RVFV病毒样颗粒的构建及其免疫原性研究

发布时间：2018-05-04 12:10

本文选题：裂谷热病毒 + 假病毒　；参考：《石河子大学》2016年博士论文

【摘要】：裂谷热(Rift Valley fever,RVF)是流行在阿拉伯半岛、非洲大陆和非洲东南印度洋几个岛屿的人兽共患病。在这些地区,其多次在动物和人间爆发。该病的病原为裂谷热病毒(Rift Valley fever virus,RVFV)。家养反刍动物,特别是绵羊对RVFV最易感,怀孕母羊感染病毒后导致几乎100%的流产和新生羔羊接近100%的死亡率。虽然牛、山羊和野生反刍动物对该病毒的敏感性稍低,但这些动物受到的威胁也是相当大。裂谷热病毒已经从30种以上的蚊子体内分离获得,其中几种在全球都有分布。这些共同的特点可以解释为什么裂谷热病毒被作为对人和动物健康威胁最严重的虫媒病毒之一。该病被世界动物卫生组织(OIE)归类为A类疫病,已列入法定报告疫病。美国疾病预防控制中心和农业部将其列为A类病原体,我国在《国家中长期动物疫病防治规划(2012—2020年)》中将RVF列入重点防范的13种外来动物疫病之一。目前,疫苗免疫注射是预防RVF的主要手段。市场上尚无特效的抗RVFV药物,也没有商品化的人用RVF疫苗。用于动物的疫苗主要有以下3种:灭活疫苗、减毒活疫苗、重组活载体疫苗。但是,这些疫苗都存在其各自的缺点,例如,灭活疫苗虽然安全,但需要重复、大剂量免疫;弱毒活疫苗虽然免疫原性较高,但存在着毒力回归的生物安全隐患。另外,RVFV能够通过接触、处理感染性材料和蚊虫叮咬等方式传播。同时,国际交流和生态环境这两个因素也严重影响着该类疾病的传播和爆发。近年来,具有免疫原性强和安全性好等优点的病毒样颗粒(virus like particles,VLPs)疫苗已经表现出了具有开发为新型疫苗的巨大潜力。目的:本研究以RVFV ZH501毒株为研究对象,为避免RVFV作为中和试验病原存在的生物安全隐患,探索建立RVFV假病毒中和试验方法。利用杆状病毒-昆虫细胞表达系统构建形态结构稳定、产量高的RVFV病毒样颗粒,并对这些颗粒在动物体内诱导的免疫应答反应进行评价。通过本研究的开展,为开发制备以病毒样颗粒为基础的免疫原性强、安全、高效的新型裂谷热疫苗提供一定的前期研发基础,为畜牧业的发展和人类的健康保驾护航。方法:(1)利用RVFV的结构蛋白(Gn、Gc)和HIV慢病毒包装系统构建RVFV假病毒。通过电镜观察和Western Blotting鉴定所构建的假病毒与HIV慢病毒相似度,检测RVFV的抗体是否能抑制RVFV假病毒的感染,建立检测RVFV抗体中和试验方法;(2)利用p ET-30a原核表达载体,构建含有截短Gn蛋白片段的重组质粒,转化BL21(DE3)感受态细胞,经IPTG诱导表达目的蛋白,通过SDS-PAGE和Western Blotting检测表达的蛋白正确与否,通过带his标签的镍柱纯化表达蛋白。将纯化后的RVFV Gn蛋白免疫小鼠,通过ELISA检测抗体效价,Western Blotting检测产生的多克隆抗体对G蛋白的特异性;(3)利用p Fast BacTMDual载体,构建同时含有M、S基因的重组质粒,转化DH10Bac获得重组Bacmid p Fast BacTMDual-M-S。重组Bacmid p Fast BacTMDual-M-S转染昆虫细胞sf9,连续盲传三代,获得重组杆状病毒r Bac-N-G,并对重组病毒进行鉴定,包括提取基因组PCR鉴定、间接免疫荧光鉴定、Western blotting、电镜观察、免疫电镜等。鉴定正确的重组杆状病毒大量扩增,分装-80℃保存;(4)将鉴定正确的第3代或4代重组杆状病毒r Bac-N-G感染Sf9细胞,通过悬浮培养大规模制备RVFV VLP,离心去细胞碎片,超速离心浓缩、蔗糖梯度纯化后,通过肌肉注射途径分别免疫Balb/c小鼠和马,检测小鼠体内抗体水平和分泌IFN-γ、IL-4等细胞因子的淋巴细胞比例与数量以及马的抗体水平等免疫指标,评价其免疫原性。结果:(1)通过电镜观察和Western Blotting鉴定结果表明,包装的假病毒在大小、结构和形状与HIV慢病毒高度相似,并且,感染抑制试验显示RVFV的抗体能抑制RVFV假病毒的感染能力,建立了检测RVFV抗体中和试验方法;(2)通过SDS-PAGE和Western Blotting检测表达蛋白,检测结果表明,目的蛋白获得成功表达。纯化后的RVFV Gn蛋白能诱导小鼠产生较高水平的Gn多克隆抗体,产生的多克隆抗体对G蛋白具有较强的特异性;(3)鉴定结果表明,成功构建了表达RVFV结构蛋白(Gn、Gc和N)的重组杆状病毒(r Bac-N-G),r Bac-N-G感染昆虫细胞sf9后表达的蛋白能够自组装成RVFV VLPs。通过间接免疫荧光、Western blotting试验鉴定表明RVFV Gn、Gc和N蛋白表达正确;通过电镜观察,所形成的病毒样颗粒在形态、结构上与RVFV相似;(4)结果表明,RVFV病毒样颗粒能有效刺激小鼠产生高水平的中和抗体和提高产生IFN-γ、IL-4等细胞因子的淋巴细胞的比例与数量;同时,VLPs也能够刺激马产生高水平的VLPs中和抗体。结论:(1)利用HIV慢病毒包装系统和RVFV的结构蛋白成功构建了RVFV的假病毒。并且建立了基于RVFV假病毒的中和试验方法,该方法能够有效评价抗体的中和活性。(2)利用PET30a载体构建了可表达RVFV截短的Gn蛋白的E.coli BL21(DE3)。通过Western blotting分析,制备的多抗对全长G蛋白的特异性较好。将纯化的蛋白免疫Balb/c小鼠制备多克隆抗体,经ELISA检测,抗体滴度较高。(3)利用杆状病毒昆虫细胞表达系统,成功的表达了RVFV病毒样颗粒;通过间接免疫荧光、Western blotting鉴定了RVFV Gn、Gc和N蛋白表达正确,通过电镜观察和免疫电镜检测,发现所形成的病毒样颗粒在大小、形状等方面与典型的RVFV病毒特征一致;通过大量培养与不连续蔗糖密度滴度离心纯化得到了RVFV病毒样颗粒;(4)获得的RVFV VLPs具有较强的免疫原性,可诱导机体产生较高水平的体液免疫和细胞免疫。RVFV病毒样颗粒能够刺激小鼠的脾细胞产生高水平的细胞因子IL-4和IFN-γ,而且产生这两种细胞因子的淋巴细胞比例和数量显著提高,并能刺激小鼠和马产生较高水平的中和抗体。
[Abstract]:Rift Valley fever (RVF) is the prevalence of zoonosis in several islands in the Arabia Peninsula, the African continent and the India ocean in southeast Africa. In these areas, it breaks out many times in animals and humans. The pathogen is the rift virus (Rift Valley fever virus, RVFV). Domestic ruminants, especially sheep, are most susceptible to RVFV, pregnant and pregnant. When the ewes infected the virus, almost 100% of the abortion and newborn lambs were close to 100%. Although cattle, goats and wild ruminants were slightly less sensitive to the virus, these animals were also threatened. Rift Valley fever virus has been separated from more than 30 species of mosquitoes, several of which are distributed globally. The common features can explain why RRV is one of the most serious human and animal health menace. The disease is classified as an epidemic disease by the World Animal Health Organization (OIE) and is listed as a statutory