表達(dá)IL-21重組狂犬病病毒免疫原性研究
發(fā)布時(shí)間：2019-05-10 08:16
【摘要】：狂犬病(Rabies)是由狂犬病病毒(Rabies virus,RABV)引起的一種人獸共患傳染病,每年約有59000人死于狂犬病,我國(guó)是狂犬病高發(fā)國(guó)家之一�？袢∫坏┌l(fā)病其致死率幾乎為100%,疫苗接種是預(yù)防狂犬病的最有效方法�？袢∫呙缭诿庖吆罂梢酝ㄟ^T細(xì)胞依賴和T細(xì)胞非依賴的途徑激活B細(xì)胞免疫應(yīng)答,從而激活機(jī)體產(chǎn)生中和抗體,預(yù)防狂犬病的發(fā)生。前期研究表明,白介素-21(Interleukin-21,IL-21)信號(hào)通路缺失后對(duì)RABV免疫后的抗體誘導(dǎo)水平產(chǎn)生影響,提示IL-21在RABV的免疫原性中起到重要作用。因此,為了進(jìn)一步探索IL-21在RABV的免疫原性中的作用機(jī)制,本研究擬通過反向遺傳操作構(gòu)建表達(dá)IL-21的重組RABV,并利用體外和體內(nèi)一系列實(shí)驗(yàn)對(duì)該重組RABV進(jìn)行研究:首先,通過反向遺傳學(xué)操作系統(tǒng)拯救表達(dá)鼠源IL-21重組RABV,命名為L(zhǎng)BNSE-IL21,通過ELISA檢測(cè)IL-21的體外表達(dá)情況,結(jié)果表明LBNSE-IL21感染BSR細(xì)胞可以表達(dá)IL-21且具有劑量依賴性;此外,LBNSE-IL21在BSR細(xì)胞和NA細(xì)胞的生長(zhǎng)曲線與親本毒株LBNSE幾乎一致,表明外源基因IL-21的插入對(duì)病毒的增殖沒有影響;在病毒致病性方面,將LBNSE-IL21腦內(nèi)注射小鼠后,小鼠無明顯臨床癥狀,且體重基本保持上升趨勢(shì),與親本毒株LBNSE體重變化曲線相似,表明IL-21的表達(dá)對(duì)致病性沒有顯著影響。進(jìn)一步研究IL-21的表達(dá)對(duì)免疫原性的影響機(jī)制,LBNSE-IL21和LBNSE感染Balb/c小鼠,利用流式細(xì)胞術(shù)檢測(cè)小鼠體液免疫應(yīng)答中各類免疫細(xì)胞的變化。結(jié)果發(fā)現(xiàn)與LBNSE相比,LBNSE-IL21不能激活淋巴結(jié)或外周血液中的樹突狀細(xì)胞(dendritic cell,DC),但可以顯著提高淋巴結(jié)中濾泡性輔助細(xì)胞(follicular helper T cells,Tfh)細(xì)胞和生發(fā)中心B細(xì)胞(germinal center B,GC B)數(shù)量,表明LBNSE-IL21免疫后表達(dá)的IL-21可以促進(jìn)Tfh細(xì)胞和GC B細(xì)胞的生成,影響生發(fā)中心的發(fā)育。進(jìn)一步研究發(fā)現(xiàn),LBNSE-IL21可以促進(jìn)B細(xì)胞向漿細(xì)胞(Plasma cells,PC)的分化,在骨髓產(chǎn)生更多數(shù)量的長(zhǎng)壽命漿細(xì)胞(Long-lasting Plasma cells),從而誘導(dǎo)產(chǎn)生高水平的中和抗體。最后,為了探究LBNSE-IL21作為疫苗的免疫效果,將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測(cè)定中和抗體效價(jià)。檢測(cè)結(jié)果顯示LBNSE-IL21在免疫后第1周便快速誘導(dǎo)機(jī)體產(chǎn)生中和抗體,并且第1-6周的抗體水平均顯著高于親本毒株LBNSE。這表明LBNSE-IL21免疫后可以更快誘導(dǎo)機(jī)體產(chǎn)生高水平的中和抗體。此外,在免疫后第三周,利用腦內(nèi)注射50 MICLD_(50)的CVS-24對(duì)小鼠進(jìn)行攻毒實(shí)驗(yàn),最終LBNSE-IL21免疫后可提供高達(dá)92%的保護(hù)率,顯著高于親本病毒免疫保護(hù)率68%。綜上所述,LBNSE-IL21免疫后可以通過促進(jìn)Tfh、GC B細(xì)胞,以及漿細(xì)胞的產(chǎn)生,從而提高中和抗體的產(chǎn)生水平,提高免疫保護(hù)。因此,IL-21可作為新型狂犬病疫苗的候選者。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65
本文編號(hào)：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【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 江馗語;;狂犬病疫苗研究進(jìn)展[J];中國(guó)工作犬業(yè);2015年11期
，本文編號(hào)：2473492
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