冷诱导RNA结合蛋白在H1N1流感病毒复制中的作用及其靶蛋白的筛选

发布时间：2018-05-21 17:24

本文选题：冷诱导RNA结合蛋白 + H1N1流感病毒　；参考：《西南民族大学》2015年硕士论文

【摘要】：冷诱导RNA结合蛋白(Cirbp)是在哺乳动物体内鉴定的首个与冷应激相关的蛋白,广泛表达于多种组织与细胞中,在脊椎动物间高度保守。在多种应激条件下Cirbp表达升高,并发挥细胞保护作用。此外,Cirbp在胚胎发育,神经发育,生物钟调节,肿瘤发生等过程中发挥重要的调节作用;近期研究表明Cirbp介导了多种病理条件下的炎症反应,如LPS刺激,脑缺血性休克,脓毒血症,酒精刺激等。本实验室前期研究结果表明,H1N1 PR-8流感病毒感染SPF小鼠后,各组织器官中Cirbp在mRNA和蛋白水平上都有不同程度的升高;过表达的Cirbp能通过上调NF-Kappa B信号通路显著促进H1N1甲型流感病毒的复制,但Cirbp对病毒复制过程的影响还不清楚。为了进一步探讨Cirbp在病毒复制过程中的作用及机制,筛选Cirbp相互作用的靶蛋白,我们进行了以下研究,主要取得了以下成果:1 H1N1流感病毒感染细胞后Cirbp表达升高并发生核质穿梭为了搞清楚Cirbp在H1N1感染的细胞内的表达规律,本试验将H1N1流感病毒以MOI=1分别接种三种细胞,其中BHK-21(叙利亚仓鼠肾细胞),和MH-S(小鼠肺泡巨噬细胞)接种人A/Puerto Rico/8/1934 H1N1,PUVEC(猪血管内皮细胞)接种猪A/Swine/GD/2/12 H1N1。分别于感染后0 h,4 h,8 h,16 h,24 h取样,用免疫组化法检测各细胞各时间点Cirbp的表达情况并用IPP6.0软件进行半定量分析。结果显示:感染PR-8 H1N1甲型流感病毒后8 h~24 h,BHK-21细胞中Cirbp的表达水平升高,达到了差异显著水平;感染PR-8 H1N1甲型流感后4 h~24 h,MH-S细胞中Cirbp的表达水平显著升高;感染猪H1N1流感病毒后8 h~24 h,PUVEC细胞中Cirbp的表达水平显著升高。为了探讨H1N1流感病毒感染下Cirbp在细胞内的亚定位,本试验将H1N1流感病毒以MOI=1感染BHK-21细胞,分别在感染后2 h,4 h,6 h,8 h对样品进行间接免疫荧光染色,并在激光共聚焦显微镜下观察结果。结果显示,H1N1流感病毒感染后2 h,Cirbp定位于细胞核与细胞浆中;感染后4 h Cirbp主要定位于细胞核中;感染后6 h Cirbp定位于细胞核与细胞浆中;感染后8 h,Cirbp主要定位于细胞核中。鉴于Cirbp在生理条件下存在于细胞核中,因此此结果表明在h1n1病毒感染条件下,cirbp可反复穿梭于细胞核和细胞浆之间。上述结果表明,h1n1流感病毒感染情况下,细胞中cirbp表达上调,并且发生了核质穿梭,为进一步研究cirbp在流感病毒复制过程中的最用奠定了基础。2敲低cirbp降低了h1n1流感病毒mrna的稳定性从而抑制病毒的复制为了证实cirbp对h1n1病毒mrna稳定性、rna合成以及蛋白合成的影响,本试验用real-timepcr方法检测了敲低cirbp的bhk21细胞(cirbp-bhk-21)感染h1n1病毒(moi=1)后病毒m和np基因mrna的半衰期,结果表明,敲低cirbp后h1n1病毒np基因和m基因mrna的相对半衰期分别约为7.5h和8h,对照组中m基因和np基因mrna的相对半衰期都超过了12h;同时用real-timepcr分析了h1n1流感病毒感染(moi=1)后3h,9h,15h,21h敲低cirbp对病毒m基因mrna合成的影响,结果显示,敲低cirbp显著(p0.05)抑制了h1n1病毒m基因mrna的合成,试验组分别是对照组的21.3%,18.6%,12.4%,7.6%,感染后15h和21h,其抑制作用达到了极显著(p0.01)水平;用real-timepcr分析了h1n1流感病毒感染(moi=1)后8h敲低cirbp对病毒m和np基因mrna,crna,vrna合成的影响,结果显示,敲低cirbp极显著(p0.01)抑制了病毒m基因crna和vrna1、vrna2的合成,试验组分别是对照组的16.9%、5.3%、11.3%;敲低cirbp极显著(p0.01)抑制了病毒np基因crna和vrna1、vrna2的合成,试验组分别是对照组的5.1%、2.2%、2.6%。进一步用间接免疫荧光技术分析了感染h1n1(moi=1)后3h和6h敲低cirbp对病毒np蛋白合成的影响,结果显示敲低cirbp抑制了病毒np蛋白的合成。最后在bhk-21和cirbp-bhk-21细胞上测定了h1n1病毒的tcid50效价。检测结果显示敲低cirbp后h1n1的tcid50效价显著高于正常bhk-21细胞的tcid50效价,试验组为对照组的57.54倍。上述结果表明,敲低cirbp降低了流感病毒np和m基因mrna的稳定性,进而抑制了病毒蛋白的合成,并影响了病毒crna和vrna的合成,最终抑制病毒的复制。分析上述结果表明敲低cirbp通过降低病毒mrna的稳定性抑制病毒的复制。本试验首次证实cirbp通过影响病毒mrna稳定性来影响病毒复制,丰富了机体因子与流感病毒互作的网络,为深入研究流感病毒的复制机理提供了资料。3过表达cirbp提高了h1n1病毒的复制效率cirbp在病毒感染情况下表达升高,为了探讨cirbp过表达对病毒复制的影响,本试验分别在bhk-21对照细胞和过表达cirbp细胞(cirbp+bhk-21细胞)上同时接种moi为0.001,0.01,2的h1n1病毒;分别在感染后3h,8h,16h,24h提取总rna,用荧光定量pcr检测h1n1m基因mrna的绝对表达量。结果显示,moi=0.001时,感染后3h,8h,16h,24h试验组m基因mrna的绝对表达量分别是对照组的1.28,30.78,79.05,26.89倍;moi=0.01时,试验组分别是对照组的0.85,26.95,83.84,47.88倍;moi=2时,试验组分别是对照组的4.07,6.46,5.01,3.98倍。结果表明过表达cirbp提高了h1n1病毒的复制效率,moi越低时,过表达cirbp使病毒复制效率的提高越明显。4过表达cirbp促进了h1n1病毒vrnp的出核转运cirbp在h1n1病毒感染情况下穿梭于细胞核和细胞浆之间,为了探讨cirbp在h1n1病毒转运过程中的作用,本试验利用间接免疫荧光技术研究了cirbp过表达对病毒粘附,穿入,入核转运和出核转运的影响。在研究cirbp过表达对病毒粘附和穿入时,以moi=50的感染量分别接种于试验组和对照组,以病毒感作后1h的时间点为0h,0h为病毒粘附和穿入的最佳时间,在此时间点固定样本,并进行间接免疫荧光染色,在激光共聚焦显微镜下观察,结果显示:感染后0h试验组和对照组细胞膜和细胞质内的病毒颗粒无明显差异,表明cirbp过表达对h1n1病毒的粘附和穿入无明显影响。研究对vrnp入核转运和出核转运时感染量为moi=1。