福尔马林灭活呼吸道合胞病毒疫苗增强疾病中细胞因子表达的初步研究及G蛋白的构建、表达和鉴定

发布时间：2018-05-07 11:05

本文选题：呼吸道合胞病毒 + 疫苗增强疾病　；参考：《浙江大学》2011年硕士论文

【摘要】：背景和目的人呼吸道合胞病毒(RSV)是婴幼儿呼吸道感染的主要病原体,先天性心脏病患者、免疫缺陷个体和老年人也是易感人群,主要引起毛细支气管炎和肺炎。呼吸道合胞病毒属副粘病毒科肺病毒属,由宿主细胞膜包裹病毒核衣壳形成球形病毒颗粒,病毒基因组为不分节段单股负链RNA,包含10个基因编码11种病毒蛋白。G糖蛋白能促进病毒对宿主细胞的吸附作用,F糖蛋白可促使病毒与宿主细胞以及宿主细胞之间的融合,是重要的保护性抗原。呼吸道合胞病毒在人群中的感染较为普遍,呈冬季季节性流行的特点。2周岁以内的婴幼儿几乎都经历过呼吸道合胞病毒感染,而且二次感染比例可达50%。在全世界范围内,每年有5‰婴幼儿由于呼吸道合胞病毒感染需住院治疗。最近的研究显示呼吸道合胞病毒在人群中的感染率和死亡率较高,造成的危害远远超过流感。接种疫苗是预防和控制传染病最有效的措施,但无法彻底解决疫苗增强疾病的难题使安全有效的呼吸道合胞病毒疫苗的研制受阻。目前仍无特异性防治方法。Chanock发现人呼吸道合胞病毒后,FI-RSV疫苗试验于十九世纪六十年代首次在婴幼儿中进行,但80%受试者自然感染RSV后出现包括肺炎,支气管炎,肺炎等较严重的呼吸道疾病,并有两名受试儿童死亡。尸检显示为伴有肺气肿和气胸的支气管肺炎。组织学检查发现肺组织广泛的炎细胞浸润和嗜酸性粒细胞增多,提示免疫病理损害可能是呼吸道合胞病毒疫苗增强疾病的主要原因。此类因疫苗免疫接种使相应病原微生物感染时致病作用增强的疾病或现象,被称之为“疫苗增强疾病”(Vaccine Enhanced Disease, VED)。疫苗增强疾病的存在给相关疾病的预防和控制带来了极大的困难。大量研究显示细胞因子在呼吸道合胞病毒疫苗增强疾病起着较重要的作用。经FI-RSV免疫的BALB/c的小鼠在感染呼吸道合胞病毒后出现强烈的Th2细胞反应,Th2相关细胞因子IL-4和IL-13分别能够促进肺嗜酸性粒细胞增多。在表达G蛋白的痘病毒重组疫苗(vacvG)免疫小鼠中,IL-13是肺嗜酸细胞增多过程所必须的,同时在趋化因子CCL11, CCL17和CCL22的作用下,Th2细胞和嗜酸性粒细胞进入肺组织。Th1细胞因子IFN-γ能抑制Th2细胞反应和肺嗜酸细胞增多症,但同时也加重了体重减轻和临床症状等系统疾病。TNF-a既能通过促进Th1细胞因子IFN-γ表达,也可单独在疫苗增强疾病过程中发挥作用。这些研究显示细胞因子和呼吸道合胞病毒疫苗增强疾病紧密相关。探索呼吸道合胞病毒疫苗免疫各阶段细胞因子的变化能为安全有效疫苗的研究提供新的角度。方法制备呼吸道合胞病毒A型FI-RSV疫苗；建立FI-RSV疫苗增强疾病BABL/c小鼠模型；逐日观察各组小鼠感染呼吸道合胞病毒后的体重和临床症状；苏木精伊红(HE)染色检查肺组织病理学变化,观察支气管周围炎、气管炎、肺泡炎和间质性肺炎；用小鼠细胞因子抗体芯片检测检测支气管肺泡灌洗液(BALF)细胞因子表达；免疫组织化学染色检查单核细胞趋化因子(MCP-1)在肺组织的分布特征。结果 FI-RSV免疫组小鼠感染呼吸道合胞病毒后体重减轻和临床症状加重。组织病理学分析显示FI-RSV免疫组小鼠感染呼吸道合胞病毒后出现更为广泛肺组织炎性病变和肺嗜酸细胞增多症,说明FI-RSV疫苗增强疾病模型成功建立。细胞因子抗体芯片检测的结果证实FI-RSV疫苗可以改变肺组织细胞因子环境,促进单核细胞趋化因子(MCP-1)的表达和分泌。应用免疫组织化学染色显示正常肺组织上皮细胞内能表达少量MCP-1,有淡黄色颗粒。FI-RSV免疫组小鼠肺组织上皮细胞染色加深,呈棕黄或棕褐色,再次证实FI-RSV疫苗免疫都能促进MCP-1的过量表达和分泌。结论本研究成功建立BALB/c小鼠FI-RSV疫苗增强疾病模型；肺组织上皮能表达少量的MCP-1可能有助于维持机体正常的免疫功能；FI-RSV疫苗能促进MCP-1表达和分泌,为深入研究疫苗增强疾病提供了线索和依据。目的构建编码呼吸道合胞病毒G蛋白基因的重组质粒pcDNA3.1/G,为深入探索RSV疫苗增强疾病奠定基础。方法利用基因重组的方法构建含RSVG蛋白基因的重组质粒,酶切和测序鉴定,Western blot检测目的基因在Hek293T细胞的表达以及目的蛋白的抗原性。结果成功构建了编码RSVG蛋白基因的重组质粒pcDNA3.1/G, Western blot证实目的蛋白在体外具有较好的抗原性。结论我们成功构建的pcDNA3.1/G能在真核细胞中表达并具有较好的抗原性。
[Abstract]:Background and purpose
