【作者】 张瑞华；

【导师】 金梅林； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 禽流感(Avian Influenza,AI)是由正粘病毒科A型流感病毒引起的一种禽类的烈性传染病。它是造成全球突发卫生事件最常见的传染病,自1878年意大利鸡群首次爆发该病以来,世界各地都相继有各种亚型的禽流感病毒(AIV)引起的AI暴发。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,尤其是H9亚型禽流感。特别是1997香港禽流感事件以及2003年末至2004年初禽流感在东南亚各国和中国的大面积暴发,引起了人们的高度重视。 A型流感病毒亚型众多,易变。目前,已经发现了15种H亚型和9种N亚型,且各亚型之间无血清学交叉反应。从而导致该病的诊断和治疗非常困难。因此,迫切需要一种较为理想的禽流感亚型诊断方法。血凝素(HA)糖蛋白为病毒最重要的表面抗原,具有亚型特异性,同时可以诱导特异性抗体的产生。因此,利用HA蛋白有望建立一种诊断禽流感亚型的方法。鉴于此,本研究利用禽流感H5、H9亚型HA蛋白作为诊断抗原建立了诊断禽流感亚型的ELISA检测方法。该方法快速、灵敏、准确、简便,可以直接检测到亚型,该方法的建立对进出口检验检疫,禽流感的防制具有十分重要的意义。主要研究内容包括: 1.H5N1、H9N2亚型禽流感病毒血凝素基因的克隆与序列分析 本研究根据GenBank收录的H5N1、H9N2亚型禽流感病毒血凝素基因序列设计并合成引物,利用RT-PCR方法从本室分离鉴定并保存A/Chicken/HuBei/326/2002(H5N1)、A/Duck/HuBei/405/2003(H9N2)禽流感病毒中扩增了血凝素基因,将两种扩增产物分别克隆进pMD18-T载体,限制性酶切及序列测定用以筛选阳性克隆子。结果发现H5N1HA、H9N2HA分别为1707bp和1683bp。 2.H5N1、H9N2亚型禽流感病毒血凝素基因的亚克隆及原核表达 基于HA蛋白的信号肽在表达中的负面作用,本研究通过基因工程手段缺失HA蛋白位于起始的信号肽的编码序列,获得了缺失HA蛋白信号肽的HA基因,并将其亚克隆到pGEX-KG中,与GST蛋白融合表达。SDS-PAGE结果显示:融合表达的蛋白分子量分别约为90KDa、92KDa。Western blot印迹表明表达蛋白具有免疫学活性,位于包涵体中。更多还原

【Abstract】 Avian influenza (AI) is an infectious disease caused by influenza A virus, a member of the family Orthomyxoviridae. It is the most common infectious disease which happens suddenly in the whole world. Since the disease have broken out in Italian chicken flocks for the first time in 1878, Avian Influenza Virus break out in succession in all parts of 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, especially the H9 AIV. In particular the 1997 Hong Kong avian influenza event and the influenza in Southeast Asia countries including China at the end of 2003 as well as the beginning of 2004, which aroused serious concern of the government and people all over the world. Avian influenza of A type has many subtypes and changes to a new strand easily. To date, 15 Hemagglutinin(HA,H)subtypes and 9 neuraminidase(NA,N) subtypes have been recognized. There are not cross -reaction among the different subtypes, which make the diagnosis and prevention very difficult. Consequently, the comparatively ideal subtype diagnosis is in the great need. Hemagglutinin (HA) is the most important surface antigen of virus and has subtype speciality, which induce the peculiar antibody. Therefore, it is feasible to set up subtype diagnosis using the HA protein. In the paper, indirect ELISA to detect antiboies against H5, H9 Avian Influenza Virus was developed using the HA protein expressed in E.coli as antigen. So the following researchs were explored:1. Cloning and sequence analysis of H5 and H9 HA genesAccording to published HA genes sequence of H5 and H9 AIV, primers were designed and synthesized. A 1683bp and 1707 bp fragments were amplified from Avian Influenza virus RNA (A/Chicken/HuBei/326/2002(H5Nl) , A/Duck/HuBei/405/2003 (H9N2)) by RT-PCR and cloned into the pMD18-T vector.The recombinant plasmid was proved to be true by enzyme analysis and sequencing. The sequence analysis showed that H5 and H9 HA gene consisted of 1683bp and 1707 bp respectively.2. Subcloning and expression of H5, H9 HA gene in E.coliBased on the negative function of signal peptide of HA gene in expression,we lackedthe signal peptide of HA gene through the genetic engineering means and obtained the HA gene with the signal peptide removed. Then the fragment of HA was subcloned into prokaryotic expression vector pGEX-KG and was expressed in E.coli B121. The expressed HA-GST fusion protein in E.coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90KDa and 92KDa protein with immunogenicity.3. Development of indirect H5HA-ELISA in Diagnosis the antibodies of H5 AIV Subtype ^ H9HA-ELISA in Diagnosis the antibodies of H9 AIV SubtypeThe fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The indirect ELISA method was established by selecting the conditions with the optimal dilution of antigen of HA protein to be 1:80, the optimal dilution of serum to be 1:80. It revealed a negative reaction with positive sera of Marek’s disease, Newcastle disease, Parainfluenza, infectious bronchitis, infectious laryngotracheitis, infectious bursal disease, aviadenovirus I, aviadenovirus III, standard negative serum and serum of avian uninfected by avian influenza. It revealed a positive reaction with some type of avian influenza standard positive serum and sera from avian influenza. In order to know reliability of the ELISA, a repetitive experiment was conducted too. The 8 sera samples was used. A total of 339 chicken sera samples was used in HA and ELISA to detect the specificity, sensitivity and reduplication. The above results indicated that the assay has good specificity, high sensitivity and excellent reduplication. It could be used to differently detect antibodies to H5 and H9 ATV. At present, at the same time, a total of 856 chicken sera samples from hubei province was detect and the result showed that the ELISA can be used in in Diagnosis the antibodies of H5 AIV Subtype and H9 AIV Subtype.更多还原