本文選題：呼吸道合胞病毒 + 疫苗增強(qiáng)疾病�。� 參考：《浙江大學(xué)》2011年碩士論文

【摘要】：背景和目的 人呼吸道合胞病毒(RSV)是嬰幼兒呼吸道感染的主要病原體,先天性心臟病患者、免疫缺陷個(gè)體和老年人也是易感人群,主要引起毛細(xì)支氣管炎和肺炎。呼吸道合胞病毒屬副粘病毒科肺病毒屬,由宿主細(xì)胞膜包裹病毒核衣殼形成球形病毒顆粒,病毒基因組為不分節(jié)段單股負(fù)鏈RNA,包含10個(gè)基因編碼11種病毒蛋白。G糖蛋白能促進(jìn)病毒對(duì)宿主細(xì)胞的吸附作用,F糖蛋白可促使病毒與宿主細(xì)胞以及宿主細(xì)胞之間的融合,是重要的保護(hù)性抗原。 呼吸道合胞病毒在人群中的感染較為普遍,呈冬季季節(jié)性流行的特點(diǎn)。2周歲以內(nèi)的嬰幼兒幾乎都經(jīng)歷過(guò)呼吸道合胞病毒感染,而且二次感染比例可達(dá)50%。在全世界范圍內(nèi),每年有5‰嬰幼兒由于呼吸道合胞病毒感染需住院治療。最近的研究顯示呼吸道合胞病毒在人群中的感染率和死亡率較高,造成的危害遠(yuǎn)遠(yuǎn)超過(guò)流感。 接種疫苗是預(yù)防和控制傳染病最有效的措施,但無(wú)法徹底解決疫苗增強(qiáng)疾病的難題使安全有效的呼吸道合胞病毒疫苗的研制受阻。目前仍無(wú)特異性防治方法。Chanock發(fā)現(xiàn)人呼吸道合胞病毒后,FI-RSV疫苗試驗(yàn)于十九世紀(jì)六十年代首次在嬰幼兒中進(jìn)行,但80%受試者自然感染RSV后出現(xiàn)包括肺炎,支氣管炎,肺炎等較嚴(yán)重的呼吸道疾病,并有兩名受試兒童死亡。尸檢顯示為伴有肺氣腫和氣胸的支氣管肺炎。組織學(xué)檢查發(fā)現(xiàn)肺組織廣泛的炎細(xì)胞浸潤(rùn)和嗜酸性粒細(xì)胞增多,提示免疫病理?yè)p害可能是呼吸道合胞病毒疫苗增強(qiáng)疾病的主要原因。此類因疫苗免疫接種使相應(yīng)病原微生物感染時(shí)致病作用增強(qiáng)的疾病或現(xiàn)象,被稱之為“疫苗增強(qiáng)疾病”(Vaccine Enhanced Disease, VED)。疫苗增強(qiáng)疾病的存在給相關(guān)疾病的預(yù)防和控制帶來(lái)了極大的困難。 大量研究顯示細(xì)胞因子在呼吸道合胞病毒疫苗增強(qiáng)疾病起著較重要的作用。經(jīng)FI-RSV免疫的BALB/c的小鼠在感染呼吸道合胞病毒后出現(xiàn)強(qiáng)烈的Th2細(xì)胞反應(yīng),Th2相關(guān)細(xì)胞因子IL-4和IL-13分別能夠促進(jìn)肺嗜酸性粒細(xì)胞增多。在表達(dá)G蛋白的痘病毒重組疫苗(vacvG)免疫小鼠中,IL-13是肺嗜酸細(xì)胞增多過(guò)程所必須的,同時(shí)在趨化因子CCL11, CCL17和CCL22的作用下,Th2細(xì)胞和嗜酸性粒細(xì)胞進(jìn)入肺組織。Th1細(xì)胞因子IFN-γ能抑制Th2細(xì)胞反應(yīng)和肺嗜酸細(xì)胞增多癥,但同時(shí)也加重了體重減輕和臨床癥狀等系統(tǒng)疾病。TNF-a既能通過(guò)促進(jìn)Th1細(xì)胞因子IFN-γ表達(dá),也可單獨(dú)在疫苗增強(qiáng)疾病過(guò)程中發(fā)揮作用。