disease. The US Centers for Disease Control and control and the Ministry of agriculture include it as a type a pathogen, and our country is in the country. The prevention and control plan of animal epidemic disease (2012 - 2020) > RVF is one of the 13 exotic animal epidemic diseases which are the key prevention. At present, vaccine immunization is the main means to prevent RVF. There are no special anti RVFV drugs in the market and no commercial people use RVF vaccine. There are 3 kinds of vaccines used in animals: inactivated vaccine and attenuated plague. However, these vaccines have their own shortcomings, for example, the inactivated vaccine, though safe, requires repeated, large doses of immunization; while the weak live vaccine has a high immunogenicity, there is a biological safety hazard with the presence of virulence. In addition, RVFV can be used to deal with infectious materials and mosquito bites through contact. At the same time, the two factors of international communication and ecological environment have also seriously affected the spread and outbreak of this kind of disease. In recent years, virus like particles (VLPs) vaccines, which have the advantages of strong immunogenicity and good safety, have shown great potential for developing new vaccines. In order to avoid the biological safety hazard of RVFV as the pathogen of neutralization test, the 01 strain was studied to establish a RVFV pseudo virus neutralization test method. By using baculovirus insect cell expression system, the RVFV virus like particles with stable morphology, high yield and high yield were constructed, and the immune responses induced by these particles in animals were carried out. Evaluation. Through this study, a new type of rift vaccine based on virus like particles is developed to provide a basis for the development of animal husbandry and to protect human health. Methods: (1) the use of RVFV structure protein (Gn, Gc) and HIV lentivirus packaging system to construct RVFV fake Virus. The similarity between the pseudo virus and the HIV lentivirus was identified by electron microscopy and Western Blotting, and whether the RVFV antibody could inhibit the infection of the RVFV pseudo virus and establish the detection method of the RVFV antibody neutralization test. (2) the recombinant plasmid containing the truncated Gn protein fragment was constructed by using the P ET-30a prokaryotic expression vector, and the BL21 (DE3) perception was transformed. The expression of target protein was induced by IPTG, and the expression protein was detected by SDS-PAGE and Western Blotting. The expression protein was purified by the his labeled nickel column. The purified RVFV Gn protein was immunized in mice, the antibody titer was detected by ELISA, and the specificity of the polyclonal antibody detected by Western Blotting was detected by the Western Blotting; (3) P Fast BacTMDual vector was used to construct a recombinant plasmid containing M, S gene, and the recombinant Bacmid P Fast BacTMDual-M-S. recombinant Bacmid P was transfected into insect cells. The recombinant baculovirus was continuously blind transmitted, and the recombinant baculovirus was obtained, and the recombinant Bacmid P was identified, including the extraction of genomic DNA. By immunofluorescence identification, Western blotting, electron microscopy, and immunoelectron microscopy, the correct recombinant baculovirus was amplified and stored at -80 C. (4) the correct third or 4 generation of recombinant baculovirus R Bac-N-G infected Sf9 cells, RVFV VLP, centrifuge cell debris, centrifugation centrifugation, sugar cane, and sugarcane were prepared by suspension