呼吸道合胞病毒疫苗增強(qiáng)性疾病（VED）模型建立及G蛋白基因重組質(zhì)粒免疫特征
發(fā)布時間：2018-07-25 17:19
【摘要】： 目的呼吸道合胞病毒(respiratory syncytial virus,RSV)是嬰幼兒下呼吸道感染的主要病原體,其感染引起嬰幼兒毛細(xì)支氣管炎和肺炎,并與兒童哮喘的發(fā)生發(fā)展有著密切關(guān)系。接種疫苗是預(yù)防和控制傳染病最為有效的措施,但RSV至今尚無安全有效的疫苗產(chǎn)生。以福爾馬林滅活RSV疫苗(FI-RSV)預(yù)防接種的嬰兒或?qū)W齡前兒童,自然感染RSV后反而導(dǎo)致更為嚴(yán)重的疫苗增強(qiáng)性疾病(Vaccineenhanced disease,VED)的產(chǎn)生。有研究表明,生化方法提純的RSV G蛋白免疫和重組G蛋白免疫均有明顯的誘導(dǎo)VED效應(yīng)。另有研究者報道,盡管G蛋白和FI-RSV在免疫病理學(xué)方面引起的免疫應(yīng)答相似,但T細(xì)胞介導(dǎo)的應(yīng)答卻不同,即兩者引起疾病加重的機(jī)理不同。提示G蛋白與VED相關(guān)性有待于進(jìn)一步研究。 本研究中我們通過建立福爾馬林滅活RSV(FI-RSV)疫苗相關(guān)VED模型并定義其主要特征,為相關(guān)免疫調(diào)控機(jī)理研究提供平臺;構(gòu)建編碼RSV G蛋白基因的重組質(zhì)粒,觀察其對RSV感染小鼠的保護(hù)性及特異性免疫效應(yīng),探討表達(dá)的G蛋白抗原與VED的相關(guān)性,為探索RSV疫苗接種后疾病加重的相關(guān)機(jī)制以及為研制安全、有效的RSV疫苗提供線索和實驗依據(jù)。 方法制備RSVA1型Long株FI-RSV疫苗;以FI-RSV免疫BALB/c小鼠后以RSV攻擊,收集攻毒前后肺組織、血清標(biāo)本;采用熒光定量RT-PCR檢測肺組織標(biāo)本中病毒滴度,HE染色法檢查肺組織病理改變;采用ELISA檢測血清標(biāo)本中RSV特異性IgG水平。利用基因重組方法構(gòu)建含RSV G蛋白編碼基因的重組質(zhì)粒,測序和酶切鑒定,Western Blot檢測目的基因經(jīng)體外表達(dá)后的免疫原性。重組質(zhì)粒免疫小鼠后以RSV感染,采用上述相同方法分別檢測肺組織標(biāo)本中RSV滴度、肺組織病理改變和血清抗體水平,同時采用雙抗體夾心ELISA測定肺泡灌洗液(BAL)內(nèi)Th1/Th2細(xì)胞因子表達(dá)量,流式細(xì)胞術(shù)檢測BAL中T淋巴細(xì)胞亞群數(shù)量及活化狀態(tài)。 結(jié)果與對照組相比,FI-RSV免疫小鼠感染RSV后肺組織病毒滴度降低,但出現(xiàn)更為嚴(yán)重的肺組織病變,主要是以肺炎、間質(zhì)性肺炎以及支氣管周圍炎為主要特征的廣泛炎癥反應(yīng),伴有局部肺氣腫。免疫小鼠血清RSV特異性IgG抗體滴度增高,但感染RSV后其滴度明顯降低。同時成功構(gòu)建了編碼RSV G蛋白基因的重組質(zhì)粒pcDNA3.1~G。Western Blot證實目的基因表達(dá)蛋白在體外具有免疫原性。被免疫小鼠感染RSV后肺組織病毒滴度降低,肺組織中未見明顯的炎性細(xì)胞浸潤。小鼠血清中產(chǎn)生較高滴度的抗RSV-G IgG。小鼠BAL細(xì)胞中CD4+CD25+T淋巴細(xì)胞比例顯著增加,并且IFN-γ(Th1)、IL-4(Th2)兩類細(xì)胞因子表達(dá)顯示平衡。 結(jié)論本研究成功地建立了FI-RSV免疫BALB/c小鼠感染RSV后VED模型,其主要特征為肺組織中出現(xiàn)與病毒滴度、血清抗體水平不一致的嚴(yán)重病理改變;編碼呼吸道合胞病毒(RSV)G蛋白基因的重組質(zhì)粒pcDNA3.1~G能誘導(dǎo)產(chǎn)生CD4+CD25+T細(xì)胞亞群,對小鼠具有明顯的保護(hù)作用,而未見明顯的VED現(xiàn)象產(chǎn)生。
[Abstract]:Objective Respiratory syncytial virus (RSV) is the main pathogen of infantile lower respiratory tract infection, which causes bronchiolitis and pneumonia in infants, and is closely related to the occurrence and development of asthma in children. Vaccination is the most effective measure to prevent and control infectious diseases, but there is no safe and effective vaccine for RSV. Infants or preschool children vaccinated with formalin inactivated RSV vaccine (FI-RSV) naturally infected with RSV resulted in more serious Vaccineenhanced disease (VED). Some studies showed that RSV G protein and recombinant G protein immunized by biochemical method could induce VED obviously. Other researchers reported that although G protein and FI-RSV have similar immune responses in immunopathology, T cell mediated responses are different, that is, the mechanisms of disease aggravation are different between them. It suggests that the correlation between G protein and VED needs further study. In this study, we established a formalin inactivated RSV (FI-RSV) vaccine related VED model and defined its main characteristics to provide a platform for the study of the related immune regulation mechanism, and to construct the recombinant plasmid encoding the RSV G protein gene. To observe the protective and specific immune effects of RSV on mice infected with RSV, to explore the correlation between G protein antigen expressed and VED, and to explore the mechanism of disease aggravation after RSV vaccination and the safety of its development. Effective RSV vaccine provides clues and experimental evidence. Methods the FI-RSV vaccine of RSVA1 Long strain was prepared, and the lung tissues and