【作者】 刘守川；

【导师】 王泽霖；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 将低致病性H9N2亚型和H5N2亚型禽流感病毒经著速离心和蔗糖密度梯度离心纯化后，免疫Balb/c小鼠，应用淋巴细胞杂交瘤技术，通过ELISA和HI筛选，建立了抗H5和H9N2亚型禽流感病毒杂交瘤细胞株3E4、2A4，其分泌抗体能力稳定，为针对HA蛋白的单抗，3E4分泌的单克隆抗体能与H5N2亚型N₂₈株发生血凝抑制反应，不与H9N2亚型Hp株发生反应：2A4与H9N2亚A／鸡／河南／A₂／99（H₉N₂）株、A／鸡／淇县／A₁／01（H₉N₂）株、A／鸡／淇县／A₁／04（H₉N₂）株AIV发生血凝抑制反应，而不与H5N2亚型AIV发生反应。两株单抗与H1N1、H3N2 SIV、NDV、IBDV、EDSV及IBV均无交叉反应，表明该单抗分别具有AIV H5和H9亚型特异性。3E4杂交瘤细胞培养上清和腹水HI效价分别为5、7（log2），ELISA效价分别为6400和12800；2A4杂交瘤细胞培养上清和腹水HI效价为8 log2和16 log2，ELISA效价为12800和256000。这两株杂交瘤细胞分泌的抗体均有良好的热稳定性。腹水经辛酸硫酸铵纯化后，蛋白含量分别7.46mg／mL、14.9 mg／mL。此两株单克隆抗体的获得为禽流感病毒快速亚型鉴别检测方法的建立，以及研究AIV H5亚型和H9亚型血凝素（HA）抗原性分析、变异规律和变异株的检测等奠定了基础。 根据我国当前禽流感H9亚型的变异和流行现状，结合国内外在HA的抗原性变异的研究情况，利用本文建立的H9亚型单克隆抗体2A4和河南农业大学禽病研究所研制保存的另三株单抗F6、H、B4，与1998～2005年间河南农大禽病研究所分离到的25个H9亚型禽流感毒株进行HI反应。25个毒株经除菌，有限稀释法纯化，特异性鉴定和RT—PCR鉴定确定为H9N2亚型。结果显示，25个毒株可以分为二类，第一类23个毒株与H6单抗的HI结果大部分都是17、18；F6单抗与25株H9AIV的HI结果大部分在15～18之间；A4单抗与25株H9AIV的HI结果也比较整齐，大部分在16～18之间；B4单抗的与25株H9AIV的HI结果则相对不是那么整齐，但大部分也都在7～11之间：另一类2个毒株即3#A／鸡／河南／A₃／98（H₉N₂）株和18#A／鸡／河南／C₄／02（H₉N₂）株与所有4株单抗都不发生反应。说明在H9N2亚型AIV表面抗原上的具有血凝活性的抗体结合位点不止一个，3#和18#毒株的该位点已经变异或缺失。四株单抗分别做病毒中和试验。发现均无中和活性，说明表面抗原上的血凝活性位点并非就是中和活性位点。本试验的研究结果进一步证实了H9N2亚型AIV血凝素抗原的变异特性，将为H9N2亚型AIV血凝素表面抗原结构与功能及变异规律的深入研究以及我国的禽流感H9亚型的流行疫苗株的选择提供了依据。更多还原

【Abstract】 The low pathogenic AIV of H9N2 and H5N2 subtypes were grown in chicken embryo eggs and purified by differential centrifugation and density centrifugation. Eight-week Balb/c mice were immunized according to Kazuhiro’s produce. Two hybridoma named 3E4、 2A4 were generated by fusion of SP20 myelomas and splenocytes from immunized mice and cloned. The two monoclonal antibodies（McAb） produced was screened by ELISA and HI. One of 3E4 against H5N2 subtype HA mouse ascetic fluid（HI 2⁷、 ELISA 12800） was purified（protein concentration:7.46mg/mL） and proved to react with only H5N2 AIV in HI and ELISA tests. The other 2A4 against H9N2 subtype mouse ascetic fluid(HI 2¹⁶、 ELISA 256000) was purified （protein concentration: 14.9mg/mL） and proved to react with only 4 H9N2 subtype strains in HI and ELISA tests. Both of them didn’t react with H1N1、 H3N2SIV、 NDV、 IBDV、 EDSV and IBV in cross-reaction of HI and ELISA. The result indicated that the two McAbs were H5N2 and H9N2 subtype-specific.According to the antigenic variation and the pandemic condition of H9N2 subtype AIV in China.It shown that the variant of H9N2 subtype AIV HA was often and universal. Four monoclonal antibodies named 2A4、 B4、 F6 and H6 react with 25 H9N2 subtype strains from different areas of the whole nation between 1998 and 2005 by the hemagglutination-inhibition （HI）tests. It was sure that the 25 strains purified by filling and limited dilution and identified by HI cross-reaction with anti-sera and RT-PCR were H9N2 subtype. The result from the HI tests with four McAbs indicate that the 25 strains could be divided into two groups. The first group, the 3^rd and the 18^th.strains, have no reaction to the 4 McAbs. Nevertheless, the second group, the rest of 23 strains, reacted to the 4 McAbs strongly. It revealed that the hemagglutination antigenic site of the 3^rd and 18^th H9N2 subtype AIV bounding to the McAbs has varied or lost.,which need further study.更多还原