狂犬病毒糖蛋白膜外區(qū)調(diào)控樹突狀細(xì)胞激活的研究
發(fā)布時(shí)間：2018-08-24 09:02
【摘要】：狂犬病(rabies)是由狂犬病毒(Rabiesvirus,RABV)引起的損傷中樞神經(jīng)系統(tǒng)的人獸共患傳染病,一旦發(fā)病后死亡率幾近100%。據(jù)統(tǒng)計(jì),全世界每年有不少于59000人死于狂犬病,我國(guó)是狂犬病高發(fā)的國(guó)家之一。RABV為單股負(fù)鏈RNA病毒,基因組全長(zhǎng)約12kb,其基因組共編碼五個(gè)結(jié)構(gòu)蛋白,按病毒RNA 3'→5'方向分別是N、P、M、G和L蛋白,其中G蛋白是唯一暴露在狂犬病毒表面的囊膜蛋白,是病毒進(jìn)入機(jī)體后引起先天性免疫應(yīng)答的最主要抗原。據(jù)報(bào)道,狂犬病毒激活DC是與其G蛋白特異相關(guān)的�？袢《竟潭ǘ局昴芤鸶咚降腄C激活,從而激起機(jī)體免疫應(yīng)答,產(chǎn)生大量的中和抗體;而街毒株狂犬病毒不能激活DC,不誘導(dǎo)機(jī)體產(chǎn)生中和抗體,從而逃逸機(jī)體先天性免疫應(yīng)答。本實(shí)驗(yàn)室之前的研究表明,狂犬病毒街毒株G蛋白結(jié)合和進(jìn)入DC的效率較低,不易引起DC激活,從而使病毒逃逸先天性免疫應(yīng)答。因此,街毒株的免疫逃逸是由病毒的G蛋白決定的。但是G蛋白上調(diào)控這種逃逸機(jī)制的結(jié)構(gòu)域或關(guān)鍵位點(diǎn)并不清楚。另外,大量研究表明狂犬病毒街毒株G蛋白的表達(dá)量比固定毒株G蛋白的表達(dá)量顯著降低。然而狂犬病毒街毒株G蛋白介導(dǎo)的免疫逃逸是否與G蛋白的表達(dá)量有關(guān)尚未得到驗(yàn)證。本研究以固定毒株B2c和街毒株SHBRV-18為研究對(duì)象,在B2c反向遺傳操作平臺(tái)的基礎(chǔ)上,將B2cG蛋白的各功能結(jié)構(gòu)域(包括信號(hào)肽、膜外區(qū)、跨膜域和膜內(nèi)區(qū))分別替換為SHBRV的對(duì)應(yīng)序列,拯救出嵌合各功能域的重組病毒。通過測(cè)定各病毒的生長(zhǎng)動(dòng)力學(xué)曲線和病毒感染細(xì)胞后的熒光斑大小,發(fā)現(xiàn)B2cG蛋白的信號(hào)肽、跨膜區(qū)、膜內(nèi)區(qū)突變?yōu)镾HBRV對(duì)應(yīng)結(jié)構(gòu)域的重組病毒感染細(xì)胞后在細(xì)胞間的擴(kuò)增能力及最高滴度沒有顯著改變;而B2c G蛋白全長(zhǎng)序列或者G蛋白膜外區(qū)突變?yōu)镾HBRV對(duì)應(yīng)結(jié)構(gòu)域的重組病毒感染細(xì)胞后在細(xì)胞間的擴(kuò)增能力及最高滴度均顯著降低。因此,狂犬病毒感染細(xì)胞和在細(xì)胞上增殖能力是由G蛋白的膜外區(qū)決定。為了研究G蛋白表達(dá)水平調(diào)控區(qū)域,將各個(gè)重組的狂犬病毒感染細(xì)胞后,對(duì)重組病毒在細(xì)胞內(nèi)或者細(xì)胞膜上的表達(dá)水平進(jìn)行定量分析。western blot結(jié)果表明,將B2c G蛋白的膜內(nèi)區(qū)突變?yōu)镾HBRV的對(duì)應(yīng)序列后,獲得的重組病毒(rB2c/SHB-Gct)在細(xì)胞內(nèi)G蛋白表達(dá)水平顯著降低。同時(shí),流式細(xì)胞術(shù)和激光共聚焦的結(jié)果也表明該重組病毒在細(xì)胞膜表面的量與SHBRV相似,均顯著低于B2c。因此,狂犬病毒G蛋白的表達(dá)水平是由G蛋白自身的膜內(nèi)區(qū)來調(diào)控的。同時(shí)利用流式細(xì)胞術(shù)對(duì)各重組病毒激活DC的水平進(jìn)行統(tǒng)計(jì)分析,發(fā)現(xiàn)B2c G蛋白膜外區(qū)替換為SHBRV對(duì)應(yīng)區(qū)域之后,得到的重組病毒(rB2c/SHB-Get)喪失了激活DC的能力。G蛋白表達(dá)量顯著降低的重組病毒rB2c/SHB-Gct激活DC的水平有一定程度的下調(diào),但與rB2c相比,無顯著差異。為進(jìn)一步研究膜外區(qū)調(diào)控DC激活的機(jī)制,對(duì)重組病毒吸附和進(jìn)入DC的能力進(jìn)行研究發(fā)現(xiàn),當(dāng)B2c G蛋白的膜外區(qū)突變之后,病毒吸附和進(jìn)入DC的能力顯著降低。因此,狂犬病毒G蛋白膜外區(qū)決定了病毒感染DC的能力,從而調(diào)控DC的激活。綜上所述,我們的研究結(jié)果闡明了狂犬病毒G蛋白的膜外區(qū)決定了病毒吸附和進(jìn)入到DC內(nèi)的能力和DC激活的水平。因此,狂犬病毒激活DC的水平主要依賴于G蛋白膜外區(qū)介導(dǎo)病毒感染DC的能力,而不是由G蛋白的表達(dá)量來決定。
[Abstract]:Rabies is a zoonotic disease caused by rabies virus (RABV) that damages the central nervous system. The mortality rate of rabies is nearly 100%. According to statistics, there are not less than 59 000 people died of rabies every year in the world. China is one of the countries with high incidence of rabies. RABV is a single strand negative strand RNA virus with a genome length of about 12 kb. The genome encodes five structural proteins, N, P, M, G and L, respectively, in the direction of virus RNA 3'5'. G protein is the only envelope protein that is exposed to rabies virus and the most important antigen that causes innate immune response after the virus enters the body. It is reported that rabies virus-activated DC is specifically related to its G protein. Fixed strains of rabies virus can induce high levels of DC activation, thus stimulating immune response and producing a large number of neutralizing antibodies; Street strain of rabies virus can not activate DC, and does not induce neutralizing antibodies to escape the innate immune response of the body. Previous studies in our laboratory have shown that the G protein of Street strain of rabies virus binds to and enters DC. Therefore, the