分别在4h,6h,8h固定样本,并进行间接免疫荧光染色,在激光共聚焦显微镜下观察,结果显示:感染后4h,少量病毒vrnp已经由细胞浆进入细胞核中,且试验组和对照组无差异;感染后6h,病毒vrnp依然存在于细胞浆和细胞核中,且试验组和对照组无差异;感染后8h,试验组病毒vrnp完全由细胞核转运至细胞浆中,而对照组病毒vrnp正在从细胞核向细胞浆内转运,因此此结果表明过表达cirbp促进了病毒vrnp的出核转运。上述结果表明,过表达cirbp促进了病毒vrnp的出核转运,而对其它过程无明显影响。为深入研究流感病毒vrnp出核转运的机制提供了线索。5h1n1流感病毒感染条件下cirbp靶蛋白的筛选与初步验证为了筛选H1N1病毒感染情况下Cirbp的靶蛋白,本试验利用免疫共沉淀(Co-IP)结合质谱的方法,在病毒感染BHK-21细胞24h后,对Cirbp结合的靶蛋白进行了筛选,结果共沉淀下来了29种Cirbp的候选靶蛋白,其中27种为宿主靶蛋白,2种为病毒靶蛋白;Pathway注释结果显示候选的宿主靶蛋白主要参与了核酸代谢和蛋白质翻译信号通路,神经调节相关信号通路,感染与免疫相关的信号通路。27种候选靶蛋白中质谱得分前5的蛋白,分别为Hsp90,hnRNP R,D1Pas1,Ddx17,Cttn,质谱得分分别为1122.72,381.43,230.4,186.49,185.49。其中Hsp90和Ddx17与流感病毒的复制密切相关。Hsp90的质谱得分最高并在流感病毒复制过程中发挥重要的作用,因此我们进一步用间接免疫荧光验证Hsp90与Cirbp发生了共定位,表明Hsp90与Cirbp至少是以复合体的形式发生了结合。Hsp90与Cirbp是否直接物理结合还需要进一步的试验验证。此外,western-blot结果显示过表达Cirbp上调了Hsp90蛋白的表达,表明Cirbp对Hsp90具有调节作用。2种候选的病毒靶蛋白中,NP的质谱得分最高,为108.68。流感病毒NP蛋白在复制过程中发挥重要的作用,它与病毒RNA,其他病毒蛋白及宿主细胞的某些大分子相互作用,影响病毒的转录、复制、装配及转运功能。进一步用间接免疫荧光验证NP与Cirbp发生了共定位,表明NP与Cirbp至少以复合体的形式发生了结合。此结果为进一步研究Cirbp与H1N1互作的分子机制提供了线索。
[Abstract]:Cold induced RNA binding protein (Cirbp) is the first protein associated with cold stress in mammals. It is widely expressed in a variety of tissues and cells, highly conserved among vertebrates. The expression of Cirbp is elevated in a variety of stressful conditions and cell protection is played. In addition, Cirbp is in embryo development, neural development, clock regulation, and swelling. The recent study showed that Cirbp mediated the inflammatory response under various pathological conditions, such as LPS stimulation, cerebral ischemic shock, sepsis, and alcohol stimulation. The results of the early study in this laboratory showed that after the H1N1 PR-8 influenza virus was infected with SPF mice, Cirbp was in mRNA and protein water in various tissues and organs. The overexpressed Cirbp can promote the replication of H1N1 influenza A virus by up regulation of the NF-Kappa B signaling pathway, but the effect of Cirbp on the replication process of the virus is not clear. In order to further explore the role and mechanism of Cirbp in the replication process of the virus and to screen the target protein of Cirbp interaction, we enter the Cirbp. Following the following research, the following results were obtained: 1 H1N1 influenza virus infected cells increased Cirbp expression and occurred nuclear shuttle in order to understand the expression of Cirbp in H1N1 infected cells. This experiment inoculated the H1N1 influenza virus with three cells respectively, including BHK-21 (Syria hamster kidney cells), and MH-S (mouse alveolus). Macrophages were inoculated with A/Puerto Rico/8/1934 H1N1, and PUVEC (porcine vascular endothelial cells) inoculated swine A/Swine/GD/2/12 H1N1. were sampled at 0 h, 4 h, 8 h, 16 h, 24 h respectively. The expression of each cell at each time point was detected by immunohistochemistry and semi quantitative analysis was performed with the software. The expression level of Cirbp increased in 8 h~24 h after the virus, and the level of Cirbp was significantly different. The expression