本文選題：狂犬病病毒 + M蛋白�。� 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：狂犬病病毒可引起中樞神經(jīng)系統(tǒng)發(fā)生病變,造成全球每年死亡人數(shù)高達(dá)70,000人,死亡率極高。目前沒有特效藥治療狂犬病,只能通過接種疫苗來預(yù)防狂犬病病毒的感染�？袢〔《臼菃喂韶�(fù)鏈不分節(jié)段的的RNA病毒,編碼N、P、M、G和L五個結(jié)構(gòu)蛋白,病毒RNA與N、P、L蛋白緊密結(jié)合在一起共同組成病毒的核糖核蛋白復(fù)合物(RNP),是病毒轉(zhuǎn)錄和復(fù)制的活性中心。本研究以廣西街毒株GX01株為研究對象,以固定弱毒株RC-HL為模板,研究GX01株M基因的生物學(xué)特性。在此前的研究中,我們發(fā)現(xiàn)GX01株M蛋白可以抑制病毒的轉(zhuǎn)錄和復(fù)制,并找到引起這種抑制作用的關(guān)鍵位點(diǎn)是M44和M46,當(dāng)對RC-HL的M蛋白進(jìn)行F44L或F44L/S46G聯(lián)合突變時,可顯著抑制病毒的轉(zhuǎn)錄及在細(xì)胞間的傳播性,但S46G的變異可增強(qiáng)病毒的轉(zhuǎn)錄過程。為闡述產(chǎn)生這種現(xiàn)象的機(jī)制,本實驗以rRC-HL(GX011M)為模板,將GX01株M蛋白44、46位氨基酸突變成RC-HL株相應(yīng)位點(diǎn)的氨基酸,得到重組病毒 rRC-HLM(L44F)、rRC-HLM(G46S)和 rRC-HLM(L44F/G46S)株,對其進(jìn)行生物學(xué)特性鑒定,檢測病毒的多步生長曲線、蛋白表達(dá)量以及mRNA水平,發(fā)現(xiàn)rRC-HLM(L44F)株的轉(zhuǎn)錄和M蛋白表達(dá)水平與親本株rRC-HL相近,病毒的生長繁殖得到恢復(fù);但拯救的rRC-HLM(G46S)株的轉(zhuǎn)錄和M蛋白表達(dá)水平稍低于rRC-HL(GXO11M)株,說明G46S突變可輕微抑制病毒的復(fù)制和轉(zhuǎn)錄;雙突變株rRC-HLM(L44F/G46S)的轉(zhuǎn)錄和M蛋白表達(dá)水平稍高于rRC-HL(GX01M)株,病毒的生長繁殖有所提高,這與之前本實驗小組得出的結(jié)論相符,結(jié)果可信。rRC-HL(F44L/S46G)株可顯著下調(diào)病毒的復(fù)制和轉(zhuǎn)錄水平,影響病毒的生長,推測與S46潛在磷酸化位點(diǎn)有關(guān),所以構(gòu)建并拯救去磷酸化突變株rRC-HL(F44L/S46A),模擬磷酸化的突變株 rRC-HL(F44L/S46D)和rRC-HL(F44L/S46E),同樣檢測拯救的突變病毒的生物學(xué)特性,發(fā)現(xiàn)突變病毒rRC-HL(F44L/S46D)和rRC-HL(F44L/S46E)株的病毒轉(zhuǎn)錄能力和M蛋白表達(dá)量與親本株rRC-HL無異,而rRC-HL(F44L/S46A)株在病毒感染的早期,轉(zhuǎn)錄水平與rRC-HL(GX01M)株相近,后期病毒生長繁殖能力提高,接近親本株rRC-HL的水平,并沒有出現(xiàn)與rRC-HL(F44L/S46G)株一樣的顯著下調(diào)病毒轉(zhuǎn)錄的現(xiàn)象,說明46位點(diǎn)的絲氨酸不是磷酸化位點(diǎn)。我們用拯救得到的所有毒株感染BSR/T7-9細(xì)胞,檢測RIG-I信號通路蛋白,發(fā)現(xiàn)狂犬病病毒M蛋白的表達(dá)量與TBK1表達(dá)水平之間存在負(fù)相關(guān)關(guān)系,隨著狂犬病病毒M蛋白量的增加TBK1的表達(dá)量下調(diào),反之亦然,從而抵抗細(xì)胞產(chǎn)生抗病毒免疫反應(yīng)。用ELISA方法檢測感染狂犬病病毒的細(xì)胞產(chǎn)生TNF-α、IL-1β、IFN-γ、IL-6和IL-12的情況。發(fā)現(xiàn)狂犬病病毒感染BSR/T7-9細(xì)胞之后,TNF-α和IL-1β水平?jīng)]有明顯上升。在病毒感染的早期,IL-12的表達(dá)量最高,發(fā)揮主要的抗病毒作用,而后期,IFN-γ、IL-6和IL-12的表達(dá)都增多,在抵抗病毒入侵的過程中,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- 偽, IL-1 尾, IFN- 緯, IL-6 and IL-12 in rabies virus infected cells. It was found that the levels of TNF- 偽 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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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