【摘要】：流感病毒是一種重要的傳染源。可以引起流感爆發(fā)流行。通用流感疫苗已經(jīng)研制多年。目前已經(jīng)發(fā)現(xiàn)一些保守抗原靶位，如HA2, M2和NP蛋白。血凝素C端的HA2形成莖部結(jié)構(gòu)錨定在病毒囊膜中,引起病毒囊膜與細(xì)胞膜的融合和病毒粒子核衣殼的釋放。血凝素C端的HA2形成莖部結(jié)構(gòu)錨定在病毒囊膜中,引起病毒囊膜與細(xì)胞膜的融合和病毒粒子核衣殼的釋放。以往的研究表明，HA2是一個(gè)重要的保守性抗原，可能用于通用的研究。 目的：本研究旨在通過選擇流感病毒保守抗原片段，獲得可對(duì)各種流感病毒具有保護(hù)作用的通用疫苗，利用優(yōu)化編碼病毒抗原密碼子策略，同時(shí)采用CD40L作為分子佐劑，獲得可顯著提高免疫效果的流感疫苗。方法：本研究擬將CD40L作為佐劑，采用病毒載體在體內(nèi)真核表達(dá)HA2基因與CD40L基因重組表達(dá)融合蛋白的策略。 CD40L序列的加入可以使融合蛋白通過CD40L與免疫效應(yīng)細(xì)胞APC和B細(xì)胞，直接激活免疫系統(tǒng)，。CD40L可與APC表面的CD40結(jié)合，從而誘導(dǎo)細(xì)胞毒性T淋巴細(xì)胞（CTLs）的活化，釋放IFN-γ，使CTLs直接殺傷感染細(xì)胞。另一方面，CD40L與B細(xì)胞表面的CD40結(jié)合，可以使刺激B細(xì)胞活化的抗原閾值降低，誘導(dǎo)B細(xì)胞針對(duì)其所攜帶抗原HA2的活化和增殖、促進(jìn)HA2特異性的免疫球蛋白類型轉(zhuǎn)換、抗體合成和分泌。此外，SHAF2CD40L復(fù)合物也可誘導(dǎo)DC細(xì)胞分泌一系列重要的細(xì)胞因子和趨化因子，進(jìn)一步上調(diào)T細(xì)胞表面CD40L的表達(dá)。 本研究首先通過PCR拼接及酶切連接的方法重組出HA2、SHA2、SHA2FCD40L、SFCD40L、HA2FCD40L、SFCD40L及S2HA2FCD40L共6個(gè)基因重組片段，其中S2HA2FCD40L為優(yōu)化密碼子的重組質(zhì)粒。將這6個(gè)重組基因通過穿梭質(zhì)粒插入腺病毒載體。 選取SHA2、SHA2FCD40L、S2HA2FCD40L及空載體對(duì)照組進(jìn)行動(dòng)物實(shí)驗(yàn)。對(duì)于每種腺病毒，小鼠體內(nèi)融合蛋白功能實(shí)驗(yàn)均包括2個(gè)部分：病毒攻擊保護(hù)實(shí)驗(yàn)和免疫效果檢測(cè)。同時(shí)為了對(duì)各組腺病毒免疫保護(hù)效果進(jìn)行全面觀察，每種腺病毒分為1×10~7PFU/只、1×10~8PFU/只、1×10~9PFU/只三個(gè)劑量組進(jìn)行免疫。 為了觀察疫苗組小鼠抵抗感病毒的保護(hù)能力，分別用5LD50H1N1（PR8株）和H9N2流感病毒進(jìn)行攻毒，觀察小鼠在不同劑量組（10~7PFU、10~8PFU、10~9PFU）的生存率、體重下降、肺部及鼻腔病毒滴度等指標(biāo)。結(jié)果：Western－blot結(jié)果表明，構(gòu)建的6種腺病毒在Hela細(xì)胞內(nèi)均可表達(dá)出目的大小的融合蛋白。用HA抗體和CD40L抗體分別檢測(cè)的結(jié)果分別證實(shí)了6種融合蛋白大小及抗原性均正確。免疫熒光的結(jié)果表明，所有6種腺病毒均可以在細(xì)胞膜上檢出，說明這6種融合蛋白具有接近天然的生物活性。 小鼠體內(nèi)免疫應(yīng)答檢測(cè)結(jié)果表明，在第一次免疫兩周后，腺病毒載體SHA2FCD40L、S2HA2FCD40L疫苗3個(gè)劑量組都能夠檢測(cè)到分泌TNF-α，IFN-γ及IL-4的T細(xì)胞。此外，細(xì)胞免疫應(yīng)答的水平與疫苗的免疫劑量呈現(xiàn)出正相關(guān)的關(guān)系，即隨著免疫劑量的增加，誘導(dǎo)機(jī)體的細(xì)胞免疫應(yīng)答的水平也隨之上升。二次免疫后，SHA2FCD40L,S2HA2FCD40L和SHA2組刺激產(chǎn)生的細(xì)胞因子明顯增多。其中Ad-S2HA2FCD40L誘導(dǎo)的細(xì)胞免疫應(yīng)答較強(qiáng)。與初次免疫相比，，二次加強(qiáng)免疫能夠顯著性地增強(qiáng)機(jī)體的細(xì)胞免疫應(yīng)答水平，并且細(xì)胞免疫應(yīng)答水平與免疫劑量呈正相關(guān)。雖然流式細(xì)胞檢測(cè)結(jié)果表明腺病毒空載體可以刺激機(jī)體產(chǎn)生免疫應(yīng)答,但綜合ELISPOT結(jié)果分析不是針對(duì)HA2蛋白產(chǎn)生特異性的免疫應(yīng)答。 體液免疫檢測(cè)結(jié)果表明，AD-SHA2、AD-SHA2FCD40L和AD-S2HA2FCD40L三種腺病毒載體疫苗組都能夠誘導(dǎo)機(jī)體產(chǎn)生體液免疫應(yīng)答。體液免疫應(yīng)答的水平與疫苗的免疫劑量呈正相關(guān)，其中后2組有更高的IgG和IgA抗體分泌， 動(dòng)物保護(hù)實(shí)驗(yàn)結(jié)果表明，10~9PFU的AD-SHA2腺病毒載體疫苗對(duì)PR8和H9N2有一定的交叉保護(hù)作用，其體內(nèi)的病毒滴度水平也相對(duì)較低。小鼠肺部和鼻腔中流感病毒滴度在有佐劑CD40L的組別SHA2FCD40L感染滴度更低，存活率明顯升高（P0.05）。優(yōu)化密碼子的S2HA2SFCD40L保護(hù)效果更好。結(jié)論：可以表達(dá)SHA2與CD40L融合蛋白的腺病毒載體，可以引起小鼠細(xì)胞及體液免疫應(yīng)答，保護(hù)小鼠抵抗不同亞型流感病毒的攻擊，為新型流感疫苗的研制打下了基礎(chǔ)。
