抗乙型流感病毒卵黄抗体的制备及其生物活性鉴定研究

发布时间：2018-08-23 08:36

【摘要】：流行性感冒是最常见的急性病毒性疾病之一,病患者会出现不同的程度的全身性症状如轻度疲劳、呼吸衰竭、甚至死亡。大约10-20%世界人口受季节性流感影响,每年导致250,000-500,000人死亡。乙型流感病毒是引起季节性流感的局部爆发的主要病原体之一。儿童感染乙型流感病毒,接受阿司匹林治疗后可能会产生一种罕见的Reye's,综合症,对大脑和肝脏均有严重影响。目前在预防和治疗乙型流感病毒感染方面,医药界已经付出很多努力。抗病毒药物,如神经氨酸酶抑制剂,被广泛用于治疗流感。然而,乙型流感病毒对这些药物日益具有耐药性。虽然接种疫苗可以提供保护作用,但是现有的疫苗几乎没有治疗方面的效果。近年来,人们基于被动免疫治疗的原理,制备特异性卵黄抗体(IgY)代替抗生素治疗病原菌和病毒引起的多种疾病,具有安全、无耐药性和成本低廉等优点。本研究以灭活乙型流感病毒免疫产卵母鸡,PEG沉淀法和硫酸铵盐析分离纯化特异性IgY抗体。然后通过SDS-PAGE、ELISA分析抗体纯度和效价；以琼脂扩散法和Western blot检测IgY对病毒颗粒及其功能蛋白的特异性反应；以血凝抑制试验和病毒空斑减数试验研究其体外抗病毒活性；通过动物实验研究其体内抗病毒活性。此外,本研究还制备了IgY抗体量子点探针,进行乙型流感病毒检测方面的探索。结果表明,首次免疫两周后,鸡卵黄中即可检测到抗体效价；初次免疫5周后效价逐步上升到峰值1：3200。经两步PEG沉淀提取和硫酸铵盐析纯化,每毫升卵黄可获得5.2mg IgY,纯度为98.2%；琼脂扩散试验和Western blot表明制备的特异性IgY不但能够沉淀病毒颗粒,而且对病毒膜蛋白神经氨酸酶和血凝素及其亚基均有特异性反应。体外抗病毒试验显示,IgY抗体血凝抑制效价为1：128,对活病毒的半数抑制浓度达到1.68mg/mL。动物试验证明,特异性IgY抗体能够清除攻毒小鼠肺部病毒从而降低病毒滴度及减轻病症。IgY量子点探针检测病毒结果表明,病毒蛋白含量与荧光强度之间存在线性关系,最低检测病毒浓度为2.834×10-7mg/mL。综上,本研究成功研制了高纯度和特异性的抗乙型流感病毒IgY抗体,该抗体在体内外均有抗病毒活性,在免疫诊断检测中有较高灵敏度。为IgY抗体作为抗流感药物和诊断试剂的应用开发奠定了基础。
[Abstract]:Influenza is one of the most common acute viral diseases with varying degrees of systemic symptoms such as mild fatigue respiratory failure and even death. About 10-20% of the world's population is affected by seasonal influenza, accounting for 2, 500-500, 000 deaths a year. Influenza B virus is one of the major pathogens of seasonal influenza outbreaks. Children infected with influenza B virus may develop a rare form of Reyez syndrome after aspirin treatment with severe effects on the brain and liver. At present, the medical industry has made a lot of efforts to prevent and treat influenza B infection. Antiviral drugs, such as neuraminidase inhibitors, are widely used in the treatment of influenza. However, influenza B virus is increasingly resistant to these drugs. While vaccination can provide protection, existing vaccines have little therapeutic effect. In recent years, based on the principle of passive immunotherapy, the preparation of specific yolk antibody (IgY) instead of antibiotics for the treatment of various diseases caused by pathogens and viruses has the advantages of safety, no drug resistance and low cost. In this study, the specific IgY antibody was isolated and purified by inactivated influenza B virus (Influenza B) immunized laying hens by PEG precipitation and ammonium sulfate saltout. Then the purity and titer of antibody were analyzed by SDS-PAGEG Elisa, the specific reaction of IgY to virus particles and its functional proteins was detected by Agar diffusion method and Western blot, the antiviral activity in vitro was studied by hemagglutination inhibition test and virus plaque reduction test. Its antiviral activity in vivo was studied by animal experiments. In addition, the IgY antibody quantum dot probe was prepared to detect influenza B virus. The results showed that antibody titers were detected in the yolk two weeks after the first immunization, and gradually increased to a peak value of 1: 3200 after the first immunization for 5 weeks. After two steps of PEG precipitation extraction and ammonium sulfate salting-out purification, 5.2mg IgYs can be obtained per milliliter of egg yolk, the purity is 98.20.Agar diffusion test and Western blot show that the specific IgY can not only precipitate virus particles, Moreover, there was a specific reaction to neuraminidase, hemagglutinin and its subunits of viral membrane protein. The antiviral test in vitro showed that the hemagglutination inhibition titer of IGY antibody was 1: 128, and the half inhibitory concentration to live virus was 1.68 mg / mL. Animal experiments showed that the specific IgY antibody could eliminate the lung virus and reduce the titer of the virus and alleviate the disease. The results showed that there was a linear relationship between the viral protein content and the fluorescence intensity. The lowest detected virus concentration was 2.834 脳 10 ~ (-7) mg / mL. In conclusion, a high purity and specificity IgY antibody against influenza B virus was successfully developed. The antibody has antiviral activity in vivo and in vitro, and has high sensitivity in immunological diagnosis. It lays a foundation for the application and development of IgY antibodies as anti-influenza drugs and diagnostic reagents.
【学位授予单位】：广东工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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