發(fā)布時間：2018-05-04 12:10

  本文選題：裂谷熱病毒 + 假病毒　； 參考：《石河子大學(xué)》2016年博士論文

【摘要】：裂谷熱(Rift Valley fever,RVF)是流行在阿拉伯半島、非洲大陸和非洲東南印度洋幾個島嶼的人獸共患病。在這些地區(qū),其多次在動物和人間爆發(fā)。該病的病原為裂谷熱病毒(Rift Valley fever virus,RVFV)。家養(yǎng)反芻動物,特別是綿羊?qū)�RVFV最易感,懷孕母羊感染病毒后導(dǎo)致幾乎100%的流產(chǎn)和新生羔羊接近100%的死亡率。雖然牛、山羊和野生反芻動物對該病毒的敏感性稍低,但這些動物受到的威脅也是相當(dāng)大。裂谷熱病毒已經(jīng)從30種以上的蚊子體內(nèi)分離獲得,其中幾種在全球都有分布。這些共同的特點可以解釋為什么裂谷熱病毒被作為對人和動物健康威脅最嚴(yán)重的蟲媒病毒之一。該病被世界動物衛(wèi)生組織(OIE)歸類為A類疫病,已列入法定報告疫病。美國疾病預(yù)防控制中心和農(nóng)業(yè)部將其列為A類病原體,我國在《國家中長期動物疫病防治規(guī)劃(2012—2020年)》中將RVF列入重點防范的13種外來動物疫病之一。目前,疫苗免疫注射是預(yù)防RVF的主要手段。市場上尚無特效的抗RVFV藥物,也沒有商品化的人用RVF疫苗。用于動物的疫苗主要有以下3種:滅活疫苗、減毒活疫苗、重組活載體疫苗。但是,這些疫苗都存在其各自的缺點,例如,滅活疫苗雖然安全,但需要重復(fù)、大劑量免疫;弱毒活疫苗雖然免疫原性較高,但存在著毒力回歸的生物安全隱患。另外,RVFV能夠通過接觸、處理感染性材料和蚊蟲叮咬等方式傳播。同時,國際交流和生態(tài)環(huán)境這兩個因素也嚴(yán)重影響著該類疾病的傳播和爆發(fā)。近年來,具有免疫原性強和安全性好等優(yōu)點的病毒樣顆粒(virus like particles,VLPs)疫苗已經(jīng)表現(xiàn)出了具有開發(fā)為新型疫苗的巨大潛力。目的:本研究以RVFV ZH501毒株為研究對象,為避免RVFV作為中和試驗病原存在的生物安全隱患,探索建立RVFV假病毒中和試驗方法。利用桿狀病毒-昆蟲細(xì)胞表達(dá)系統(tǒng)構(gòu)建形態(tài)結(jié)構(gòu)穩(wěn)定、產(chǎn)量高的RVFV病毒樣顆粒,并對這些顆粒在動物體內(nèi)誘導(dǎo)的免疫應(yīng)答反應(yīng)進(jìn)行評價。通過本研究的開展,為開發(fā)制備以病毒樣顆粒為基礎(chǔ)的免疫原性強、安全、高效的新型裂谷熱疫苗提供一定的前期研發(fā)基礎(chǔ),為畜牧業(yè)的發(fā)展和人類的健康保駕護(hù)航。方法:(1)利用RVFV的結(jié)構(gòu)蛋白(Gn、Gc)和HIV慢病毒包裝系統(tǒng)構(gòu)建RVFV假病毒。通過電鏡觀察和Western Blotting鑒定所構(gòu)建的假病毒與HIV慢病毒相似度,檢測RVFV的抗體是否能抑制RVFV假病毒的感染,建立檢測RVFV抗體中和試驗方法;(2)利用p ET-30a原核表達(dá)載體,構(gòu)建含有截短Gn蛋白片段的重組質(zhì)粒,轉(zhuǎn)化BL21(DE3)感受態(tài)細(xì)胞,經(jīng)IPTG誘導(dǎo)表達(dá)目的蛋白,通過SDS-PAGE和Western Blotting檢測表達(dá)的蛋白正確與否,通過帶his標(biāo)簽的鎳柱純化表達(dá)蛋白。將純化后的RVFV Gn蛋白免疫小鼠,通過ELISA檢測抗體效價,Western Blotting檢測產(chǎn)生的多克隆抗體對G蛋白的特異性;(3)利用p Fast BacTMDual載體,構(gòu)建同時含有M、S基因的重組質(zhì)粒,轉(zhuǎn)化DH10Bac獲得重組Bacmid p Fast BacTMDual-M-S。重組Bacmid p Fast BacTMDual-M-S轉(zhuǎn)染昆蟲細(xì)胞sf9,連續(xù)盲傳三代,獲得重組桿狀病毒r Bac-N-G,并對重組病毒進(jìn)行鑒定,包括提取基因組PCR鑒定、間接免疫熒光鑒定、Western blotting、電鏡觀察、免疫電鏡等。鑒定正確的重組桿狀病毒大量擴(kuò)增,分裝-80℃保存;(4)將鑒定正確的第3代或4代重組桿狀病毒r Bac-N-G感染Sf9細(xì)胞,通過懸浮培養(yǎng)大規(guī)模制備RVFV VLP,離心去細(xì)胞碎片,超速離心濃縮、蔗糖梯度純化后,通過肌肉注射途徑分別免疫Balb/c小鼠和馬,檢測小鼠體內(nèi)抗體水平和分泌IFN-γ、IL-4等細(xì)胞因子的淋巴細(xì)胞比例與數(shù)量以及馬的抗體水平等免疫指標(biāo),評價其免疫原性。結(jié)果:(1)通過電鏡觀察和Western Blotting鑒定結(jié)果表明,包裝的假病毒在大小、結(jié)構(gòu)和形狀與HIV慢病毒高度相似,并且,感染抑制試驗顯示RVFV的抗體能抑制RVFV假病毒的感染能力,建立了檢測RVFV抗體中和試驗方法;(2)通過SDS-PAGE和Western Blotting檢測表達(dá)蛋白,檢測結(jié)果表明,目的蛋白獲得成功表達(dá)。純化后的RVFV Gn蛋白能誘導(dǎo)小鼠產(chǎn)生較高水平的Gn多克隆抗體,產(chǎn)生的多克隆抗體對G蛋白具有較強的特異性;(3)鑒定結(jié)果表明,成功構(gòu)建了表達(dá)RVFV結(jié)構(gòu)蛋白(Gn、Gc和N)的重組桿狀病毒(r Bac-N-G),r Bac-N-G感染昆蟲細(xì)胞sf9后表達(dá)的蛋白能夠自組裝成RVFV VLPs。通過間接免疫熒光、Western blotting試驗鑒定表明RVFV Gn、Gc和N蛋白表達(dá)正確;通過電鏡觀察,所形成的病毒樣顆粒在形態(tài)、結(jié)構(gòu)上與RVFV相似;(4)結(jié)果表明,RVFV病毒樣顆粒能有效刺激小鼠產(chǎn)生高水平的中和抗體和提高產(chǎn)生IFN-γ、IL-4等細(xì)胞因子的淋巴細(xì)胞的比例與數(shù)量;同時,VLPs也能夠刺激馬產(chǎn)生高水平的VLPs中和抗體。結(jié)論:(1)利用HIV慢病毒包裝系統(tǒng)和RVFV的結(jié)構(gòu)蛋白成功構(gòu)建了RVFV的假病毒。并且建立了基于RVFV假病毒的中和試驗方法,該方法能夠有效評價抗體的中和活性。(2)利用PET30a載體構(gòu)建了可表達(dá)RVFV截短的Gn蛋白的E.coli BL21(DE3)。通過Western blotting分析,制備的多抗對全長G蛋白的特異性較好。將純化的蛋白免疫Balb/c小鼠制備多克隆抗體,經(jīng)ELISA檢測,抗體滴度較高。(3)利用桿狀病毒昆蟲細(xì)胞表達(dá)系統(tǒng),成功的表達(dá)了RVFV病毒樣顆粒;通過間接免疫熒光、Western blotting鑒定了RVFV Gn、Gc和N蛋白表達(dá)正確,通過電鏡觀察和免疫電鏡檢測,發(fā)現(xiàn)所形成的病毒樣顆粒在大小、形狀等方面與典型的RVFV病毒特征一致;通過大量培養(yǎng)與不連續(xù)蔗糖密度滴度離心純化得到了RVFV病毒樣顆粒;(4)獲得的RVFV VLPs具有較強的免疫原性,可誘導(dǎo)機體產(chǎn)生較高水平的體液免疫和細(xì)胞免疫。RVFV病毒樣顆粒能夠刺激小鼠的脾細(xì)胞產(chǎn)生高水平的細(xì)胞因子IL-4和IFN-γ,而且產(chǎn)生這兩種細(xì)胞因子的淋巴細(xì)胞比例和數(shù)量顯著提高,并能刺激小鼠和馬產(chǎn)生較高水平的中和抗體。