immune escape of Street Virus strains is determined by the G protein of the virus. However, the domain or key site of G protein regulating this escape mechanism is not clear. In addition, a large number of studies have shown that the expression of G protein of Street Virus strains is more than that of fixed virus. The expression of G protein in Street Virus strain B 2 C and Street Virus strain SHBRV-18 was studied. The functional domains (including signal peptides) of B2cG protein were identified on the basis of the reverse genetic manipulation platform of B2c. By measuring the growth kinetics curves of the viruses and the fluorescence spot size of the infected cells, it was found that the signal peptide of B2cG protein, transmembrane region and intramembrane region mutated into the corresponding domain of SHBRV. There was no significant change in the cell-to-cell amplification ability and the highest titer after infection; however, the cell-to-cell amplification ability and the highest titer of the recombinant virus infected with the full-length sequence of B2c G protein or the outer-membrane region of G protein mutated into the corresponding domain of SHBRV were significantly decreased. In order to study the regulatory region of G protein expression, the expression levels of recombinant rabies virus in cells or cell membranes were quantitatively analyzed. Western blot results showed that the intramembrane region of B2c G protein was mutated into the corresponding sequence of SHBRV. The expression level of G protein in cells was significantly decreased by rB2c/SHB-Gct. The results of flow cytometry and confocal laser scanning also showed that the amount of the recombinant virus on the surface of cell membrane was similar to that of SHBRV and significantly lower than that of B2c. Cytological analysis showed that the recombinant virus (rB2c/SHB-Get) lost the ability to activate DC after replacing the outer membrane region of the B2c G protein with the corresponding SHBRV region. The expression of G protein in the DCs activated by the recombinant virus rB2c/SHB-Gct was down-regulated to a certain extent, but not compared with rB2c. In order to further study the mechanism of extramembrane region regulating DC activation, the ability of recombinant virus to adsorb and enter DC was studied. It was found that the ability of virus to adsorb and enter DC was significantly reduced after the extramembrane region of B2c G protein was mutated. In summary, our results clarify that the extramembrane region of rabies virus G protein determines the ability of the virus to adsorb and enter DC and the level of DC activation.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65
本文編號(hào)：2200297
[Abstract]:Rabies is a zoonotic disease caused by rabies virus (RABV) that damages the central nervous system. The mortality rate of rabies is nearly 100%. According to statistics, there are not less than 59 000 people died of rabies every year in the world. China is one of the countries with high incidence of rabies. RABV is a single strand negative strand RNA virus with a genome length of about 12 kb. The genome encodes five structural proteins, N, P, M, G and L, respectively, in the direction of virus RNA 3'5'. G protein is the only envelope protein that is exposed to rabies virus and the most important antigen that causes innate immune response after the virus enters the body. It is reported