Human respiratory syncytial virus (RSV) is the main pathogen of respiratory tract infection in infants and infants. Congenital heart disease, immune deficiency and old people are also susceptible, which are mainly caused by bronchiolitis and pneumonia. The respiratory syncytial virus belongs to the paramyxovirus of the family of the paramyxovirus, which is formed by the virus nucleocapsid of the host cell membrane to form a spherical virus. The virus genome is an insegmental single strand negative strand RNA, which contains 10 genes encoding 11 kinds of viral protein.G glycoproteins that can promote the adsorption of the virus to the host cells. F glycoprotein can promote the fusion between the virus and host cells and the host cells, and is an important protective antigen.
The infection of respiratory syncytial virus (RSV) in the population is more common, and the characteristics of the seasonal epidemic in winter are characterized by respiratory syncytial virus infection in infants within.2 years of the year, and the proportion of two infantile infections can reach 50%. all over the world, and 5 per thousand infants and young children need to be hospitalized every year because of the infection of the virus. Research shows that respiratory syncytial virus infection and mortality in the crowd are higher, causing much more harm than influenza.
Vaccination is the most effective measure to prevent and control infectious diseases, but the problem of vaccine enhancement can not be solved thoroughly. The development of a safe and effective respiratory syncytial virus vaccine is blocked. There is still no specific prevention and control method.Chanock for the first time in 1860s after the discovery of the human respiratory syncytial virus (FI-RSV). The 80% subjects were naturally infected with RSV, including pneumonia, bronchitis, pneumonia and other severe respiratory diseases, and two of the children died. Autopsy showed bronchopneumonia accompanied by emphysema and pneumothorax. Histological examination found extensive inflammatory cell infiltration and eosinophils in the lung tissue. The pathological damage of the pestilence may be the main cause of the respiratory syncytial virus vaccine, which is known as "Vaccine Enhanced Disease, VED" because of the vaccine immunization that causes the pathogenic bacteria to be infected by the pathogenic microorganism. The existence of the epidemic Miao Zengqiang disease is the prevention of the related diseases and the prevention and prevention of the disease. Control has brought great difficulties.
