H9N2亚型禽流感病毒样颗粒的构建与鉴定

发布时间：2018-05-28 09:51

  本文选题：流感 + 病毒样颗粒　； 参考：《山东农业大学》2017年硕士论文

【摘要】：禽流感(Avian influenza,AI)是危害我国养禽业的重大传染病,是目前和今后禽病防控的重点。我国禽群中流行的禽流感病毒(Avian influenza virus,AIV)以低致病性H9N2亚型病毒为主。H9N2病毒的感染可导致鸡不同程度的呼吸道症状,产蛋鸡产蛋量下降约10-20%,而且可造成鸡免疫抵抗力下降,感染鸡容易并发或继发其它各种细菌和病毒性疾病。H9N2病毒可以在种间传播,给人类的生命安全造成严重的威胁。H9N2亚型AIVs还是感染人类的新型流感病毒的“母病毒”,为国内流行的鸡源H5N1和H7N9病毒提供了6个内部基因片段。这些含H9N2病毒基因的新病毒对哺乳动物的感染性增强,公共卫生学意义越来越明显。因此,做好家禽H9N2亚型AIVs感染的防控工作意义重大。目前,疫苗免疫是我国防控禽流感的主要手段。AIVs亚型众多,变异频繁,给疫苗的研发带来了巨大的困难。病毒样颗粒(Virus-like particles,VLPs)是新型流感疫苗研究的一种策略,该疫苗不依赖于传统的鸡胚生产系统,而采用体外细胞培养制备病毒抗原。VLPs具有与病毒粒子相似的天然构象,因其不含有病毒的基因组,因此具有更高的安全性。本研究以H9N2亚型禽流感病毒中的57基因型为研究对象,选取HA、NA、M1共表达的方式进行VLP的构建。本研究首先构建重组转移载体pFBDHA-NA-M1共表达载体,使用p FastBac Dual载体,将M1基因克隆到P10启动子下游,将HA、NA两个基因克隆到PpH启动子下游,即pFBD-HA-NA-M1。重组转移质粒p FBD-HA-NA-M1转化至E.coli DH10Bac感受肽细胞,通过转座将插入的目的片段转移到重组杆状病毒DNA上。然后,通过蓝白斑筛选及PCR鉴定后便得到了重组杆粒rBacmid-HA-NA-M1。将杆粒转染至Sf21昆虫细胞,27℃培养72小时后收获上清。将上清盲传3代后,通过Western-blot、电镜观察等方法进行蛋白表达及VLP组装的鉴定。Western-blot检测发现:在62KD、54KD、30KD处检测出明显的蛋白条带,电镜观察结果表明,本研究获得了与病毒颗粒外观相似的,大小在100 nm左右的球状颗粒。本研究成功利用昆虫杆状病毒表达系统,以三种结构蛋白组装成了H9N2亚型的病毒样颗粒,为今后的疫苗研究奠定了基础。
[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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