that rabies virus-activated DC is specifically related to its G protein. Fixed strains of rabies virus can induce high levels of DC activation, thus stimulating immune response and producing a large number of neutralizing antibodies; Street strain of rabies virus can not activate DC, and does not induce neutralizing antibodies to escape the innate immune response of the body. Previous studies in our laboratory have shown that the G protein of Street strain of rabies virus binds to and enters DC. Therefore, the immune escape of Street Virus strains is determined by the G protein of the virus. However, the domain or key site of G protein regulating this escape mechanism is not clear. In addition, a large number of studies have shown that the expression of G protein of Street Virus strains is more than that of fixed virus. The expression of G protein in Street Virus strain B 2 C and Street Virus strain SHBRV-18 was studied. The functional domains (including signal peptides) of B2cG protein were identified on the basis of the reverse genetic manipulation platform of B2c. By measuring the growth kinetics curves of the viruses and the fluorescence spot size of the infected cells, it was found that the signal peptide of B2cG protein, transmembrane region and intramembrane region mutated into the corresponding domain of SHBRV. There was no significant change in the cell-to-cell amplification ability and the highest titer after infection; however, the cell-to-cell amplification ability and the highest titer of the recombinant virus infected with the full-length sequence of B2c G protein or the outer-membrane region of G protein mutated into the corresponding domain of SHBRV were significantly decreased. In order to study the regulatory region of G protein expression, the expression levels of recombinant rabies virus in cells or cell membranes were quantitatively analyzed. Western blot results showed that the intramembrane region of B2c G protein was mutated into the corresponding sequence of SHBRV. The expression level of G protein in cells was significantly decreased by rB2c/SHB-Gct. The results of flow cytometry and confocal laser scanning also showed that the amount of the recombinant virus on the surface of cell membrane was similar to that of SHBRV and significantly lower than that of B2c. Cytological analysis showed that the recombinant virus (rB2c/SHB-Get) lost the ability to activate DC after replacing the outer membrane region of the B2c G protein with the corresponding SHBRV region. The expression of G protein in the DCs activated by the recombinant virus rB2c/SHB-Gct was down-regulated to a certain extent, but not compared with rB2c. In order to further study the mechanism of extramembrane region regulating DC activation, the ability of recombinant virus to adsorb and enter DC was studied. It was found that the ability of virus to adsorb and enter DC was significantly reduced after the extramembrane region of B2c G protein was mutated. In summary, our results clarify that the extramembrane region of rabies virus G protein determines the ability of the virus to adsorb and enter DC and the level of DC activation.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Shani Gluska;Stefan Finke;Eran Perlson;;Receptor-mediated increase in rabies virus axonal transport[J];Neural Regeneration Research;2015年06期
，本文編號(hào)：2200297
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