[Abstract]:Influenza virus is an important source of infection. The outbreak of the flu can be caused. The universal influenza vaccine has been developed for many years. Some conserved antigen targets, such as HA2, M2 and NP proteins, have been found. The HA2-forming stem structure at the C-terminus of the hemagglutinin is anchored in the viral envelope, resulting in the fusion of the viral envelope to the cell membrane and the release of the viral particle nucleocapsid. The HA2-forming stem structure at the C-terminus of the hemagglutinin is anchored in the viral envelope, resulting in the fusion of the viral envelope to the cell membrane and the release of the viral particle nucleocapsid. Previous studies have shown that HA2 is an important conservative antigen and may be used in general research. Objective: The purpose of this study was to obtain a universal vaccine with a protective effect on various influenza viruses by selecting the conservative antigen fragment of influenza virus. the agent is used for obtaining the influenza epidemic disease which can remarkably improve the immune effect, Methods: CD40L was used as an adjuvant, and the expression of the fusion protein was expressed by the recombinant expression of the HA2 gene and the CD40L gene in the body by using the viral vector. The addition of CD40L sequence can enable the fusion protein to directly activate the immune system through the CD40L and the immune effector cells APC and B cells. CD40L can be combined with CD40 on the APC surface to induce activation of cytotoxic T lymphocytes (CTLs), release of IFN-1, and direct the direct killing of CTLs. The CD40L, on the other hand, binds to the CD40 on the surface of the B cell, which reduces the antigen threshold that stimulates the activation of the B-cell, induces the activation and proliferation of the B-cell against the antigen HA2, promotes the type conversion of the HA2-specific immunoglobulin, the synthesis of the antibody, and the separation. In addition, the SHAF2CD40L complex can also induce a series of important cytokines and chemokines in the DC cell, and further up-regulate the surface of the T-cell surface CD40L. In this study,6 gene recombinant fragments of HA2, SHA2, SHA2FCD40L, SFCD40L, HA2FCD40L, SFCD40L and S2HA2FCD40L were recombined by PCR and enzyme digestion. The 6 recombinant genes were inserted into the adenosis through the shuttle plasmid. Toxic carrier. SHA2, SHA2FCD40L, S2HA2FCD40L and empty vector control group were selected. Animal experiments were performed. For each of the adenoviruses, the mouse in vivo fusion protein function test consisted of two parts: virus attack protection experiment and maintenance-free in order to make a comprehensive observation on the effect of the immune protection of each group, each adenovirus is divided into 1-10-7 PFU/ only,1-10-8 PFU/ only,1-10-9 PFU/ only three doses The mice were challenged with 5LD50H1N1 (PR8 strain) and H9N2 influenza virus, and the survival rate, weight loss, lung and nasal cavity of the mice were observed in different dose groups (10-7PFU,10-8PFU,10-9PFU). Results: The results of Western-blot show that the six adenoviruses can be expressed in Hela cells. The size of the fusion protein was confirmed by the results of the detection by the HA antibody and the CD40L antibody, respectively. The results of the immunofluorescence showed that all six kinds of adenoviruses can be detected on the cell membrane, indicating that the six fusion proteins are close to each other. The results of immune response in mice showed that, after the first immunization for two weeks, the recombinant adenovirus vector SHA2FCD40L and S2HA2FCD40L vaccine can detect the secretion of TNF-1, IFN-1, And the level of the cellular immune response is positively correlated with the immune dose of the vaccine, that is, the cellular immunity of the induction body should be induced with the increase of the immune dose. The level of response of SHA2FCD40L, S2HA2FCD40L and SHA2 was also increased after secondary immunization. The cytokines of Ad-S2HA2FCD40L induced by Ad-S2HA2FCD40L were significantly increased. The cellular immune response of the cell was stronger than that of the primary immunization, and the level of cellular immune response of the body was significantly enhanced and the cellular immune response level was increased. The results of flow cytometry show that the adenovirus empty vector can stimulate the immune response of the body, but the analysis of the comprehensive ELISPOT results is not for the production of the HA2 protein. The results of humoral immunity test showed that the three adenoviral vector vaccine groups of AD-SHA2, AD-SHA2FCD40L and AD-S2HA2FCD40L could be induced The level of humoral immune response is positively related to the immune dose of the vaccine, and the latter group is higher. IgG and IgA antibody secretion, animal protection experiment results show that the AD-SHA2 adenovirus vector vaccine of 10-9PFU is used for PR8 and H 9N2 has a certain cross-protection effect, The level of virus titer in mouse lung and nasal cavity was also relatively low. The titer of influenza virus in mouse lung and nasal cavity was lower than that of group SHA2FCD40L with adjuvant CD40L. Obvious increase (P0.05). The optimized codon of S2HA2 Conclusion: The adenovirus vector of SHA2 and CD40L fusion protein can be expressed, and the immune response of mouse and humoral immune response can be induced.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1

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