柯薩奇病毒B組3型在ECV304中持續(xù)感染模型的建立及其持續(xù)感染機制的初步研究
發(fā)布時間：2018-07-26 10:04
【摘要】： 盡管CVB3是常見的病毒性心肌炎等急性疾病的病原體，然而也有許多證據(jù)表明CVB3也參與了許多嚴重的慢性疾病的病理過程。有研究表明CVB3與擴張型心肌病關(guān)系密切，說明病毒可能在心血管系統(tǒng)中持續(xù)存在，本文旨在通過建立CVB3(Nancy)在ECV304細胞中持續(xù)感染的模型，初步探索CVB3持續(xù)感染的機制。 以10~(-1) TCID50，10~(-2) TCID50，10~(-3) TCID50 CVB3感染ECV304，建立持續(xù)感染細胞模型，穩(wěn)定傳代120天以上，上清液及細胞裂解液接種敏感細胞Vero沒有CPE，核酸檢測結(jié)果陽性，從細胞裂解液中提取病毒RNA，進行RT反應(yīng)，反轉(zhuǎn)錄出基因組cDNA，將整個基因組劃分為5段進行PCR擴增，連接目的片段至PGM-T載體，進行測序，分析可能存在的變異是否與持續(xù)感染狀態(tài)的形成有關(guān)。 結(jié)果發(fā)現(xiàn)持續(xù)感染細胞中CVB3與建模前的CVB3存在16處核苷酸變異，1處位于5’NTR：16R-G，其余15處位于病毒的ORF，其中有7個核苷酸變異造成了氨基酸的改變，分別是：P1-A區(qū)16P-G、18K-N，P3-AB區(qū)1486H-Y，P3-C區(qū)1696E-K，P3-D區(qū)1745R-K、1849A-T、2098P-L。經(jīng)過對文獻中病毒基因的分析P3-AB區(qū)、P3-D區(qū)的改變最可能影響病毒的毒力，致病毒表現(xiàn)出一種低復(fù)制的表型，這一結(jié)論尚需要反向遺傳學(xué)驗證。
[Abstract]:Although CVB3 is a common pathogen of acute diseases such as viral myocarditis, there is a lot of evidence that CVB3 is also involved in the pathological process of many serious chronic diseases. Some studies have shown that CVB3 is closely related to dilated cardiomyopathy, indicating that the virus may persist in the cardiovascular system. This paper aims to explore the mechanism of CVB3 persistent infection by establishing a model of CVB3 (Nancy) persistent infection in ECV304 cells. ECV304 was infected with 10 ~ (-1) TCID _ (50) ~ (-2) TCID _ (50) ~ (-3) TCID50 CVB3. The cell model of persistent infection was established. The supernatant and cell lysate were inoculated with sensitive cell Vero for more than 120 days. The results of nucleic acid detection were positive, virus RNAs were extracted from the cell lysis fluid and RT reaction was performed. The whole genome was divided into 5 segments for PCR amplification. The target fragment was ligated into PGM-T vector and sequenced to analyze whether the possible mutation was related to the formation of persistent infection. The results showed that there were 16 nucleotide mutations in CVB3 and CVB3 before modeling, one was located at 5 NTR: 16R-Gand the other 15 were located at ORF of virus, among which 7 nucleotide mutations caused amino acid changes. They are respectively: 1: P1-A region 16P-Gnn 18K-NCU P3-AB region 1486H-YP3-C region 1696E-KN P3-C region 1745R-Knn 1849A-Tnn 2098P-L. By analyzing the viral genes in the literature, the change of the P3-D region in the P3-AB region is most likely to affect the virulence of the virus, and the virus exhibits a low replication phenotype. This conclusion needs to be verified by reverse genetics.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373
本文編號：2145645
[Abstract]:Although CVB3 is a common pathogen of acute diseases such as viral myocarditis, there is a lot of evidence that CVB3 is also involved in the pathological process of many serious chronic diseases. Some studies have shown that CVB3 is closely related to dilated cardiomyopathy, indicating that the virus may persist in the cardiovascular system. This paper aims to explore the mechanism of CVB3 persistent infection by establishing a model of CVB3 (Nancy) persistent infection in ECV304 cells. ECV304 was infected with 10 ~ (-1) TCID _ (50) ~ (-2) TCID _ (50) ~ (-3) TCID50 CVB3. The cell model of persistent infection was established. The supernatant and cell lysate were inoculated with sensitive cell Vero for more than 120 days. The results of nucleic acid detection were positive, virus RNAs were extracted from the cell lysis fluid and RT reaction was performed. The whole genome was divided into 5 segments for PCR amplification. The target fragment was ligated into PGM-T vector and sequenced to analyze whether the possible mutation was related to the formation of persistent infection. The results showed that there were 16 nucleotide mutations in CVB3 and CVB3 before modeling, one was located at 5 NTR: 16R-Gand the other 15 were located at ORF of virus, among which 7 nucleotide mutations caused amino acid changes. They are respectively: 1: P1-A region 16P-Gnn 18K-NCU P3-AB region 1486H-YP3-C region 1696E-KN P3-C region 1745R-Knn 1849A-Tnn 2098P-L. By analyzing the viral genes in the literature, the change of the P3-D region in the P3-AB region is most likely to affect the virulence of the virus, and the virus exhibits a low replication phenotype. This conclusion needs to be verified by reverse genetics.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373
【參考文獻】
相關(guān)期刊論文 前1條
1 蘇琦華,訾自強,張霞,李曉眠,徐燕;柯薩奇B組3型、5型病毒在人臍靜脈血管內(nèi)皮細胞中持續(xù)感染模型的建立[J];中華微生物學(xué)和免疫學(xué)雜志;2004年10期
，本文編號：2145645
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