流感病毒聚合酶陷阱系统抑制流感病毒复制的研究

发布时间：2018-06-22 08:55

本文选题：流感病毒 + RNA聚合酶陷阱系统　；参考：《中国农业大学》2015年博士论文

【摘要】：流感病毒是引起动物和人呼吸道疾病的重要病原体,对畜牧生产和公共卫生安全构成了巨大的威胁。控制流感病毒在畜禽中的感染和传播,对畜牧生产和人类健康具有重要意义。随着动物转基因技术的迅速发展,通过基因修饰的方法培育抗流感病毒的畜禽新品种,为在畜牧业中有效防治流感疫情提供了新的方法。流感病毒基因组末端具有保守的5'和3'非翻译区,特别是5'非翻译区最末端的13个碱基在流感病毒基因组所有片段中全部相同,3'非翻译区最末端的12个碱基只有一个位置的碱基会发生变化。保守的非翻译区包含有流感病毒RNA复制和转录的顺式作用元件,能够被流感病毒聚合酶识别,在病毒的复制、转录和包装中起到重要作用。本研究设计了一个流感病毒聚合酶陷阱系统(Influenza A Virus RNA Polymerase Decoy System, IVPDS),该系统载体能够表达类似于流感病毒RNA (viral RNA like, vRNA-like)序列,该序列两侧带有流感病毒基因组5'和3'非翻译区。vRNA-like序列竞争性结合流感病毒的聚合酶,一方面抑制聚合酶对流感病毒的复制,另一方面vRNA-like序列在聚合酶的作用下进行复制和转录,表达外源蛋白,激活宿主的免疫反应,增加宿主对病毒的抗性。在本研究中,构建了由RNA聚合酶Ⅰ启动子转录的表达GFP的IVPDS报告载体,pIVPDS-I-G。瞬时转染pIVPDS-I-G的细胞或稳定整合pIVPDS-I-G-Neo的细胞(简称IVPDS-G细胞)都能够转录出含有GFP的类似于流感病毒的序列vRNA-GFP(-)。在瞬转pIVPDS-I-G或稳定整合的IVPDS-G细胞中转染病毒聚合酶和NP表达载体,检测到了 mRNA-GFP(+)以及GFP蛋白的表达。流感病毒感染上述细胞后可以得到相似的结果,但与转染病毒聚合酶和NP表达载体相比,细胞感染流感病毒后诱导表达的mRNA-GFP(+)以及GFP蛋白的表达量显著降低。在转染pIVPDS-I-G的DF-1细胞中,共转流感病毒聚合酶和NP表达载体能提高I型干扰素以及I型干扰素下游基因mRNA水平,说明IVPDS载体被诱导后产生的双链RNA能激活Ⅰ型干扰素通路。为了分析pIVPDS-I-G对流感病毒复制的影响,检测了感染流感病毒的细胞内病毒mRNA的表达量和细胞培养液中的病毒滴度。与空白对照组相比,瞬转pIVPDS-I-G的细胞或IVPDS-G细胞内流感病毒Martix基因mRNA水平均显著降低,但是胞外上清中病毒滴度没有显著差异。此外,本研究中还构建了表达人MxA和小鼠Mx (总称Mx)的IVPDS载体,pIVPDS-I-Mx-Neo。在 293T 和 DF-1 细胞瞬时转染 pIVPDS-I-Mx-Neo 能够转录 vRNA-Mx(-),共转染pIVPDS-I-Mx-Neo、流感病毒聚合酶和NP蛋白质粒时,检测到细胞中mRNA-Mx(+)和Mx蛋白质的表达。在瞬时转染pIVPDS-I-Mx-Neo的DF-1细胞感染流感病毒后,检测细胞上清中病毒滴度,结果显示在24 hpi和36 hpi转染pIVPDS-I-MxA-Neo的细胞上清中的病毒滴度显著低于空载体对照组,转染pIVPDS-I-Mx1-Neo的细胞上清中的病毒滴度与空载体对照组相比并没有显著的差异。综上所述,本研究成功构建了 IVPDS,该系统载体表达的vRNA-like序列能够被流感病毒聚合酶识别进行转录和翻译,达到了诱导性表达外源基因的目的,转染pIVPDS-I-MxA-Neo的细胞对流感病毒产生了一定抗性。本研究验证了流感病毒聚合酶陷阱系统抑制流感病毒复制策略的可行性,为下一步的研究和应用该系统进行抗流感病毒育种奠定基础并提供了新的思路。
[Abstract]:Influenza virus is an important pathogen causing animal and human respiratory diseases, which poses a great threat to animal production and public health safety. Controlling infection and transmission of influenza virus in livestock and poultry is of great significance to animal production and human health. With the rapid development of transgenic techniques in animals, the gene modified Fang Fapei has been developed. A new breed of livestock and poultry resistant to influenza virus provides a new method for effective prevention and control of influenza in animal husbandry. The end of the genome of the influenza virus has a conservative 5'and 3' non translation area, especially the 13 bases at the most end of the 5'non translation region in all segments of the genome of the influenza virus genome, and the most terminal 12 base of the non translated region of the 3' The base of only one location changes. The conservative non translation area contains the cis acting element with the replication and transcription of influenza virus RNA, which can be identified by influenza virus polymerase and plays an important role in the replication, transcription and packaging of the virus. A influenza virus polymerase trap system (Influenza A Virus RNA P) is designed in this study. Olymerase Decoy System, IVPDS), the system vector can express the sequence of influenza virus RNA (viral RNA like, vRNA-like), which combines the polymerase of influenza virus genome 5'and 3' untranslated region.VRNA-like sequences, on the one hand inhibit the replication of the polymerase to influenza virus, on the other