[Abstract]:Rift Valley fever (RVF) is the prevalence of zoonosis in several islands in the Arabia Peninsula, the African continent and the India ocean in southeast Africa. In these areas, it breaks out many times in animals and humans. The pathogen is the rift virus (Rift Valley fever virus, RVFV). Domestic ruminants, especially sheep, are most susceptible to RVFV, pregnant and pregnant. When the ewes infected the virus, almost 100% of the abortion and newborn lambs were close to 100%. Although cattle, goats and wild ruminants were slightly less sensitive to the virus, these animals were also threatened. Rift Valley fever virus has been separated from more than 30 species of mosquitoes, several of which are distributed globally. The common features can explain why RRV is one of the most serious human and animal health menace. The disease is classified as an epidemic disease by the World Animal Health Organization (OIE) and is listed as a statutory disease. The US Centers for Disease Control and control and the Ministry of agriculture include it as a type a pathogen, and our country is in the country. The prevention and control plan of animal epidemic disease (2012 - 2020) > RVF is one of the 13 exotic animal epidemic diseases which are the key prevention. At present, vaccine immunization is the main means to prevent RVF. There are no special anti RVFV drugs in the market and no commercial people use RVF vaccine. There are 3 kinds of vaccines used in animals: inactivated vaccine and attenuated plague. However, these vaccines have their own shortcomings, for example, the inactivated vaccine, though safe, requires repeated, large doses of immunization; while the weak live vaccine has a high immunogenicity, there is a biological safety hazard with the presence of virulence. In addition, RVFV can be used to deal with infectious materials and mosquito bites through contact. At the same time, the two factors of international communication and ecological environment have also seriously affected the spread and outbreak of this kind of disease. In recent years, virus like particles (VLPs) vaccines, which have the advantages of strong immunogenicity and good safety, have shown great potential for developing new vaccines. In order to avoid the biological safety hazard of RVFV as the pathogen of neutralization test, the 01 strain was studied to establish a RVFV pseudo virus neutralization test method. By using baculovirus insect cell expression system, the RVFV virus