本文選題：表達(dá) + 呼吸道�。� 參考：《中國疾病預(yù)防控制中心》2009年博士論文

【摘要】： 人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)廣泛分布于世界各地,是導(dǎo)致嬰幼兒嚴(yán)重下呼吸道感染的最重要的病毒病原之一,老年人和免疫缺陷病人也易遭受RSV引起的嚴(yán)重感染。RSV屬于副粘病毒科肺病毒屬,基因組為15.2 Kb的單股負(fù)鏈RNA,編碼11種蛋白,其中G、F系包膜糖蛋白,為RSV中和抗原。與G蛋白相比,F蛋白不但具有B細(xì)胞抗原表位也有CD8+細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte,CTL)抗原表位,且其抗原性極為保守,可產(chǎn)生交叉保護(hù)性免疫。 從福爾馬林滅活疫苗開始,人們已經(jīng)研制了多種形式的RSV疫苗,但由于安全性和免疫原性等問題,至今尚未有能夠用于預(yù)防人RSV感染的疫苗問世,因此世界衛(wèi)生組織將發(fā)展RSV疫苗列為二十一世紀(jì)最優(yōu)先發(fā)展的疫苗項目之一。 非復(fù)制型第一代腺病毒載體(first generation replication deficient recombinantadenoviral vector,FGAd)具有繁殖滴度高、易純化、能夠感染分裂和非分裂細(xì)胞等優(yōu)點(diǎn)而被廣泛用于疫苗研究。FGAd曾被用于表達(dá)經(jīng)基因工程改造的RSV G蛋白,在動物實驗中獲得了較好的免疫保護(hù)作用,然而以FGAd為基礎(chǔ)開展RSVF蛋白疫苗研究尚未見報道。與FGAd相比,輔助病毒依賴型腺病毒載體(helper-dependent adenoviral vector,HDAd)可大大降低腺病毒(Adenovirus,Ad)特異性的細(xì)胞免疫反應(yīng),增強(qiáng)轉(zhuǎn)染DC細(xì)胞能力,且轉(zhuǎn)基因表達(dá)時間更加持久。以HDAd作為黏膜疫苗載體,開展RSV F蛋白疫苗研究有待深入研究。 本課題首先用表達(dá)RSV F蛋白的FGAd重組腺病毒(FGAd-F)滴鼻免疫BALB/c小鼠,觀察免疫保護(hù)效果和保護(hù)作用特點(diǎn)。結(jié)果表明:FGAd-F滴鼻免疫BALB/c小鼠能夠誘導(dǎo)產(chǎn)生RSV特異性的血清IgG、黏膜IgA和CD8+CTL,抗體亞類分析顯示誘導(dǎo)產(chǎn)生了Th1和Th2平衡的CD4+T細(xì)胞免疫應(yīng)答,沒有產(chǎn)生疾病增強(qiáng),對RSV攻擊具有一定的免疫保護(hù)作用。 隨后,我們用表達(dá)EGFP蛋白的HDAd重組腺病毒(HDAd-EGFP)滴鼻免疫BALB/c小鼠,探討HDAd作為黏膜疫苗載體的可行性和合理性。結(jié)果表明:盡管與等量的FGAd-EGFP誘導(dǎo)產(chǎn)生相似的anti-Ad抗體,但HDAd-EGFP經(jīng)鼻免疫小鼠可誘導(dǎo)產(chǎn)生更高的EGFP特異性的血清IgG、黏膜IgA和細(xì)胞免疫,且抗體亞類分析顯示誘導(dǎo)產(chǎn)生了Th1和Th2平衡的CD4+T細(xì)胞免疫應(yīng)答。另外HDAd-EGFP同源加強(qiáng)免疫也能提高EGFP特異性的體液免疫和細(xì)胞免疫。 在上述研究的基礎(chǔ)上,我們用表達(dá)RSV F蛋白的HDAd重組腺病毒(HDAd-F)經(jīng)滴鼻途徑免疫BALB/c小鼠,初步探討了HDAd-F的免疫效果。結(jié)果表明:與等量的FGAd-F相比,HDAd-F經(jīng)鼻兩次免疫小鼠可誘導(dǎo)更好的RSV F特異性的黏膜免疫,HDAd-F經(jīng)鼻一次免疫小鼠可誘導(dǎo)更高的RSV F特異性的血清抗體。 綜上所述,FGAd-F能夠誘導(dǎo)有效的免疫保護(hù)作用,以FGAd為載體的疫苗是預(yù)防RSV等通過黏膜途徑感染的病毒疫苗的一個重要方法。HDAd是一種理想的黏膜疫苗載體,適于經(jīng)呼吸道黏膜感染的RSV疫苗的研制,通過提高轉(zhuǎn)基因表達(dá)量、降低載體用量等措施,有望獲得較好的免疫效果和免疫保護(hù)作用。
[Abstract]:Human respiratory syncytial virus (Human Respiratory Syncytial Virus, RSV) is widely distributed all over the world. It is one of the most important viral pathogens causing severe lower respiratory infection in infants and young children. The elderly and immunodeficiency patients are also vulnerable to RSV caused by severe infection of.RSV belonging to the paramyxovirus family, a single strand of 15.2 Kb. Negative chain RNA, encoding 11 proteins, of which G, F envelope glycoprotein is RSV neutralizing antigen. Compared with G protein, F protein not only has the epitopes of B cell antigen but also CD8+ cell toxic T lymphocyte (cytotoxic T lymphocyte) antigen epitope, and its antigenicity is extremely conservative, and can produce cross protective immunity.
Since the formalin inactivated vaccine has begun, many forms of RSV vaccines have been developed, but because of the safety and immunogenicity of the vaccine, there has not yet been a vaccine to prevent human RSV infection. Therefore, the WHO is developing the RSV vaccine as one of the top priority vaccine projects in twenty-first Century.
