H5亚型禽流感病毒HA蛋白单克隆抗体的制备与鉴定  

Preparation and characterization of monoclonal antibodies against HA protein of H5 subtype avian influenza virus

作　　者：卢昆鹏[1,2] 崔鹏飞[2] 张芳[2] 王晶[1,2] 肖红波[1] 邓国华[2] 陈化兰[2] 

机构地区：[1]湖南农业大学动物医学学院,湖南长沙410128 [2]中国农业科学院哈尔滨兽医研究所国家禽流感参考实验室/兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2015年第5期453-457,共5页Chinese Veterinary Science

基　　金：兽医生物技术国家重点实验室研究项目(SKLVBP201405)

摘　　要：为制备Clade 2.3.2分支H5亚型禽流感病毒HA蛋白的单克隆抗体,以灭活的超速离心浓缩纯化的H5N1亚型禽流感病毒A/duck/Guangdong/S1322/2010(DK/GD/S1322/2010)免疫6~8周龄的雌性BALB/c小鼠。取免疫后小鼠脾细胞,使其与骨髓瘤细胞(SP2/0)融合,通过血凝抑制(HI)和ELISA方法筛选,共获得2株能够稳定分泌HA蛋白特异性单克隆抗体的杂交瘤细胞株A4和D11。2株单克隆抗体的重链均为IgG1,轻链均为κ链。经HI检测,A4和D11的细胞培养上清和腹水的抗体效价分别为23、22和211、211。HI和中和试验结果显示,这2株单克隆抗体与Clade 2.3.2.B分支和Clade 2.3.2.C分支的病毒反应性良好,而与Clade 2.3.4和Clade 7分支的病毒均不反应。间接免疫荧光试验结果表明,这2株单克隆抗体能够识别MDCK细胞内感染的H5亚型禽流感病毒DK/GD/S1322/2010。结果表明,本研究制备的2株单克隆抗体为H5亚型禽流感病毒的检测和诊断奠定了物质基础。To prepare monoclonal antibodies(McAbs)against HA protein of Clade 2.3.2 H5 subtype avian influenza virus(AIV),six to eight-week old female BALB/c mice were immunized with the inactivated AIV A/duck/Guangdong/S1322/2010(DK/GD/S1322/2010),which was purified by ultracentrifugation.Mouse splenic cells were fused with SP2/0cells.By means of HI and ELISA tests,we got two hybridoma cell strains,then designated as A4 and D11,respectively.The heavy chain of the 2McAbs belongs to IgG1 subclass,and the light chain belongs toκ.By HI test,the antibody titers of the cell supernatants and ascites of A4 and D11were 23,22 and 211,211,respectively.HI and neutralizing activity tests showed that the2 McAbs reacted well with the H5 subtype AIVs of Clade 2.3.2.B and Clade 2.3.2.C,but did not reacted with the H5 subtype AIVs of Clade 2.3.4and Clade 7.Indirect immunofluorescence assay indicated that the 2 McAbs reacted well with H5 subtype AIV DK/GD/S1322/2010 in MDCK cells.The results showed that the McAbs would be useful for the surveillance and diagnosis of H5 subtype AIVs.

关 键 词：禽流感病毒 单克隆抗体 血凝素 

分 类 号：S852.65[农业科学—基础兽医学]