serum samples were collected after BALB/c mice were immunized with FI-RSV before and after attack with RSV, and the pathological changes of lung tissues were detected by fluorescence quantitative RT-PCR and HE staining. ELISA was used to detect the level of RSV specific IgG in serum samples. The recombinant plasmid containing RSV G protein encoding gene was constructed by gene recombination method. The immunogenicity of the target gene expressed in vitro was determined by sequencing and restriction endonuclease digestion. Mice immunized with recombinant plasmids were infected with RSV. The titer of RSV, the pathological changes of lung tissue and the level of serum antibody were detected by the same method mentioned above. The expression of Th1/Th2 cytokines in alveolar lavage fluid (BAL) was determined by double antibody sandwich ELISA, and the number and activation of T lymphocyte subsets in BAL were detected by flow cytometry. Results compared with the control group, the viral titers of lung tissue in mice immunized with FI-RSV decreased after infection with RSV, but more serious lung tissue lesions occurred, mainly characterized by extensive inflammatory reactions characterized by pneumonia, interstitial pneumonia and bronchitis. Accompanied by local emphysema. The titer of serum RSV specific IgG antibody increased in immunized mice, but decreased significantly after RSV infection. At the same time, the recombinant plasmid pcDNA3.1~G.Western Blot encoding RSV G protein gene was successfully constructed to confirm the immunogenicity of the expressed protein in vitro. The viral titer of lung tissue decreased after RSV infection in immunized mice, and no inflammatory cell infiltration was found in the lung tissue. A high titer of anti RSV-G IgG was produced in mouse serum. The proportion of CD4 CD25 T lymphocytes in mouse BAL cells increased significantly, and the expression of IFN- 緯 (Th1) IL-4 (Th2) cytokines showed a balance. Conclusion the VED model of FI-RSV immunized BALB/c mice after infection with RSV was successfully established. The main characteristics of the model were severe pathological changes in lung tissues which were inconsistent with virus titer and serum antibody level. The recombinant plasmid pcDNA3.1~G encoding the (RSV) G protein gene of respiratory syncytial virus can induce the production of CD4 CD25 T cell subsets, which has obvious protective effect on mice, but no obvious VED phenomenon.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R186
本文編號：2144505
[Abstract]:Objective Respiratory syncytial virus (RSV) is the main pathogen of infantile lower respiratory tract infection, which causes bronchiolitis and pneumonia in infants, and is closely related to the occurrence and development of asthma in children. Vaccination is the most effective measure to prevent and control infectious diseases, but there is no safe and effective vaccine for RSV. Infants or preschool children vaccinated with formalin inactivated RSV vaccine (FI-RSV) naturally infected with RSV resulted in more serious Vaccineenhanced disease (VED). Some studies showed that RSV G protein and recombinant G protein immunized by biochemical method could induce VED obviously. Other researchers reported that although G protein and FI-RSV have similar immune responses in immunopathology, T cell mediated responses are different, that is, the mechanisms of disease aggravation are different between them. It suggests that the correlation