like particles with stable morphology, high yield and high yield were constructed, and the immune responses induced by these particles in animals were carried out. Evaluation. Through this study, a new type of rift vaccine based on virus like particles is developed to provide a basis for the development of animal husbandry and to protect human health. Methods: (1) the use of RVFV structure protein (Gn, Gc) and HIV lentivirus packaging system to construct RVFV fake Virus. The similarity between the pseudo virus and the HIV lentivirus was identified by electron microscopy and Western Blotting, and whether the RVFV antibody could inhibit the infection of the RVFV pseudo virus and establish the detection method of the RVFV antibody neutralization test. (2) the recombinant plasmid containing the truncated Gn protein fragment was constructed by using the P ET-30a prokaryotic expression vector, and the BL21 (DE3) perception was transformed. The expression of target protein was induced by IPTG, and the expression protein was detected by SDS-PAGE and Western Blotting. The expression protein was purified by the his labeled nickel column. The purified RVFV Gn protein was immunized in mice, the antibody titer was detected by ELISA, and the specificity of the polyclonal antibody detected by Western Blotting was detected by the Western Blotting; (3) P Fast BacTMDual vector was used to construct a recombinant plasmid containing M, S gene, and the recombinant Bacmid P Fast BacTMDual-M-S. recombinant Bacmid P was transfected into insect cells. The recombinant baculovirus was continuously blind transmitted, and the recombinant baculovirus was obtained, and the recombinant Bacmid P was identified, including the extraction of genomic DNA. By immunofluorescence identification, Western blotting, electron microscopy, and immunoelectron microscopy, the correct recombinant baculovirus was amplified and stored at -80 C. (4) the correct third or 4 generation of recombinant baculovirus R Bac-N-G infected Sf9 cells, RVFV VLP, centrifuge cell debris, centrifugation centrifugation, sugar cane, and sugarcane were prepared by suspension culture. After the sugar gradient was purified, the Balb/c mice and horses were immunized by intramuscular injection. The antibody level in the mice and the proportion and quantity of lymphocytes secreting IFN- gamma, IL-4 and other cytokines, as well as the antibody level of the horse, were evaluated. The results were as follows: (1) the results of electron microscopy and Western Blotting identification showed that the results of the immunogenicity were indicated by the results of electron microscopy and Western Blotting identification. The size, structure and shape of the packaged pseudo virus were similar to the HIV lentivirus, and the infection inhibition test showed that the antibody of RVFV could inhibit the infection ability of RVFV pseudo virus. The detection of RVFV antibody