這些研究顯示細(xì)胞因子和呼吸道合胞病毒疫苗增強(qiáng)疾病緊密相關(guān)。探索呼吸道合胞病毒疫苗免疫各階段細(xì)胞因子的變化能為安全有效疫苗的研究提供新的角度。 方法 制備呼吸道合胞病毒A型FI-RSV疫苗；建立FI-RSV疫苗增強(qiáng)疾病BABL/c小鼠模型；逐日觀察各組小鼠感染呼吸道合胞病毒后的體重和臨床癥狀；蘇木精伊紅(HE)染色檢查肺組織病理學(xué)變化,觀察支氣管周?chē)�、氣管炎、肺泡炎和間質(zhì)性肺炎；用小鼠細(xì)胞因子抗體芯片檢測(cè)檢測(cè)支氣管肺泡灌洗液(BALF)細(xì)胞因子表達(dá)；免疫組織化學(xué)染色檢查單核細(xì)胞趨化因子(MCP-1)在肺組織的分布特征。 結(jié)果 FI-RSV免疫組小鼠感染呼吸道合胞病毒后體重減輕和臨床癥狀加重。組織病理學(xué)分析顯示FI-RSV免疫組小鼠感染呼吸道合胞病毒后出現(xiàn)更為廣泛肺組織炎性病變和肺嗜酸細(xì)胞增多癥,說(shuō)明FI-RSV疫苗增強(qiáng)疾病模型成功建立。細(xì)胞因子抗體芯片檢測(cè)的結(jié)果證實(shí)FI-RSV疫苗可以改變肺組織細(xì)胞因子環(huán)境,促進(jìn)單核細(xì)胞趨化因子(MCP-1)的表達(dá)和分泌。應(yīng)用免疫組織化學(xué)染色顯示正常肺組織上皮細(xì)胞內(nèi)能表達(dá)少量MCP-1,有淡黃色顆粒。FI-RSV免疫組小鼠肺組織上皮細(xì)胞染色加深,呈棕黃或棕褐色,再次證實(shí)FI-RSV疫苗免疫都能促進(jìn)MCP-1的過(guò)量表達(dá)和分泌。 結(jié)論 本研究成功建立BALB/c小鼠FI-RSV疫苗增強(qiáng)疾病模型；肺組織上皮能表達(dá)少量的MCP-1可能有助于維持機(jī)體正常的免疫功能；FI-RSV疫苗能促進(jìn)MCP-1表達(dá)和分泌,為深入研究疫苗增強(qiáng)疾病提供了線索和依據(jù)。 目的 構(gòu)建編碼呼吸道合胞病毒G蛋白基因的重組質(zhì)粒pcDNA3.1/G,為深入探索RSV疫苗增強(qiáng)疾病奠定基礎(chǔ)。 方法 利用基因重組的方法構(gòu)建含RSVG蛋白基因的重組質(zhì)粒,酶切和測(cè)序鑒定,Western blot檢測(cè)目的基因在Hek293T細(xì)胞的表達(dá)以及目的蛋白的抗原性。 結(jié)果 成功構(gòu)建了編碼RSVG蛋白基因的重組質(zhì)粒pcDNA3.1/G, Western blot證實(shí)目的蛋白在體外具有較好的抗原性。 結(jié)論 我們成功構(gòu)建的pcDNA3.1/G能在真核細(xì)胞中表達(dá)并具有較好的抗原性。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 彭慧琴,葉招明,錢(qián)景,陶君;呼吸道合胞病毒感染對(duì)細(xì)胞凋亡及FasL、Fas、Bcl-2、Bax基因表達(dá)的研究[J];中國(guó)病理生理雜志;2005年09期

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本文編號(hào)：1856674