狂犬病毒糖蛋白DNA疫苗的研究

发布时间：2018-06-03 05:11

本文选题：狂犬病毒 + 糖蛋白　；参考：《吉林大学》2017年硕士论文

【摘要】：狂犬病是由狂犬病毒引起的人兽共患的全球性传染疾病,存在于全球150多个国家和地区,根据世界卫生组织WHO 2016的统计数据,全球每年大约有59000人死于该病,其中95%发生在亚洲和非洲等发展中国家。狂犬病是一种在出现临床症状后几乎100%会致命的病毒性传染病,因此研制狂犬疫苗对于预防与控制狂犬病毒传播是非常必要的。糖蛋白(G)是狂犬病病毒唯一能诱导宿主产生中和抗体的蛋白,也能有效的刺激T淋巴细胞增殖,并决定病毒的毒力。近些年的实验研究显示,天然的狂犬病毒糖蛋白(G)是以三聚体形式存在的跨膜糖蛋白,然而可溶性G蛋白单体的免疫原性和保护性效果并不十分理性,表明狂犬病毒G蛋白的有效免疫原性是非常依赖G蛋白三聚体空间构象的完整性。Fibritin foldon(Fd)是T4噬菌体fibritin蛋白质的结构域,能够辅助fibritin蛋白质形成三聚体,并通过氢键、疏水相互作用和盐桥的方式来稳定天然三聚体结构。利用foldon的这种性质,融合可溶性蛋白质形成三聚体,帮助并稳定目的蛋白形成正确的、免疫原性更高的构象,从而达到更好的治疗与预防效果。目前已被广泛应用于流感病毒糖蛋白,HIV-1,RSV糖蛋白等,并获得理想的效果。自1994年以来,DNA疫苗已被提议作为用于预防传染性疾病便宜、有效的策略,且其可行性已经在许多动物模型中被验证,并被用于人类病毒疫苗临床试验中。经过二十多年的研究,证实了 DNA疫苗对多种感染性(病毒,细菌和寄生虫)和非感染性(过敏和肿瘤)疾病能引起有效的细胞免疫和体液免疫。同时DNA疫苗具有成本低,设计和管理简单,生产相对较快的优势,为DNA狂犬疫苗领域的发展注入新的动力。本文以真核VR1012质粒为载体,分别构建了 G蛋白全长(mRVG)质粒,G蛋白截断型(包含有分泌信号肽和胞外区,mRVGst)质粒,具有foldon结构域的G蛋白融合(tRVGst-LI、tRVGst-IQG8)质粒。重组质粒能表达不同大小的G蛋白,以CpG为免疫佐剂,对BALB/c小鼠进行免疫。经过3次免疫后,结果显示,小鼠产生了较强的IgG水平,mRVG(全长型抗原)免疫组抗体IgG是mRVGst(截短型抗原)免疫组抗体水平的1.6倍,是tRVGst-LI、tRVGst-IQG8(融合型抗原)免疫组的1.4倍;tRVGst-LI与tRVGst-IQG8免疫组分泌的IgG抗体是mRVGst(截短型抗原)免疫组的1.4倍。经过抗体分型以及细胞因子检测后,实验结果显示本实验DNA疫苗产生了较强的Th1细胞免疫应答。通过快速荧光灶抑制试验(RFFIT)检测中和抗体(RVNA)活性,并通过颅内攻毒实验研究该DNA疫苗的保护力。结果显示,融合型抗原tRVGst-LI、tRVGst-IQG8中和抗体水平是9.3 IU/ml和12.7 IU/ml,保护力是50%～67%,免疫效果优于截短型抗原mRVGst的4.3 IU/ml,16.7%,但是弱于全长型抗原mRVG的17.4 IU/ml,保护力83.3%。表明foldon结构域在一定程度上能增加可溶性融合G蛋白的免疫原性,但是G蛋白的免疫原性不仅与胞外区有关,还与跨膜区及胞外区有关,即更依赖于结构的完整性。
[Abstract]:Rabies is a global infectious disease caused by rabies virus. It exists in more than 150 countries and regions around the world. According to the World Health Organization (WHO) WHO 2016 statistics, about 59000 people die of the disease every year in the world. Of these, 95 per cent occurred in developing countries such as Asia and Africa. Rabies is a viral infectious disease that can kill almost 100% of the patients after clinical symptoms. So it is necessary to develop rabies vaccine to prevent and control the spread of rabies virus. Glycoprotein G) is the only protein of rabies virus that can induce the host to produce neutralizing antibody. It can also effectively stimulate the proliferation of T lymphocytes and determine the virulence of the virus. Experimental studies in recent years have shown that natural rabies virus glycoprotein G) is a transmembrane glycoprotein in the form of trimer, but the immunogenicity and protective effect of soluble G protein monomer are not very rational. The results indicate that the effective immunogenicity of rabies virus G protein is highly dependent on the integrity of G protein trimer spatial conformation. Fibritin foldonnin Fd) is the domain of fibritin protein of T4 phage, which can assist fibritin protein to form trimer and through hydrogen bond. Hydrophobic interactions and salt bridges are used to stabilize the natural trimer structure. By using this property of foldon, the soluble protein is fused to form trimer, which helps and stabilizes the conformation of the target protein with higher immunogenicity and helps to achieve a better therapeutic and preventive effect. At present, it has been widely used in influenza virus glycoprotein, such as HIV-1 RSV glycoprotein, and obtained ideal results. Since 1994, DNA vaccine has been proposed as a cheap and effective strategy for the prevention of infectious diseases, and its feasibility has been verified in many animal models and used in clinical trials of human virus vaccine. After more than 20 years of research, it has been proved that DNA vaccine can induce effective cellular and humoral immunity against various infectious (viruses, bacteria and parasites) and non-infectious (allergy and tumor) diseases. At the same time, DNA vaccine has the advantages of low cost, simple design and management, and relatively fast production, which provides a new impetus for the development of DNA rabies vaccine field. In this paper, eukaryotic VR1012 plasmids were used to construct G protein full-length mRVGs, G protein truncated plasmids (including secretory signal peptide and extracellular region mRVGst1) plasmids, and G protein fusion plasmids with foldon domain. The recombinant plasmid could express G protein of different sizes and immunize BALB/c mice with CpG as immune adjuvant. After three times of immunization, the results showed that the antibody IgG of mRVG immunized mice was 1.6 times higher than that of mRVGST-immunized mice, and the results showed that the antibody level of mRVG immunized mice was 1.6-fold higher than that of mRVGST-immunized mice. The IgG antibody secreted by tRVGst-LI and tRVGst-IQG8 immunized group was 1.4 times higher than that of mRVGstimmunized group. After antibody typing and cytokine detection, the results showed that the DNA vaccine produced a strong Th1 cellular immune response. The activity of neutralization antibody was detected by rapid fluorescence foci inhibition test (RFFIT), and the protective effect of DNA vaccine was studied by intracranial attack test. The results showed that the neutralizing antibody levels of fusion antigen tRVGst-LItRVGst-IQG8 were 9.3 IU/ml and 12.7 IU/ml / ml, and the protective power was 50 ~ (67)%. The immune effect was better than that of truncated antigen mRVGst (4.3% / ml ~ 16.7g), but weaker than that of full-length antigen mRVG (17.4%), and the protective power was 83.3%. The results showed that foldon domain could increase the immunogenicity of soluble fusion G protein to some extent, but the immunogenicity of G protein was not only related to the extracellular domain, but also related to the transmembrane region and extracellular domain, which was more dependent on the integrity of the structure.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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