between G protein and VED needs further study. In this study, we established a formalin inactivated RSV (FI-RSV) vaccine related VED model and defined its main characteristics to provide a platform for the study of the related immune regulation mechanism, and to construct the recombinant plasmid encoding the RSV G protein gene. To observe the protective and specific immune effects of RSV on mice infected with RSV, to explore the correlation between G protein antigen expressed and VED, and to explore the mechanism of disease aggravation after RSV vaccination and the safety of its development. Effective RSV vaccine provides clues and experimental evidence. Methods the FI-RSV vaccine of RSVA1 Long strain was prepared, and the lung tissues and serum samples were collected after BALB/c mice were immunized with FI-RSV before and after attack with RSV, and the pathological changes of lung tissues were detected by fluorescence quantitative RT-PCR and HE staining. ELISA was used to detect the level of RSV specific IgG in serum samples. The recombinant plasmid containing RSV G protein encoding gene was constructed by gene recombination method. The immunogenicity of the target gene expressed in vitro was determined by sequencing and restriction endonuclease digestion. Mice immunized with recombinant plasmids were infected with RSV. The titer of RSV, the pathological changes of lung tissue and the level of serum antibody were detected by the same method mentioned above. The expression of Th1/Th2 cytokines in alveolar lavage fluid (BAL) was determined by double antibody sandwich ELISA, and the number and activation of T lymphocyte subsets in BAL were detected by flow cytometry. Results compared with the control group, the viral titers of lung tissue in mice immunized with FI-RSV decreased after infection with RSV, but more serious lung tissue lesions occurred, mainly characterized by extensive inflammatory reactions characterized by pneumonia, interstitial pneumonia and bronchitis. Accompanied by local emphysema. The titer of serum RSV specific IgG antibody increased in immunized mice, but decreased significantly after RSV infection. At the same time, the recombinant plasmid pcDNA3.1~G.Western Blot encoding RSV G protein gene was successfully constructed to confirm the immunogenicity of the expressed protein in vitro. The viral titer of lung tissue decreased after RSV infection in immunized mice, and no inflammatory cell infiltration was found in the lung tissue. A high titer of anti RSV-G IgG was produced in mouse serum. The proportion of CD4 CD25 T lymphocytes in mouse BAL cells increased significantly, and the expression of IFN- 緯 (Th1) IL-4 (Th2) cytokines showed a balance. Conclusion the VED model of FI-RSV immunized BALB/c mice after infection with RSV was successfully established. The main characteristics of the model were severe pathological changes in lung tissues which were inconsistent with virus titer and serum antibody level. The recombinant plasmid pcDNA3.1~G encoding the (RSV) G protein gene of respiratory syncytial virus can induce the production of CD4 CD25 T cell subsets, which has obvious protective effect on mice, but no obvious VED phenomenon.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R186
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Harvey Cantor;;Generation and Regulation of CD8~+ Regulatory T Cells[J];Cellular & Molecular Immunology;2008年06期
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