culture. After the sugar gradient was purified, the Balb/c mice and horses were immunized by intramuscular injection. The antibody level in the mice and the proportion and quantity of lymphocytes secreting IFN- gamma, IL-4 and other cytokines, as well as the antibody level of the horse, were evaluated. The results were as follows: (1) the results of electron microscopy and Western Blotting identification showed that the results of the immunogenicity were indicated by the results of electron microscopy and Western Blotting identification. The size, structure and shape of the packaged pseudo virus were similar to the HIV lentivirus, and the infection inhibition test showed that the antibody of RVFV could inhibit the infection ability of RVFV pseudo virus. The detection of RVFV antibody neutralization test method was established. (2) the expression protein was detected by SDS-PAGE and Western Blotting, and the results showed that the target protein was successful. The purified RVFV Gn protein can induce a high level of Gn polyclonal antibody in mice, and the polyclonal antibody produced by the polyclonal antibody has strong specificity to the G protein. (3) the identification results showed that the recombinant baculovirus expressing the RVFV structural protein (Gn, Gc and N) was successfully constructed, and the protein expressed after the R Bac-N-G was infected by the insect cells could be expressed. Self-assembled RVFV VLPs. through indirect immunofluorescence, Western blotting test showed that RVFV Gn, Gc and N protein were expressed correctly. By electron microscopy, the formed virus like particles were similar to RVFV in morphology and structure. (4) the results showed that RVFV virus like particles could effectively stimulate the production of high level neutralization antibodies and increase the production IFN- in mice. The proportion and quantity of lymphocytes of gamma, IL-4 and other cytokines, and VLPs can also stimulate a high level of VLPs neutralization antibody in horses. Conclusion: (1) the pseudo virus of RVFV was successfully constructed by using the HIV lentivirus packaging system and the structural protein of RVFV. And a neutralization test based on the RVFV pseudo virus was established. This method can be effectively evaluated. The neutralization activity of antibody. (2) E.coli BL21 (DE3), which can express RVFV truncated Gn protein, was constructed using PET30a vector. Through Western blotting analysis, the specificity of the polyclonal antibody to full length G protein was better. The purified protein was immunized with Balb/c mice to prepare polyclonal antibody, and the antibody titer was higher by ELISA detection. (3) baculovirus Kunming was used. RVFV virus like particles were successfully expressed in the insect cell expression system, and the expression of RVFV Gn, Gc and N protein was correctly identified by indirect immunofluorescence. The expression of RVFV Gn, Gc and N protein was detected by electron microscopy and immunoelectron microscopy. RVFV virus like particles were obtained by discontinuous sucrose density titer centrifugation; (4) the obtained RVFV VLPs had strong immunogenicity, which could induce high level of body humoral immunity and cell immune.RVFV virus like particles to stimulate the high level of cytokine IL-4 and IFN- gamma in the spleen cells of mice, and produce these two kinds of fine particles. Lymphocyte ratio and number of cytokines increased significantly, and could stimulate mice and horses to produce a higher level of neutralizing antibodies.

【学位授予单位】：石河子大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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