表达IL-21重组狂犬病病毒免疫原性研究
发布时间：2019-05-10 08:16
【摘要】：狂犬病(Rabies)是由狂犬病病毒(Rabies virus,RABV)引起的一种人兽共患传染病,每年约有59000人死于狂犬病,我国是狂犬病高发国家之一。狂犬病一旦发病其致死率几乎为100%,疫苗接种是预防狂犬病的最有效方法。狂犬病疫苗在免疫后可以通过T细胞依赖和T细胞非依赖的途径激活B细胞免疫应答,从而激活机体产生中和抗体,预防狂犬病的发生。前期研究表明,白介素-21(Interleukin-21,IL-21)信号通路缺失后对RABV免疫后的抗体诱导水平产生影响,提示IL-21在RABV的免疫原性中起到重要作用。因此,为了进一步探索IL-21在RABV的免疫原性中的作用机制,本研究拟通过反向遗传操作构建表达IL-21的重组RABV,并利用体外和体内一系列实验对该重组RABV进行研究:首先,通过反向遗传学操作系统拯救表达鼠源IL-21重组RABV,命名为LBNSE-IL21,通过ELISA检测IL-21的体外表达情况,结果表明LBNSE-IL21感染BSR细胞可以表达IL-21且具有剂量依赖性;此外,LBNSE-IL21在BSR细胞和NA细胞的生长曲线与亲本毒株LBNSE几乎一致,表明外源基因IL-21的插入对病毒的增殖没有影响;在病毒致病性方面,将LBNSE-IL21脑内注射小鼠后,小鼠无明显临床症状,且体重基本保持上升趋势,与亲本毒株LBNSE体重变化曲线相似,表明IL-21的表达对致病性没有显著影响。进一步研究IL-21的表达对免疫原性的影响机制,LBNSE-IL21和LBNSE感染Balb/c小鼠,利用流式细胞术检测小鼠体液免疫应答中各类免疫细胞的变化。结果发现与LBNSE相比,LBNSE-IL21不能激活淋巴结或外周血液中的树突状细胞(dendritic cell,DC),但可以显著提高淋巴结中滤泡性辅助细胞(follicular helper T cells,Tfh)细胞和生发中心B细胞(germinal center B,GC B)数量,表明LBNSE-IL21免疫后表达的IL-21可以促进Tfh细胞和GC B细胞的生成,影响生发中心的发育。进一步研究发现,LBNSE-IL21可以促进B细胞向浆细胞(Plasma cells,PC)的分化,在骨髓产生更多数量的长寿命浆细胞(Long-lasting Plasma cells),从而诱导产生高水平的中和抗体。最后,为了探究LBNSE-IL21作为疫苗的免疫效果,将LBNSE-IL21和LBNSE免疫ICR小鼠,采集免疫后第1-7周的小鼠血清,利用FAVN测定中和抗体效价。检测结果显示LBNSE-IL21在免疫后第1周便快速诱导机体产生中和抗体,并且第1-6周的抗体水平均显著高于亲本毒株LBNSE。这表明LBNSE-IL21免疫后可以更快诱导机体产生高水平的中和抗体。此外,在免疫后第三周,利用脑内注射50 MICLD_(50)的CVS-24对小鼠进行攻毒实验,最终LBNSE-IL21免疫后可提供高达92%的保护率,显著高于亲本病毒免疫保护率68%。综上所述,LBNSE-IL21免疫后可以通过促进Tfh、GC B细胞,以及浆细胞的产生,从而提高中和抗体的产生水平,提高免疫保护。因此,IL-21可作为新型狂犬病疫苗的候选者。
[Abstract]:Rabies (Rabies) is a zoonotic infectious disease caused by rabies virus (Rabies virus,RABV). About 59000 people die of rabies every year. China is one of the countries with high incidence of rabies. Once rabies occurs, the mortality rate is almost 100%. Vaccination is the most effective method to prevent rabies. After immunization, rabies vaccine can activate B cell immune response through T cell dependent and T cell independent pathway, so as to activate the body to produce neutralizing antibody and prevent the occurrence of rabies. Previous studies have shown that the loss of interleukin-21 (Interleukin-21,IL-21) signaling pathway affects the level of antibody induction after RABV immunization, suggesting that IL-21 plays an important role in the immunogenicity of RABV. Therefore, in order to further explore the mechanism of IL-21 in the immunogenicity of RABV, the recombinant RABV, expressing IL-21 was constructed by reverse genetic manipulation and the recombinant RABV was studied by a series of experiments in vitro and in vivo. The recombinant mouse IL-21 RABV, was named LBNSE-IL21, by reverse genetic operating system. The results showed that LBNSE-IL21 infected BSR cells could express IL-21 in a dose-dependent manner, and the expression of IL-21 in vitro was detected by ELISA. In addition, the growth curve of LBNSE-IL21 in BSR cells and NA cells was almost the same as that of the parent strain LBNSE, indicating that the insertion of exogenous gene IL-21 had no effect on the proliferation of the virus. In terms of virus pathogenicity, after intracerebral injection of LBNSE-IL21 into the brain of mice, there were no obvious clinical symptoms, and the body weight of mice basically maintained an upward trend, which was similar to that of parent strain LBNSE, indicating that the expression of IL-21 had no significant effect on pathogenicity. The mechanism of the effect of IL-21 expression on immunogenicity was further studied. Balb/c mice were infected with LBNSE-IL21 and LBNSE. The changes of various immune cells in humoral immune response of mice were detected by flow cytometry. The results showed that compared with LBNSE, LBNSE-IL21 could not activate dendritic cells (dendritic cell,DC) in lymph nodes or peripheral blood, but could significantly increase the (follicular helper T cells, of follicular helper cells in lymph nodes. The number of (germinal center B and GC B cells in Tfh) indicated that IL-21 expressed after LBNSE-IL21 immunization could promote the production of Tfh cells and GC B cells and affect the development of germatogenic centers. Further studies showed that LBNSE-IL21 could promote the differentiation of B cells into plasma cells (Plasma cells,PC) and produce more long-lived plasma cells (Long-lasting Plasma cells),) in bone marrow, thus inducing the production of high levels of neutralizing antibodies. Finally, in order to explore the immune effect of LBNSE-IL21 as vaccine, ICR mice were immunized with LBNSE-IL21 and LBNSE. The serum of ICR mice was collected from the 1st to 7th week after immunization, and the neutralizing antibody titer was determined by FAVN. The results showed that LBNSE-IL21 rapidly induced the production of neutralizing antibody at the first week after immunization, and the antibody level at the 1st to 6th week was significantly higher than that of the parent strain LBNSE.. This suggests that LBNSE-IL21 immunization can induce the body to produce a high level of neutralizing antibody more quickly. In addition, in the third week after immunization, the mice were challenged with 50 MICLD_ (50) CVS-24 into the brain, and the protective rate of LBNSE-IL21 was up to 92% after immunization, which was significantly higher than that of the parent virus (68%). In conclusion, LBNSE-IL21 immunization can promote the production of Tfh,GC B cells and plasma cells, so as to improve the production level of neutralizing antibody and improve immune protection. Therefore, IL-21 can be used as a candidate for a new rabies vaccine.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：2473492
