【作者】 刘芳芳；

【导师】 金梅林； 陈焕春；

【作者基本信息】 华中农业大学 ， 生化与分子生物学， 2006， 硕士

【摘要】 禽流感（AI）是由A型流感病毒引起的一种禽类的烈性传染病。自1878年意大利首先发现至今，世界各地都有特定毒株引起的禽流感爆发和流行。尤其是高致病力禽流感可导致高达100％发病率和／或死亡率。并可直接感染人并致人死亡。而低致病力禽流感病毒同样可以给养禽业带来灭顶之灾，对人类的威胁同样严重，目前，高致病性禽流感（HPAI）已被国际兽疫局规定为A类动物传染病。其防制已直接关系到养禽业的持续稳定发展和人民的身体健康。 猪流感病毒（SIV）最突出的流行病学特点是同时具有感染人、禽的潜力，而且猪可最大限度地被AIV和人流感病毒感染。因此，对SI的监测作为预测和防止流感大暴发的最重要手段早已在欧美国家引起关注并达成共识。 A型流感病毒亚型众多，易变。目前，已经发现了16种H亚型和10种N亚型，从而导致该病的诊断和治疗非常困难。国内对流感病毒的临床诊断主要是对抗体的检测。我国许多养鸡场使用禽流感灭活苗，如何区分免疫鸡群与感染鸡群却成为当前面临的一大难题因此，迫切需要一种较为理想的禽流感诊断方法。流感病毒的HA和NA蛋白容易变异，但NP蛋白却相对保守。因此，利用NP蛋白和HA蛋白分别有望建立一种所有亚型禽流感病毒的检测方法和诊断流感病毒亚型的方法。本研究利用杂交瘤技术获得了抗禽流感病毒NP蛋白和H3 HA的单抗，为对流感病毒的进一步研究和建立针对流感病毒的检测方法奠定基础。主要研究内容包括： 分别用甲醛灭活的和Triton x-100裂解的禽流感H9N2病毒，禽流感病毒NP基因的原核表达产物免疫Balb／c小鼠，取免疫脾细胞与SP2／0骨髓瘤细胞融合。经间接ELISA筛选，3次有限稀释法克隆。共得到6株能稳定分泌抗禽流感病毒NP蛋白抗体的杂交瘤细胞株，分别命名为4F4、1C3、1G11、1C2、1D10、2E7。经3次亚克隆后，100％的杂交瘤细胞保持了分泌抗禽流感病毒NP蛋白抗体的能力。染色体计数结果表明所有杂交瘤细胞确实为脾细胞与SP2／0细胞的杂交产物。间接免疫荧光说明单抗的结合活性很好。其中1G11、1D10单克隆抗体小鼠腹水的效价最高，ELISA效价分别为2¹³×100和2¹⁴×100。单克隆抗体1G11、1D10分别为IgG1、IgG2a亚类。 研究结果表明，1G11、1D10单克隆抗体与禽流感病毒的NP蛋白能发生特异性反应，而不与新城疫病毒（NDV）、传染性支气管炎病毒（IBV）、传染性喉气管炎病毒（ILTV）、传染性法氏囊病毒（IBDV）、产蛋下降综合征病毒（EDS-76）、禽腺病毒1型（APMV-1）发生反应。单克隆抗体的获得为禽流感病毒的研究及快速诊断方法的建立奠定了基础。 用H3N8病毒免疫Balb／c小鼠，取免疫脾细胞与SP2／0骨髓瘤细胞融合。经血凝抑制（HI）筛选，3次有限稀释法克隆。共得到3株能稳定分泌抗禽流感病毒HA蛋白抗体的杂交瘤细胞株，分别命名为287、1C11、4G7。经3次亚克隆后，100％的杂交更多还原

【Abstract】 Avian Influenza (AI) is one of fatal infectious disease caused by influenza A virus in avian. It was first reported in Italy in 1878. Subsequently, different strains were found and caused enormous economic losses to the avian industry throughout the world. Especially the Highly Pathogenic Avian Influenza Virus, which can cause up to 100% outbreak rate with or death rate and even can infect mankind. The threat of low pathogenic Avian Influenza Virus is equally serious .At present, highly pathogenic avian influenza (HPAI) is listed in a infectious diseases by World Organization for Animal Health (OIE). Recently, since late 2003, H5N1 HPAIV broke out among poultry throughout Asia, And from late 2005, it has being reported that AI lead to death of fowls and even human beings from various places of the world .Leading to not only tremendous economic losses in poultry industry, but also human infections and deaths. It shocked mankind and the govements of the world. The virus was confirmed in Russia, Turkey, South America ,many European countries and Africa. A common viewpoint is that prevention of AI is closely linked to persistent and steady development of avian industry and humankind’s health.The most outstanding characteristic of Swine influenza virus in epidemiology is that SIV can infect both human and avian contemporary, and pigs as host can be infected furthest by AIV and human IV. So it had been reached a final consensus that detection on SI is the most important way to forecast and prevent IV breaking out.Influenza virus of A type has many subtypes and changes to a new strand easily. Till now, 16 Hemagglutinin (HA,H) subtypes and 10 neuraminidase (NA,N) subtypes have been recognized, which make the diagnosis and prevention very difficult. Domestic diagnostic methods against IV are detection of antibodies and It is also known that a common practice to vaccinate chickens against AIV and monitor AIV more closely. The wide presence of AIV warrants a diagnostic method to differentiate AIV infected and vaccinated birds. As HA-protein of IV changes easily while the NP-protein is conser-vative so it is feasible to set up diagnosis against all Avian Influenza virus of A type using the NP-protein and subtype diagnosis using the HA-protein.This dissertation mainly targets gaining Mab against NP-protein and HA-protein of H3 subtype IV by using hybridoma technique to get further study on IV and set up a usable diagnosis method to detect IV. So the following researchs were explored :Six monoclonal antibodies against NP- protein of avian influenza virus (AIV) were developed by fusing SP2/0 myeloma cells with spleen cells of Balb/c mice immunized更多还原