狂犬病病毒M蛋白与转录相关的功能性位点鉴定

发布时间：2018-06-26 12:33

本文选题：狂犬病病毒 + M蛋白　；参考：《广西大学》2017年硕士论文

【摘要】：狂犬病病毒可引起中枢神经系统发生病变,造成全球每年死亡人数高达70,000人,死亡率极高。目前没有特效药治疗狂犬病,只能通过接种疫苗来预防狂犬病病毒的感染。狂犬病病毒是单股负链不分节段的的RNA病毒,编码N、P、M、G和L五个结构蛋白,病毒RNA与N、P、L蛋白紧密结合在一起共同组成病毒的核糖核蛋白复合物(RNP),是病毒转录和复制的活性中心。本研究以广西街毒株GX01株为研究对象,以固定弱毒株RC-HL为模板,研究GX01株M基因的生物学特性。在此前的研究中,我们发现GX01株M蛋白可以抑制病毒的转录和复制,并找到引起这种抑制作用的关键位点是M44和M46,当对RC-HL的M蛋白进行F44L或F44L/S46G联合突变时,可显著抑制病毒的转录及在细胞间的传播性,但S46G的变异可增强病毒的转录过程。为阐述产生这种现象的机制,本实验以rRC-HL(GX011M)为模板,将GX01株M蛋白44、46位氨基酸突变成RC-HL株相应位点的氨基酸,得到重组病毒 rRC-HLM(L44F)、rRC-HLM(G46S)和 rRC-HLM(L44F/G46S)株,对其进行生物学特性鉴定,检测病毒的多步生长曲线、蛋白表达量以及mRNA水平,发现rRC-HLM(L44F)株的转录和M蛋白表达水平与亲本株rRC-HL相近,病毒的生长繁殖得到恢复;但拯救的rRC-HLM(G46S)株的转录和M蛋白表达水平稍低于rRC-HL(GXO11M)株,说明G46S突变可轻微抑制病毒的复制和转录;双突变株rRC-HLM(L44F/G46S)的转录和M蛋白表达水平稍高于rRC-HL(GX01M)株,病毒的生长繁殖有所提高,这与之前本实验小组得出的结论相符,结果可信。rRC-HL(F44L/S46G)株可显著下调病毒的复制和转录水平,影响病毒的生长,推测与S46潜在磷酸化位点有关,所以构建并拯救去磷酸化突变株rRC-HL(F44L/S46A),模拟磷酸化的突变株 rRC-HL(F44L/S46D)和rRC-HL(F44L/S46E),同样检测拯救的突变病毒的生物学特性,发现突变病毒rRC-HL(F44L/S46D)和rRC-HL(F44L/S46E)株的病毒转录能力和M蛋白表达量与亲本株rRC-HL无异,而rRC-HL(F44L/S46A)株在病毒感染的早期,转录水平与rRC-HL(GX01M)株相近,后期病毒生长繁殖能力提高,接近亲本株rRC-HL的水平,并没有出现与rRC-HL(F44L/S46G)株一样的显著下调病毒转录的现象,说明46位点的丝氨酸不是磷酸化位点。我们用拯救得到的所有毒株感染BSR/T7-9细胞,检测RIG-I信号通路蛋白,发现狂犬病病毒M蛋白的表达量与TBK1表达水平之间存在负相关关系,随着狂犬病病毒M蛋白量的增加TBK1的表达量下调,反之亦然,从而抵抗细胞产生抗病毒免疫反应。用ELISA方法检测感染狂犬病病毒的细胞产生TNF-α、IL-1β、IFN-γ、IL-6和IL-12的情况。发现狂犬病病毒感染BSR/T7-9细胞之后,TNF-α和IL-1β水平没有明显上升。在病毒感染的早期,IL-12的表达量最高,发挥主要的抗病毒作用,而后期,IFN-γ、IL-6和IL-12的表达都增多,在抵抗病毒入侵的过程中,三者都起到一定的抵抗作用。
[Abstract]:Rabies virus can cause diseases in the central nervous system, resulting in 70000 deaths per year and a very high mortality rate. At present, there is no specific drug to treat rabies, only through vaccination to prevent rabies virus infection. Rabies virus (RNP) is a single-stranded RNA virus with no segments. It encodes five structural proteins, namely, NP-PnMU G and L, and the ribonucleoprotein complex (RNP) of the virus is composed of viral RNA and NP-PnL protein together, which is the active center of viral transcription and replication. In this study, the biological characteristics of M gene of GX01 strain were studied by using the fixed attenuated strain RC-HL as the template. In previous studies, we found that the M protein of GX01 strain could inhibit the transcription and replication of the virus, and found that the key sites for this inhibition were M44 and M46, when F44L or F44L / S46G co-mutation of M protein in RC-HL was carried out. The transcriptional and intercellular transmissibility of the virus was significantly inhibited, but the S46G mutation enhanced the transcriptional process of the virus. In order to elucidate the mechanism of this phenomenon, rRC-HL (GX011M) was used as a template to mutate the 446 amino acids of M protein from GX01 strain into the corresponding amino acids of RC-HL strain. The recombinant viruses rRC-HLM (G46S) and rRC-HLM (L44FrG46S) were obtained and their biological characteristics were identified. The multistep growth curve, protein expression and mRNA level of rRC-HLM (L44F) strain were detected. The transcription and M protein expression levels of rRC-HLM (L44F) strain were similar to those of parent strain rRC-HL, and the growth and reproduction of rRC-HLM (L44F) strain were recovered. However, the transcription and M protein expression level of the rescued rRC-HLM (G46S) strain was slightly lower than that of rRC-HL (GXO11M) strain, indicating that G46S mutation could slightly inhibit the replication and transcription of the virus, while the transcription and M protein expression level of the double mutant rRC-HLM (L44F / G46S) strain was slightly higher than that of rRC-HL (GX01M) strain, and the growth and reproduction of the virus increased. This is consistent with the previous conclusions of our team. The results suggest that the strain of .rRC-HL (F44L / S46G) can significantly down-regulate the replication and transcription levels of the virus and affect the growth of the virus, presumably related to the potential phosphorylation site of S46. So we constructed and saved the dephosphorylation mutant rRC-HL (F44L / S46A), the mimic phosphorylation mutant rRC-HL (F44L / S46D) and rRC-HL (F44L / S46E), and we also tested the biological characteristics of the rescued mutant virus. It was found that the transcriptional ability and M protein expression of mutant virus rRC-HL (F44L / S46D) and rRC-HL (F44L / S46E) were similar to those of parent rRC-HL, while the transcription level of rRC-HL (F44L / S46A) strain was similar to that of rRC-HL (GX01M) strain at the early stage of infection. There was no significant down-regulation of viral transcription as in rRC-HL (F44L / S46G) strain, indicating that the serine at locus 46 was not phosphorylated. We infected BSR-T7-9 cells with all the strains we saved, detected RIG-I signaling pathway proteins, and found that there was a negative correlation between the expression of rabies virus M protein and the expression level of TBK1. With the increase of M protein of rabies virus, the expression of TBK1 was down-regulated, and vice versa, which prevented the cells from producing anti-virus immune response. Elisa was used to detect the production of TNF- 伪, IL-1 尾, IFN- 纬, IL-6 and IL-12 in rabies virus infected cells. It was found that the levels of TNF- 伪 and IL-1 尾 did not increase significantly after rabies virus infection in BSR-T7-9 cells. In the early stage of virus infection, the expression of IL-12 was the highest, which played a major role in antiviral activity, while the expression of IL-6 and IL-12 increased in the later stage of infection, which played a certain role in resisting virus invasion.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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