A large number of studies have shown that cytokines play an important role in enhancing the respiratory syncytial virus vaccine. The BALB/c mice immunized with FI-RSV have a strong Th2 cell response after infection of the respiratory syncytial virus. Th2 related cytokines, IL-4 and IL-13, can promote the increase of pulmonary eosinophils respectively. In the expression of G protein, pox disease In the immunized recombinant vaccine (vacvG), IL-13 is necessary for the pulmonary eosinophilia process. At the same time, the entry of Th2 cells and eosinophils into the lung.Th1 cytokine IFN- gamma can inhibit the Th2 cell response and pulmonary eosinophilia under the action of chemokine CCL11, CCL17 and CCL22, but it also aggravates weight loss and progression. A systemic disease, such as bed symptoms,.TNF-a can be used both by promoting the expression of Th1 cytokine IFN- gamma, but also in the process of vaccine enhancement. These studies have shown that cytokines and respiratory syncytial virus vaccines are closely related to the disease. The study of effective vaccines provides a new perspective.
Method
The FI-RSV vaccine of respiratory syncytial virus type A was prepared; the FI-RSV vaccine was established to enhance the model of the disease BABL/c mice; the weight and clinical symptoms of the mice infected with respiratory syncytial virus were observed day by day; the pathological changes of lung tissue were examined by hematoxylin eosin (HE) staining, and the peribronchitis, bronchitis, alveolitis and interstitial pneumonia were observed. Cytokine antibody chip was used to detect the expression of cytokine in bronchoalveolar lavage fluid (BALF), and immunohistochemical staining was used to examine the distribution of monocyte chemoattractant (MCP-1) in the lung tissue.
Result
The weight loss and clinical symptoms of FI-RSV immunized mice infected with respiratory syncytial virus were increased. Histopathological analysis showed that FI-RSV immunized mice infected with respiratory syncytial virus and had more extensive pulmonary inflammatory diseases and pulmonary eosinophilia, indicating that the FI-RSV vaccine enhanced disease model was successfully established. The results of microarray detection confirmed that FI-RSV vaccine could change the cytokine environment of lung tissue and promote the expression and secretion of monocyte chemoattractant factor (MCP-1). The immunohistochemical staining showed that the normal lung tissue epithelial cells could express a small amount of MCP-1 in the normal lung tissue, and the lung tissue epithelial cells of the yellowish granule.FI-RSV immunization group were dyed to deepen. Brown or brown again confirmed that FI-RSV vaccine immunization can promote the over expression and secretion of MCP-1.
conclusion
This study has successfully established the FI-RSV vaccine enhanced disease model of BALB/c mice; the expression of a small amount of MCP-1 in the lung tissue may help to maintain the normal immune function of the body, and the FI-RSV vaccine can promote the expression and secretion of MCP-1, and provide clues and evidence for the in-depth study of vaccine enhanced diseases.
objective
The recombinant plasmid pcDNA3.1/G encoding G protein gene of respiratory syncytial virus was constructed to lay the foundation for further exploration of RSV vaccine to enhance disease.
Method
The recombinant plasmid containing RSVG protein gene was constructed by gene recombination, and the recombinant plasmid was identified by enzyme digestion and sequencing. Western blot was used to detect the expression of the target gene in Hek293T cells and the antigenicity of the target protein.
Result
The recombinant plasmid pcDNA3.1/G encoding RSVG protein gene was successfully constructed. Western blot confirmed that the target protein had good antigenicity in vitro.
conclusion
Our successfully constructed pcDNA3.1/G can be expressed in eukaryotic cells and has good antigenicity.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【参考文献】