减毒鼠伤寒沙门氏菌为载体的人呼吸道合胞病毒DNA疫苗免疫保护作用研究

发布时间：2018-01-08 02:00

本文关键词：减毒鼠伤寒沙门氏菌为载体的人呼吸道合胞病毒DNA疫苗免疫保护作用研究　出处：《安徽医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染的最重要的病毒病原。目前尚无特异性防治方法,世界卫生组织将发展RSV疫苗列为最优先发展的疫苗项目之一。RSV的11种蛋白中,融合蛋白(Fusion Protein,F)是中和抗原,免疫动物后,可以产生具有免疫保护作用的中和抗体,为新型疫苗候选抗原。本实验中我们用减毒鼠伤寒沙门菌(Salmonella typhimurium aroA strain SL7207,SL7207)为载体携带能够表达F蛋白的DNA疫苗,经黏膜途径,开展体内免疫效果和免疫保护作用研究,以期更好了解该候选疫苗免疫生物学特征。方法:本研究中我们将能够表达RSV F蛋白的真核表达载体pcDNA3.1/F转化到SL7207,构建重组的减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/F,同时用pcDNA3.1空载体做对照。分别于0、2、6周三次口服免疫小鼠,通过体液免疫(IgG)、黏膜免疫(sIgA)、细胞免疫(CTL、T淋巴细胞增殖实验)等指标分析免疫后小鼠RSV特异性免疫应答产生情况。最后,利用RSV病毒经鼻腔攻击小鼠,并分别于攻毒后3、5和7天处死小鼠进行肺组织病毒含量及肺组织病理学动态分析了解免疫保护作用的效果。结果:取已转化pcDNA3.1/F的SL7207并提取质粒,经限制性内切酶分析与预期一致。经口服免疫BALB/c小鼠,与对照组相比,实验组小鼠血清及肺泡灌洗液(bronchoalveolar lavage,BAL)中分别产生了有效的血清IgG及sIgA(p0.01),血清中抗体亚类分析显示IgG1和IgG2a的比值为0.96。实验组CTL反应(p0.01,效靶比值为100:1和50:1)和T淋巴细胞增殖实验(p0.05)均明显增强。攻毒实验中,病毒攻击后3天和5天的小鼠肺部病毒载量,实验组比对照组明显降低(p0.05),组织病理学分析结果也显示在炎性细胞浸润和肺泡壁厚度方面两组有显著性差异(p0.01)。结论:获得的重组减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/F,能够刺激机体产生有效的体液免疫和细胞免疫及局部免疫,抗体亚类分析显示诱导产生了Th1和Th2平衡的免疫应答,没有产生疾病增强作用,对RSV感染具有一定的免疫保护作用。因此我们认为以减毒伤寒沙门菌为载体的口服DNA疫苗兼具黏膜免疫及DNA疫苗的双重优点,是RSV等通过黏膜途径感染的病毒疫苗研究的重要方向,为今后RSV疫苗免疫策略等研究奠定了坚实基础。
[Abstract]:Objective: human respiratory syncytial virus (Human Respiratory Syncytial Virus, RSV) is widely distributed in the world, is the leading cause of infant viral pathogens of severe lower respiratory tract infection is the most important. There is no specific treatment, the WHO will RSV vaccine development 11 protein as the priority development project of.RSV vaccine, fusion protein (Fusion Protein F) is a neutralizing antigen, immune animal, can induce neutralizing antibody immune protective effect, a new type of vaccine candidate antigen. In this experiment we used Salmonella typhimurium (Salmonella typhimurium aroA strain SL7207, SL7207) to carry the expression of F protein DNA vaccine as the carrier, by way of mucosa and carry out the protective immunity and its mechanism research, in order to better understand the biological characteristics of the immune vaccine candidate.
Methods: the eukaryotic expression vector pcDNA3.1/F was transformed into SL7207 in this study, we will be able to express RSV F protein, attenuated Salmonella typhimurium vaccine recombinant SL7207/pcDNA3.1/F reduction, at the same time with the pcDNA3.1 empty vector as control. 0,2,6 respectively in the three week oral immunization, the humoral immunity, mucosal immunity (IgG) (sIgA). Immune cells (CTL, T lymphocyte proliferation assay) index analysis of the formation of RSV specific immune responses in mice. Finally, using the RSV virus nasal attack mice, respectively after infection of 3,5 and mice were sacrificed 7 days to learn the dynamic analysis to understand the immune effect of lung tissue and lung tissue content of virus.
Results: pcDNA3.1/F and SL7207 have been transformed by extraction of plasmid, restriction endonuclease analysis is consistent with that expected. After oral immunization of BALB/c mice, compared with the control group, the serum and bronchoalveolar lavage fluid in the experimental group (bronchoalveolar lavage, BAL) respectively generate effective serum IgG and sIgA (P0.01), serum antibody subgroup analysis showed that the ratio of IgG1 and IgG2a as the experimental group 0.96. CTL reaction (P0.01, 100:1 and 50:1 ratio target) and T lymphocyte proliferation assay (P0.05) were significantly increased. The poison attack experiment, virus attack after 3 days and 5 days of small rat lung virus load, the experimental group was significantly lower than the control group (P0.05). The analysis results also show that the infiltration of inflammatory cells and alveolar wall thickness had significant difference between the two groups (P0.01). The pathological
Conclusion: the recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/F can induce humoral and cellular immunity and effective local immunity, antibody subtype analysis showed that induced Th1 and Th2 balance of immune response, no disease has enhanced immune protective effect on RSV infection. Therefore we believe in attenuated Salmonella typhimurium as the carrier of the oral DNA vaccine has dual advantages of mucosal immunity and DNA vaccine, RSV vaccine research is an important direction through mucosal infection, laid a solid foundation for the future study of RSV vaccine immunization strategies.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【相似文献】