level of Cirbp in MH-S cells was significantly increased after the infection of PR-8 H1N1 A influenza A, and 8 of the infected pigs H1N1 influenza virus. In the subcellular localization, the H1N1 influenza virus was infected with MOI=1 in BHK-21 cells, 2 h, 4 h, 6 h, 8 h after infection, and the results were observed under the laser confocal microscope. The results showed that H1N1 influenza virus infection was 2 h after infection, Cirbp was located in the nucleus and cytoplasm; 4 h Cirbp was after infection. 6 h Cirbp was located in the nucleus and cytoplasm after infection; after infection, 8 h, Cirbp was located mainly in the nucleus. Since Cirbp exists in the nucleus under physiological conditions, this result indicates that cirbp can repeatedly shuttle between the nucleus and cytoplasm under the condition of H1N1 virus infection. The above results show that H1 In the case of N1 influenza virus infection, the expression of cirbp in the cells was up-regulated, and the nuclear shuttle was taken, which laid the foundation for the most use of cirbp in the replication process of influenza virus to reduce the stability of the H1N1 influenza virus mRNA and inhibit the replication of the virus, so as to verify the stability of cirbp against H1N1 virus mRNA, and to synthesize the RNA synthesis. The real-timepcr method was used to detect the half-life of M and NP gene mRNA after cirbp BHK21 cells (cirbp-bhk-21) infected with H1N1 virus (moi=1). The relative half-life was more than 12h, and the effect of 3H, 9h, 15h and 21h on mRNA synthesis of the virus M gene after H1N1 influenza virus infection (moi=1) was analyzed with real-timepcr. The results showed that the knock down cirbp significantly inhibited the synthesis of the virus, and the experimental group was 21.3%, 18.6%, 12.4%, 7.6% of the control group, respectively. And 21h, the inhibitory effect reached very significant (P0.01) level; the effect of 8h knockout cirbp on M and NP gene mRNA, cRNA, vRNA synthesis after H1N1 influenza virus infection (moi=1) was analyzed with real-timepcr. 5.3%, 11.3%; the knock low cirbp extremely significant (P0.01) inhibited the virus NP gene cRNA and vrna1, vrna2 synthesis, the test group was 5.1%, 2.2% of the control group, respectively, 2.6%. further used indirect immunofluorescence technique to analyze the effect of H1N1 (moi=1) 3H and 6h cirbp on the synthesis of egg white. The TCID50 titer of H1N1 virus was finally measured on BHK-21 and cirbp-bhk-21 cells. The test results showed that the TCID50 titer of H1N1 was significantly higher than the TCID50 titer of normal BHK-21 cells after the knock low cirbp, and the test group was 57.54 times as high as that of the control group. The results showed that the knock low cirbp reduced the stability of the influenza virus NP and the M gene. It inhibits the synthesis of viral proteins and affects the synthesis of virus cRNA and vRNA, and eventually inhibits the replication of the virus. Analysis of the above results shows that knocking low cirbp