【摘要】：流行性感冒是最常見的急性病毒性疾病之一,病患者會(huì)出現(xiàn)不同的程度的全身性癥狀如輕度疲勞、呼吸衰竭、甚至死亡。大約10-20%世界人口受季節(jié)性流感影響,每年導(dǎo)致250,000-500,000人死亡。乙型流感病毒是引起季節(jié)性流感的局部爆發(fā)的主要病原體之一。兒童感染乙型流感病毒,接受阿司匹林治療后可能會(huì)產(chǎn)生一種罕見的Reye's,綜合癥,對(duì)大腦和肝臟均有嚴(yán)重影響。 目前在預(yù)防和治療乙型流感病毒感染方面,醫(yī)藥界已經(jīng)付出很多努力�？共《舅幬�,如神經(jīng)氨酸酶抑制劑,被廣泛用于治療流感。然而,乙型流感病毒對(duì)這些藥物日益具有耐藥性。雖然接種疫苗可以提供保護(hù)作用,但是現(xiàn)有的疫苗幾乎沒有治療方面的效果。近年來,人們基于被動(dòng)免疫治療的原理,制備特異性卵黃抗體(IgY)代替抗生素治療病原菌和病毒引起的多種疾病,具有安全、無耐藥性和成本低廉等優(yōu)點(diǎn)。 本研究以滅活乙型流感病毒免疫產(chǎn)卵母雞,PEG沉淀法和硫酸銨鹽析分離純化特異性IgY抗體。然后通過SDS-PAGE、ELISA分析抗體純度和效價(jià)；以瓊脂擴(kuò)散法和Western blot檢測(cè)IgY對(duì)病毒顆粒及其功能蛋白的特異性反應(yīng)；以血凝抑制試驗(yàn)和病毒空斑減數(shù)試驗(yàn)研究其體外抗病毒活性；通過動(dòng)物實(shí)驗(yàn)研究其體內(nèi)抗病毒活性。此外,本研究還制備了IgY抗體量子點(diǎn)探針,進(jìn)行乙型流感病毒檢測(cè)方面的探索。 結(jié)果表明,首次免疫兩周后,雞卵黃中即可檢測(cè)到抗體效價(jià)；初次免疫5周后效價(jià)逐步上升到峰值1：3200。經(jīng)兩步PEG沉淀提取和硫酸銨鹽析純化,每毫升卵黃可獲得5.2mg IgY,純度為98.2%；瓊脂擴(kuò)散試驗(yàn)和Western blot表明制備的特異性IgY不但能夠沉淀病毒顆粒,而且對(duì)病毒膜蛋白神經(jīng)氨酸酶和血凝素及其亞基均有特異性反應(yīng)。體外抗病毒試驗(yàn)顯示,IgY抗體血凝抑制效價(jià)為1：128,對(duì)活病毒的半數(shù)抑制濃度達(dá)到1.68mg/mL。動(dòng)物試驗(yàn)證明,特異性IgY抗體能夠清除攻毒小鼠肺部病毒從而降低病毒滴度及減輕病癥。IgY量子點(diǎn)探針檢測(cè)病毒結(jié)果表明,病毒蛋白含量與熒光強(qiáng)度之間存在線性關(guān)系,最低檢測(cè)病毒濃度為2.834×10-7mg/mL。 綜上,本研究成功研制了高純度和特異性的抗乙型流感病毒IgY抗體,該抗體在體內(nèi)外均有抗病毒活性,在免疫診斷檢測(cè)中有較高靈敏度。為IgY抗體作為抗流感藥物和診斷試劑的應(yīng)用開發(fā)奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 李向忠;吳新偉;劉維斯;王鳴;陳藝韻;伍業(yè)健;;廣州2006年乙型流感病毒血凝素和神經(jīng)氨酸酶基因特性分析[J];病毒學(xué)報(bào);2007年05期

2 李瑤琛;趙志剛;李康生;;RNA干擾對(duì)流感病毒NP和PA基因表達(dá)及病毒增殖的抑制[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2006年08期

3 曹康,張衛(wèi)東,施橋發(fā),李虹,蔣中華,李明遠(yuǎn);2種純化流感病毒方法的比較研究[J];河北醫(yī)科大學(xué)學(xué)報(bào);2005年03期

4 陳偉;沈鶴柏;陳艷;周麗佳;徐文濤;;新型熒光物質(zhì)——量子點(diǎn)在生命科學(xué)領(lǐng)域的應(yīng)用研究進(jìn)展[J];化學(xué)通報(bào);2007年06期

5 趙湖珊,呂利雄,朱順和;早期流感應(yīng)用磷酸奧司他韋的療效觀察[J];中國臨床醫(yī)學(xué);2004年05期

6 王桂平;廖洪映;劉新艷;韋敏;周瓊;;抗蝰蛇毒卵黃抗體的制備及其免疫特性研究[J];中國熱帶醫(yī)學(xué);2008年08期

7 潘泓;陳程;詹迪深;陳學(xué)文;孔天翰;;抗中華眼鏡蛇毒雞卵黃抗體IgY的動(dòng)物保護(hù)作用研究[J];蛇志;2009年02期

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