn)錄載體的構(gòu)建與檢測。采用Overlap PCR方法構(gòu)建5'端上游有T7-RNA聚合酶啟動子、下游有HDV核酶序列與T7-RNA聚合酶終止子的vp7基因,構(gòu)建vp7轉(zhuǎn)錄載體pGEMvp7-Rz。該質(zhì)粒轉(zhuǎn)染預(yù)先感染vTF7-3的哺乳動物細(xì)胞后,在哺乳動物細(xì)胞中獲得具有正確的正確5'與3'末端的vp7+sRNA。 第四章反向遺傳學(xué)方法構(gòu)建A組輪狀病毒重配株。采用補骨質(zhì)素-紫外線A使重組痘病毒vTF7-3失去感染性,但仍保持表達(dá)T7-RNA聚合酶的活性。將重組痘病毒vTF7-3感染Vero細(xì)胞,采用G1型A組輪狀病毒vp7的全長cNDA轉(zhuǎn)錄載體轉(zhuǎn)染感染了vTF7-3的Vero細(xì)胞,在細(xì)胞內(nèi)獲得具有正確5'與3'端的輪狀病毒vp7基因+sRNA。然后將另一株G10型A組輪狀病毒感染輪狀病毒敏感的vp7細(xì)胞,在病毒的包裝過程中vp7發(fā)生重配。然后使用單克隆抗體對重配病毒進(jìn)行篩選。
[Abstract]:Rotavirus (Rotavirus,RV) belongs to the family Reoviridae and has been identified as the most common cause of acute gastroenteritis in infants and children in the world. Rotavirus infection causes diarrhea in about 125 million infants and young children in the world every year. Rotavirus diarrhea causes deaths of children under 5 years of age in the world every year, which seriously affects the health of infants and children and brings a huge burden of disease to the whole world. There is no effective drug to treat rotavirus-induced diarrhea, and vaccine is the only effective method to prevent diarrhea caused by rotavirus. This thesis includes the isolation of CC-1 and the cloning and analysis of vp7 gene of group A rotavirus (GARV) strain G9 in Wuhan region, the construction and detection of vp7 gene transcription vector of group A rotavirus. Construction of Group A rotavirus Recombinant. The main contents are as follows: in the first chapter, the morphology, physicochemical properties, genome structure and encoded protein of rotavirus, culture in vitro, epidemiology of group A rotavirus and the status of rotavirus vaccine were summarized. The epidemic characteristics of G 9 rotavirus and the reverse genetics of rotavirus were discussed. Chapter 2 isolation of G9 rotavirus and cloning and sequence Analysis of vp7 Gene in Wuhan region CC-1 strain was isolated from fecal samples by MA104 cells. Vp7 gene was obtained by RT-PCR and cloned into pGEMT-easy vector to obtain plasmid pGEMvp7-G9.. The plasmid insert fragment was sequenced and sequenced. Chapter 3: construction and detection of full-length vp7 transcription vector of group A rotavirus. The vp7 gene with HDV ribozyme sequence and T7-RNA polymerase Terminator was constructed by Overlap PCR method. The vp7 transcription vector pGEMvp7-Rz. was constructed. After transfection of the plasmid into mammalian cells preinfected with vTF7-3, the correct 5 'and 3' terminal vp7 sRNA. were obtained in mammalian cells. Chapter 4 reverse genetics method was used to construct group A rotavirus recombinant strain. The recombinant poxvirus (vTF7-3) was infected by UVA, but the activity of T7-RNA polymerase was maintained. The recombinant poxvirus vTF7-3 was infected with Vero cells. The Vero cells infected with vTF7-3 were transfected with the full-length cNDA transcriptional vector of group G1 rotavirus vp7. The 5 'and 3'end rotavirus vp7 gene sRNA. was obtained in the cells. Then another G10 group A rotavirus strain was infected with rotavirus sensitive vp7 cells and vp7 reassorted during the packaging of the virus. Then monoclonal antibodies were used to screen the reassorted virus.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R373
本文編號：2372140
[Abstract]:Rotavirus (Rotavirus,RV) belongs to the family Reoviridae and has been identified as the most common cause of acute gastroenteritis in infants and children in the world. Rotavirus infection causes diarrhea in about 125 million infants and young children in the world every year. Rotavirus diarrhea causes deaths of children under 5 years of age in the world every year, which seriously affects the health of infants and children and brings a huge burden of disease to the whole world. There is no effective drug to treat rotavirus-induced diarrhea, and vaccine is the only effective method to prevent diarrhea caused by rotavirus. This thesis includes the isolation of CC-1 and the cloning and analysis of vp7 gene of group A rotavirus (GARV) strain G9 in Wuhan region, the construction and detection of vp7 gene transcription vector of group A rotavirus. Construction of Group A rotavirus Recombinant. The main contents are as follows: in the first chapter, the morphology, physicochemical properties, genome structure and encoded protein of rotavirus, culture in vitro, epidemiology of group A rotavirus and the status of rotavirus vaccine were summarized. The epidemic characteristics of G 9 rotavirus and the reverse genetics of rotavirus were discussed. Chapter 2 isolation of G9 rotavirus and cloning and sequence Analysis of vp7 Gene in Wuhan region CC-1 strain was isolated from fecal samples by MA104 cells. Vp7 gene was obtained by RT-PCR and cloned into pGEMT-easy vector to obtain plasmid pGEMvp7-G9.. The plasmid insert fragment was sequenced and sequenced. Chapter 3: construction and detection of full-length vp7 transcription vector of group A rotavirus. The vp7 gene with HDV ribozyme sequence and T7-RNA polymerase Terminator was constructed by Overlap PCR method. The vp7 transcription vector pGEMvp7-Rz. was constructed. After transfection of the plasmid into mammalian cells preinfected with vTF7-3, the correct 5 'and 3' terminal vp7 sRNA. were obtained in mammalian cells. Chapter 4 reverse genetics method was used to construct group A rotavirus recombinant strain. The recombinant poxvirus (vTF7-3) was infected by UVA, but the activity of T7-RNA polymerase was maintained. The recombinant poxvirus vTF7-3 was infected with Vero cells. The Vero cells infected with vTF7-3 were transfected with the full-length cNDA transcriptional vector of group G1 rotavirus vp7. The 5 'and 3'end rotavirus vp7 gene sRNA. was obtained in the cells. Then another G10 group A rotavirus strain was infected with rotavirus sensitive vp7 cells and vp7 reassorted during the packaging of the virus. Then monoclonal antibodies were used to screen the reassorted virus.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 錢淵，袁麗娟，，熊朝暉，張又，劉軍，關(guān)德華，王之梁;在我國腹瀉患兒中發(fā)現(xiàn)G9型輪狀病毒感染[J];病毒學(xué)報;1994年03期
2 童志禮;嬰幼兒輪狀病毒流行的新趨勢——G9型輪狀病毒全球流行綜述[J];國外醫(yī)學(xué).病毒學(xué)分冊;2002年04期
3 溫樂英;輪狀病毒蛋白的研究進(jìn)展[J];國外醫(yī)學(xué).病毒學(xué)分冊;1996年03期
4 汪洋;白萱;余暉;孫紅;楊繼紅;;2006年武漢地區(qū)A組輪狀病毒VP7基因型初步研究[J];黃岡師范學(xué)院學(xué)報;2006年06期
5 石長信;王長安;王湛;周偉;洪濤;;A組輪狀病毒外殼蛋白VP4的原核表達(dá)[J];中華實驗和臨床病毒學(xué)雜志;1996年02期
本文編號：2372140
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2372140.html
最近更新
教材專著
