狂犬病毒糖蛋白膜外区调控树突状细胞激活的研究
发布时间：2018-08-24 09:02
【摘要】：狂犬病(rabies)是由狂犬病毒(Rabiesvirus,RABV)引起的损伤中枢神经系统的人兽共患传染病,一旦发病后死亡率几近100%。据统计,全世界每年有不少于59000人死于狂犬病,我国是狂犬病高发的国家之一。RABV为单股负链RNA病毒,基因组全长约12kb,其基因组共编码五个结构蛋白,按病毒RNA 3'→5'方向分别是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,是病毒进入机体后引起先天性免疫应答的最主要抗原。据报道,狂犬病毒激活DC是与其G蛋白特异相关的。狂犬病毒固定毒株能引起高水平的DC激活,从而激起机体免疫应答,产生大量的中和抗体;而街毒株狂犬病毒不能激活DC,不诱导机体产生中和抗体,从而逃逸机体先天性免疫应答。本实验室之前的研究表明,狂犬病毒街毒株G蛋白结合和进入DC的效率较低,不易引起DC激活,从而使病毒逃逸先天性免疫应答。因此,街毒株的免疫逃逸是由病毒的G蛋白决定的。但是G蛋白上调控这种逃逸机制的结构域或关键位点并不清楚。另外,大量研究表明狂犬病毒街毒株G蛋白的表达量比固定毒株G蛋白的表达量显著降低。然而狂犬病毒街毒株G蛋白介导的免疫逃逸是否与G蛋白的表达量有关尚未得到验证。本研究以固定毒株B2c和街毒株SHBRV-18为研究对象,在B2c反向遗传操作平台的基础上,将B2cG蛋白的各功能结构域(包括信号肽、膜外区、跨膜域和膜内区)分别替换为SHBRV的对应序列,拯救出嵌合各功能域的重组病毒。通过测定各病毒的生长动力学曲线和病毒感染细胞后的荧光斑大小,发现B2cG蛋白的信号肽、跨膜区、膜内区突变为SHBRV对应结构域的重组病毒感染细胞后在细胞间的扩增能力及最高滴度没有显著改变;而B2c G蛋白全长序列或者G蛋白膜外区突变为SHBRV对应结构域的重组病毒感染细胞后在细胞间的扩增能力及最高滴度均显著降低。因此,狂犬病毒感染细胞和在细胞上增殖能力是由G蛋白的膜外区决定。为了研究G蛋白表达水平调控区域,将各个重组的狂犬病毒感染细胞后,对重组病毒在细胞内或者细胞膜上的表达水平进行定量分析。western blot结果表明,将B2c G蛋白的膜内区突变为SHBRV的对应序列后,获得的重组病毒(rB2c/SHB-Gct)在细胞内G蛋白表达水平显著降低。同时,流式细胞术和激光共聚焦的结果也表明该重组病毒在细胞膜表面的量与SHBRV相似,均显著低于B2c。因此,狂犬病毒G蛋白的表达水平是由G蛋白自身的膜内区来调控的。同时利用流式细胞术对各重组病毒激活DC的水平进行统计分析,发现B2c G蛋白膜外区替换为SHBRV对应区域之后,得到的重组病毒(rB2c/SHB-Get)丧失了激活DC的能力。G蛋白表达量显著降低的重组病毒rB2c/SHB-Gct激活DC的水平有一定程度的下调,但与rB2c相比,无显著差异。为进一步研究膜外区调控DC激活的机制,对重组病毒吸附和进入DC的能力进行研究发现,当B2c G蛋白的膜外区突变之后,病毒吸附和进入DC的能力显著降低。因此,狂犬病毒G蛋白膜外区决定了病毒感染DC的能力,从而调控DC的激活。综上所述,我们的研究结果阐明了狂犬病毒G蛋白的膜外区决定了病毒吸附和进入到DC内的能力和DC激活的水平。因此,狂犬病毒激活DC的水平主要依赖于G蛋白膜外区介导病毒感染DC的能力,而不是由G蛋白的表达量来决定。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65
【参考文献】
相关期刊论文 前1条
1 Shani Gluska;Stefan Finke;Eran Perlson;;Receptor-mediated increase in rabies virus axonal transport[J];Neural Regeneration Research;2015年06期
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