本文選題：流感 + 病毒樣顆粒��； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：禽流感(Avian influenza,AI)是危害我國(guó)養(yǎng)禽業(yè)的重大傳染病,是目前和今后禽病防控的重點(diǎn)。我國(guó)禽群中流行的禽流感病毒(Avian influenza virus,AIV)以低致病性H9N2亞型病毒為主。H9N2病毒的感染可導(dǎo)致雞不同程度的呼吸道癥狀,產(chǎn)蛋雞產(chǎn)蛋量下降約10-20%,而且可造成雞免疫抵抗力下降,感染雞容易并發(fā)或繼發(fā)其它各種細(xì)菌和病毒性疾病。H9N2病毒可以在種間傳播,給人類的生命安全造成嚴(yán)重的威脅。H9N2亞型AIVs還是感染人類的新型流感病毒的“母病毒”,為國(guó)內(nèi)流行的雞源H5N1和H7N9病毒提供了6個(gè)內(nèi)部基因片段。這些含H9N2病毒基因的新病毒對(duì)哺乳動(dòng)物的感染性增強(qiáng),公共衛(wèi)生學(xué)意義越來(lái)越明顯。因此,做好家禽H9N2亞型AIVs感染的防控工作意義重大。目前,疫苗免疫是我國(guó)防控禽流感的主要手段。AIVs亞型眾多,變異頻繁,給疫苗的研發(fā)帶來(lái)了巨大的困難。病毒樣顆粒(Virus-like particles,VLPs)是新型流感疫苗研究的一種策略,該疫苗不依賴于傳統(tǒng)的雞胚生產(chǎn)系統(tǒng),而采用體外細(xì)胞培養(yǎng)制備病毒抗原。VLPs具有與病毒粒子相似的天然構(gòu)象,因其不含有病毒的基因組,因此具有更高的安全性。本研究以H9N2亞型禽流感病毒中的57基因型為研究對(duì)象,選取HA、NA、M1共表達(dá)的方式進(jìn)行VLP的構(gòu)建。本研究首先構(gòu)建重組轉(zhuǎn)移載體pFBDHA-NA-M1共表達(dá)載體,使用p FastBac Dual載體,將M1基因克隆到P10啟動(dòng)子下游,將HA、NA兩個(gè)基因克隆到PpH啟動(dòng)子下游,即pFBD-HA-NA-M1。重組轉(zhuǎn)移質(zhì)粒p FBD-HA-NA-M1轉(zhuǎn)化至E.coli DH10Bac感受肽細(xì)胞,通過(guò)轉(zhuǎn)座將插入的目的片段轉(zhuǎn)移到重組桿狀病毒DNA上。然后,通過(guò)藍(lán)白斑篩選及PCR鑒定后便得到了重組桿粒rBacmid-HA-NA-M1。將桿粒轉(zhuǎn)染至Sf21昆蟲細(xì)胞,27℃培養(yǎng)72小時(shí)后收獲上清。將上清盲傳3代后,通過(guò)Western-blot、電鏡觀察等方法進(jìn)行蛋白表達(dá)及VLP組裝的鑒定。Western-blot檢測(cè)發(fā)現(xiàn):在62KD、54KD、30KD處檢測(cè)出明顯的蛋白條帶,電鏡觀察結(jié)果表明,本研究獲得了與病毒顆粒外觀相似的,大小在100 nm左右的球狀顆粒。本研究成功利用昆蟲桿狀病毒表達(dá)系統(tǒng),以三種結(jié)構(gòu)蛋白組裝成了H9N2亞型的病毒樣顆粒,為今后的疫苗研究奠定了基礎(chǔ)。
[Abstract]:Avian influenza A (AI) is a major infectious disease that endangers poultry industry in China, and is the focus of prevention and control of avian diseases at present and in the future. Avian influenza virus (Ave), which is a prevalent avian influenza virus in China, is mainly caused by low pathogenic H9N2 subtype virus. H9N2 virus infection can lead to different respiratory symptoms in chickens, and the laying quantity of laying hens decreases about 10-20 percent, and the immune resistance of chickens decreases. It is easy to infect chicken with or secondary to other bacterial and viral diseases. H9N2 virus can be transmitted between species, which poses a serious threat to human life and safety. H9N2 subtype AIVs is also a "mother virus" of a new influenza virus infected with human beings. Six internal gene fragments were provided for H5N1 and H7N9 viruses in China. The public health implications of these new viruses containing the H9N2 gene are increasing in mammals. Therefore, it is of great significance to prevent and control AIVs infection of H9N2 subtype in poultry. At present, vaccine immunization is the main method to prevent and control avian influenza in China. AIVs subtypes are numerous and frequently mutated, which brings great difficulties to the research and development of vaccine. Virus-like virus like VLPsis a strategy in the study of novel influenza vaccine. The vaccine does not depend on the traditional chicken embryo production system, but uses in vitro cell culture to prepare virus antigen. VLPs have a natural conformation similar to that of virus particles. Because it does not contain the genome of the virus, it has higher safety. In this study, 57 genotypes of H9N2 subtype avian influenza virus were selected to construct VLP. In this study, the recombinant transfer vector pFBDHA-NA-M1 co-expression vector was constructed. M1 gene was cloned into the downstream of P10 promoter using p FastBac Dual vector, and two genes of Hana were cloned into the downstream of PpH promoter, that is, pFBD-HA-NA-M1. The recombinant transfer plasmid p FBD-HA-NA-M1 was transformed into E.coli DH10Bac receptive peptide cells and the inserted target fragment was transferred to the recombinant baculovirus DNA by transposition. Then, the recombinant rBacmid-HA-NA-M1 was obtained by blue and white spot screening and PCR identification. Sf21 insect cells were cultured at 27 鈩，

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