neutralization test method was established. (2) the expression protein was detected by SDS-PAGE and Western Blotting, and the results showed that the target protein was successful. The purified RVFV Gn protein can induce a high level of Gn polyclonal antibody in mice, and the polyclonal antibody produced by the polyclonal antibody has strong specificity to the G protein. (3) the identification results showed that the recombinant baculovirus expressing the RVFV structural protein (Gn, Gc and N) was successfully constructed, and the protein expressed after the R Bac-N-G was infected by the insect cells could be expressed. Self-assembled RVFV VLPs. through indirect immunofluorescence, Western blotting test showed that RVFV Gn, Gc and N protein were expressed correctly. By electron microscopy, the formed virus like particles were similar to RVFV in morphology and structure. (4) the results showed that RVFV virus like particles could effectively stimulate the production of high level neutralization antibodies and increase the production IFN- in mice. The proportion and quantity of lymphocytes of gamma, IL-4 and other cytokines, and VLPs can also stimulate a high level of VLPs neutralization antibody in horses. Conclusion: (1) the pseudo virus of RVFV was successfully constructed by using the HIV lentivirus packaging system and the structural protein of RVFV. And a neutralization test based on the RVFV pseudo virus was established. This method can be effectively evaluated. The neutralization activity of antibody. (2) E.coli BL21 (DE3), which can express RVFV truncated Gn protein, was constructed using PET30a vector. Through Western blotting analysis, the specificity of the polyclonal antibody to full length G protein was better. The purified protein was immunized with Balb/c mice to prepare polyclonal antibody, and the antibody titer was higher by ELISA detection. (3) baculovirus Kunming was used. RVFV virus like particles were successfully expressed in the insect cell expression system, and the expression of RVFV Gn, Gc and N protein was correctly identified by indirect immunofluorescence. The expression of RVFV Gn, Gc and N protein was detected by electron microscopy and immunoelectron microscopy. RVFV virus like particles were obtained by discontinuous sucrose density titer centrifugation; (4) the obtained RVFV VLPs had strong immunogenicity, which could induce high level of body humoral immunity and cell immune.RVFV virus like particles to stimulate the high level of cytokine IL-4 and IFN- gamma in the spleen cells of mice, and produce these two kinds of fine particles. Lymphocyte ratio and number of cytokines increased significantly, and could stimulate mice and horses to produce a higher level of neutralizing antibodies.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.65

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6 王，

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