[Abstract]:Rabies (Rabies) is a zoonotic infectious disease caused by rabies virus (Rabies virus,RABV). About 59000 people die of rabies every year. China is one of the countries with high incidence of rabies. Once rabies occurs, the mortality rate is almost 100%. Vaccination is the most effective method to prevent rabies. After immunization, rabies vaccine can activate B cell immune response through T cell dependent and T cell independent pathway, so as to activate the body to produce neutralizing antibody and prevent the occurrence of rabies. Previous studies have shown that the loss of interleukin-21 (Interleukin-21,IL-21) signaling pathway affects the level of antibody induction after RABV immunization, suggesting that IL-21 plays an important role in the immunogenicity of RABV. Therefore, in order to further explore the mechanism of IL-21 in the immunogenicity of RABV, the recombinant RABV, expressing IL-21 was constructed by reverse genetic manipulation and the recombinant RABV was studied by a series of experiments in vitro and in vivo. The recombinant mouse IL-21 RABV, was named LBNSE-IL21, by reverse genetic operating system. The results showed that LBNSE-IL21 infected BSR cells could express IL-21 in a dose-dependent manner, and the expression of IL-21 in vitro was detected by ELISA. In addition, the growth curve of LBNSE-IL21 in BSR cells and NA cells was almost the same as that of the parent strain LBNSE, indicating that the insertion of exogenous gene IL-21 had no effect on the proliferation of the virus. In terms of virus pathogenicity, after intracerebral injection of LBNSE-IL21 into the brain of mice, there were no obvious clinical symptoms, and the body weight of mice basically maintained an upward trend, which was similar to that of parent strain LBNSE, indicating that the expression of IL-21 had no significant effect on pathogenicity. The mechanism of the effect of IL-21 expression on immunogenicity was further studied. Balb/c mice were infected with LBNSE-IL21 and LBNSE. The changes of various immune cells in humoral immune response of mice were detected by flow cytometry. The results showed that compared with LBNSE, LBNSE-IL21 could not activate dendritic cells (dendritic cell,DC) in lymph nodes or peripheral blood, but could significantly increase the (follicular helper T cells, of follicular helper cells in lymph nodes. The number of (germinal center B and GC B cells in Tfh) indicated that IL-21 expressed after LBNSE-IL21 immunization could promote the production of Tfh cells and GC B cells and affect the development of germatogenic centers. Further studies showed that LBNSE-IL21 could promote the differentiation of B cells into plasma cells (Plasma cells,PC) and produce more long-lived plasma cells (Long-lasting Plasma cells),) in bone marrow, thus inducing the production of high levels of neutralizing antibodies. Finally, in order to explore the immune effect of LBNSE-IL21 as vaccine, ICR mice were immunized with LBNSE-IL21 and LBNSE. The serum of ICR mice was collected from the 1st to 7th week after immunization, and the neutralizing antibody titer was determined by FAVN. The results showed that LBNSE-IL21 rapidly induced the production of neutralizing antibody at the first week after immunization, and the antibody level at the 1st to 6th week was significantly higher than that of the parent strain LBNSE.. This suggests that LBNSE-IL21 immunization can induce the body to produce a high level of neutralizing antibody more quickly. In addition, in the third week after immunization, the mice were challenged with 50 MICLD_ (50) CVS-24 into the brain, and the protective rate of LBNSE-IL21 was up to 92% after immunization, which was significantly higher than that of the parent virus (68%). In conclusion, LBNSE-IL21 immunization can promote the production of Tfh,GC B cells and plasma cells, so as to improve the production level of neutralizing antibody and improve immune protection. Therefore, IL-21 can be used as a candidate for a new rabies vaccine.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【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 江馗语;;狂犬病疫苗研究进展[J];中国工作犬业;2015年11期
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