by reducing the stability of the virus mRNA inhibits the replication of the virus. It is the first time that cirbp has been proved to affect the replication of the virus by affecting the stability of the virus mRNA and enriching the body factor and flow. The network of virus interacting with each other provides information on the replication mechanism of influenza virus,.3 overexpression cirbp enhances the replication efficiency of H1N1 virus, cirbp increases in virus infection. In order to explore the effect of cirbp overexpression on virus replication, this experiment is in BHK-21 control cells and over expressed cirbp cells (cirbp+bhk-21), respectively. Cells were inoculated with H1N1 virus of 0.001,0.01,2 at the same time, and the total RNA was extracted from 3h, 8h, 16h, 24h after infection, and the absolute expression of h1n1m gene mRNA was detected by fluorescence quantitative PCR. The results showed that when moi=0.001, the absolute expression of MOI was twice as much as that of the control group; When the test group was 0.85,26.95,83.84,47.88 times of the control group, the test group was 4.07,6.46,5.01,3.98 times of the control group respectively. The results showed that the overexpression of cirbp increased the replication efficiency of the H1N1 virus, the lower the MOI, the more the overexpressed cirbp enhanced the replication efficiency of the virus, the more obvious the.4 overexpressed cirbp promoted the nucleation of H1N1 virus vrnp. In order to explore the role of cirbp in the process of H1N1 virus transport, the transshipment cirbp was shuttle between the nucleus and cytoplasm of H1N1 virus infection. The effect of cirbp overexpression on the adhesion, penetration, nuclear transfer and nuclear transfer of the virus was studied by indirect immunofluorescence. In the study of cirbp overexpression, the adhesion and penetration of the virus to the virus was studied. At the time of entry, the infection of moi=50 was inoculated in the experimental group and the control group respectively. The time point of 1h after the virus feeling was 0h, and the 0h was the best time for the adhesion and penetration of the virus. The samples were fixed at this time point, and the indirect immunofluorescence staining was carried out under the laser confocal microscope. The results showed that the cell membrane of the 0h test group and the control group after infection was revealed. There is no obvious difference between the virus particles in the cytoplasm, indicating that the overexpression of cirbp has no obvious effect on the adhesion and penetration of H1N1 virus. The infection amount of vrnp in nuclear transfer and nuclear transfer was moi=1. in 4h, 6h, 8h fixed samples, and the indirect immunofluorescence staining was carried out under the light confocal microscope. The results showed: 4h after infection. A small amount of virus vrnp has come into the nucleus from the cytoplasm, and there is no difference between the test group and the control group. After infection 6h, the