hand, -like sequences replicate and transcribe under the action of polymerase, express foreign proteins, activate the host immune response, and increase host resistance to the virus. In this study, a IVPDS reporter vector expressing GFP, a transcription of RNA polymerase I promoter, was constructed, pIVPDS-I-G. was transiently transfected to pIVPDS-I-G cells or stable integrated pIVPDS-I-G-Neo The cells (IVPDS-G cells) can transcribe the sequence vRNA-GFP (-) containing GFP, which are similar to influenza viruses. Virus polymerase and NP expression vectors are transfected into pIVPDS-I-G or stable IVPDS-G cells. The expression of mRNA-GFP (+) and GFP protein is detected. Influenza virus infection can be similar to those of the above cells. However, compared with the transfected virus polymerase and NP expression vector, the expression of mRNA-GFP (+) and GFP protein in the infected cells infected with influenza virus decreased significantly. In the DF-1 cells transfected with pIVPDS-I-G, the influenza virus polymerase and NP expression vector could improve the mRNA level of the I type interferon and the downstream gene of the type I interferon. In order to analyze the effect of pIVPDS-I-G on the replication of influenza virus, the expression of intracellular virus mRNA and the titer of virus in cell culture fluid were detected by the double stranded RNA induced by the induced IVPDS vector. In contrast to the blank control group, the transient pIVPDS-I-G cell or the intracellular flow of IVPDS-G cells was compared with that of the blank control group. The mRNA level of the Martix gene of the virus was significantly decreased, but there was no significant difference in the titer of the virus in the supernatant. In addition, the IVPDS vector expressing human MxA and mouse Mx (general Mx) was also constructed. The transient transfection of pIVPDS-I-Mx-Neo to pIVPDS-I-Mx-Neo. in 293T and DF-1 cells could transcribe vRNA-Mx (-) and co transfect pIVPDS-I-Mx-Neo, The expression of mRNA-Mx (+) and Mx protein in cells was detected when influenza virus polymerase and NP protein particles were detected. The virus titer in cell supernatant was detected after infected with influenza virus from DF-1 cells transfected instantaneously, and the results showed that the titer of the cell supernatant of 24 HPI and 36 HPI transfected pIVPDS-I-MxA-Neo was significantly lower than that of the empty body. In the control group, the virus titer in the cell supernatant transfected with pIVPDS-I-Mx1-Neo had no significant difference compared with the empty vector control group. In summary, the study successfully constructed the IVPDS. The vRNA-like sequence expressed by the system can be transcribed and translated by influenza virus polymerase, and the inducible expression of foreign genes is achieved. Objective: the transfected pIVPDS-I-MxA-Neo cells have a certain resistance to influenza virus. This study verified the feasibility of influenza virus polymerase trap system to inhibit the replication strategy of influenza virus, and provided a new idea for the next research and application of the system for the breeding of influenza virus.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

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