The non replicating first generation adenovirus vector (first generation replication deficient recombinantadenoviral vector, FGAd) has the advantages of high breeding titer, easy purification, and the ability to infect split and non cleavage cells and is widely used in vaccine research..FGAd has been used to express the RSV G protein that has been genetically engineered. It has been obtained in animal experiments. Better immune protection has been achieved. However, the study of RSVF vaccine based on FGAd has not yet been reported. Compared with FGAd, the auxiliary virus dependent adenovirus vector (helper-dependent adenoviral vector, HDAd) can greatly reduce the specific cellular immune response of adenovirus (Adenovirus, Ad), enhance the transfection of DC cells, and turn the base of the transfected DC cells. Because of the longer expression time, the study of RSV F protein vaccine needs to be further studied with HDAd as a mucosal vaccine carrier.
In this study, we first immunized BALB/c mice with FGAd recombinant adenovirus (FGAd-F) expressing RSV F protein (FGAd-F), and observed the protective effects and protective effects of BALB/c. The results showed that BALB/c mice immunized with FGAd-F nose drops could induce RSV specific serum IgG, mucous membrane IgA and CD8+CTL. Antibody subclass analysis showed that the inducible production of Th1 and equilibrium was induced by the antibody subclass analysis. CD4+T cell immune response did not produce disease enhancement, and had a certain immune protection against RSV attack.
Subsequently, we immunized the BALB/c mice with the recombinant adenovirus expressing EGFP protein (HDAd-EGFP) in the nose drops to explore the feasibility and rationality of HDAd as a mucosal vaccine carrier. The results showed that, despite the similar anti-Ad antibody induced by the equivalent FGAd-EGFP induction, the HDAd-EGFP transnasal immune mice could induce higher EGFP specific blood. IgG, mucosal IgA and cellular immunity, and the antibody subclass analysis showed that the induced CD4+T cell immune response was induced by the balance of Th1 and Th2. In addition, HDAd-EGFP homologous immunization could also improve the humoral and cellular immunity of EGFP specific body.
On the basis of the above study, we immunized BALB/c mice with the HDAd recombinant adenovirus expressing RSV F protein (HDAd-F) through the nasal drip route. The immune effect of HDAd-F was preliminarily discussed. The results showed that the two mice immunized with HDAd-F through the nose could induce better RSV F specific mucosal immunity compared with the equal amount of FGAd-F, and the HDAd-F via the nose was immune to one time. Mice could induce higher RSV F specific serum antibodies.
To sum up, FGAd-F can induce effective immune protection. FGAd based vaccine is an important way to prevent RSV and other viral vaccines through mucosal pathway..HDAd is an ideal carrier for mucosal vaccine. It is suitable for the research of RSV vaccine that is infected by respiratory tract mucosa. By improving the expression of transgene and reducing the carrier use And other measures are expected to achieve better immune effects and immune protection.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392.1

【共引文獻(xiàn)】

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1 關(guān)瑞;腫瘤相關(guān)基因ZNF403的功能研究[D];中南大學(xué);2012年

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