virus vrnp still exists in the cytoplasm and nucleus, and there is no difference between the test group and the control group. After infection, the test group of virus vrnp is completely transferred from the nucleus to the cytoplasm, and the control group of the virus vrnp is from the nucleus to the nucleus. Intracellular transport, therefore, the results showed that overexpression of cirbp promoted the nuclear transfer of viral vrnp. The above results showed that overexpression of cirbp promoted the nuclear transfer of viral vrnp, but had no obvious effect on other processes. It provided a clue to the mechanism of.5h1n1 influenza virus infection in cirbp targets for further study of the mechanism of nucleation and transport of influenza virus vrnp. Screening and preliminary verification of protein in order to screen the target protein of Cirbp in H1N1 virus infection, this experiment uses immunoprecipitation (Co-IP) binding mass spectrometry. After the virus infected BHK-21 cell 24h, the target protein of Cirbp binding is screened. The results have precipitated 29 candidate target proteins of Cirbp, of which 27 are the host target eggs. The 2 species are virus target proteins; the Pathway annotation results show that the candidate host target proteins are mainly involved in nucleic acid metabolism and protein translation signal pathway, neuromodulation related signaling pathway, and the first 5 egg white of the candidate target proteins of the signal pathway related to the immune related signaling pathway, which are Hsp90, hnRNP R, D1Pas1, Ddx17, Cttn, mass spectrometry, respectively. The scores were 1122.72381.43230.4186.49185.49. and Hsp90 and Ddx17 were closely related to the replication of influenza virus and the.Hsp90 score was highest and played an important role in the replication process of influenza virus. Therefore, we further demonstrated that Hsp90 and Cirbp were Co located by indirect immunofluorescence, indicating that Hsp90 and Cirbp are at least the same. Further experimental verification was needed in the form of a combination of.Hsp90 and Cirbp directly. In addition, the Western-blot results showed that overexpression Cirbp increased the expression of Hsp90 protein, indicating that Cirbp has the highest score of the NP mass spectrum in the viral target protein of the.2 candidate for Hsp90, which is 108.68. influenza virus NP. Protein plays an important role in the process of replication. It interacts with the virus RNA, other viral proteins and some large molecules of the host cell. It affects the transcription, replication, assembly and transport functions of the virus. Further indirect immunofluorescence is used to verify the co localization of NP and Cirbp, indicating that NP and Cirbp are at least in the form of complex complexes. These results provide clues for further studying the molecular mechanism of Cirbp and H1N1 interactions.
【学位授予单位】：西南民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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