摘要
为了制备H9N2亚型禽流感病毒(AIV)的单克隆抗体,从而为建立AIV的诊断方法提供有效试剂,试验将纯化后的H9N2亚型AIV血凝素(HA)蛋白作为免疫原注射至Balb/c小鼠腹腔中,取符合融合标准的小鼠脾脏制成悬液,同小鼠骨髓瘤细胞(SP2/0)进行融合,制备杂交瘤细胞;建立单克隆细胞株的筛选方法以得到能够稳定分泌抗体的单克隆细胞株,并对其分泌的单克隆抗体进行纯化,测定效价及浓度,并分别检测其与H9N2标准病毒的反应性及特异性。结果表明:经过筛选后共获得3株可持续分泌抗H9N2 AIV HA的单克隆细胞株,分别以2D10D10、1F10E7和5A10C3命名。其中1F10E7细胞株抗体效价可达到1∶128000,浓度最高可达到2.4319 mg/mL;经亚型鉴定,以上3株杂交瘤细胞均为IgG1型。通过特异性试验检测,制备所得HA单克隆抗体只与纯化后的AIV HA抗原结合且与H9N2标准病毒能够发生反应。说明试验制备了针对AIV HA蛋白的单克隆抗体,可为后续禽流感检测方法的研究提供有效试剂。
In order to prepare monoclonal antibodies(McAb)against H9 N2 subtype avian Influenza A virus(AIV)and to provide an effective reagent for the establishment of AIV diagnostic methods,in the experiment,the purified H9 N2 subtype AIV hemagglutinin(HA)protein was injected into the abdominal cavity of Balb/c mice as an immunogen;the mouse spleen that met the fusion standard was scraped to make a suspension,which was fused with mouse myeloma cells(SP2/0)to prepare hybridoma cells.The monoclonal cell line screening method was established to obtain the monoclonal cell line that could secrete antibody stably;the secretory monoclonal antibody was purified,and the concentration and titer were determined;and the reaction and specificity with the subtype and H9 N2 standard virus were identified.The results showed that three monoclonal cell lines that could continuously secrete anti-H9 N2 AIV HA were obtained after screening,named 2 D10 D10,1 F10 E7 and 5 A10 C3.Among them,the antibody titer of 1 F10 E7 cell line was 1∶128000;the maximum concentration was 2.4319 mg/mL.By subtype identification,the above three hybridoma cells were all of IgG1 type.Through specificity test detection,the prepared HA monoclonal antibody only binded to the purified AIV HA antigen and could react with H9 N2 standard virus.The results suggested that the monoclonal antibodies against the AIV HA protein were prepared,and an effective reagent was provided for the subsequent research on the detection method of avian influenza.
作者
侯力嘉
吴胜昔
龚吕鸿
刘姿怡
杨溪
郭宇
梁望旺
HOU Lijia;WU Shengxi;GONG Lyuhong;LIU Ziyi;YANG Xi;GUO Yu;LIANG Wangwang(College of Pharmacy and Biological Engineering,Chongqing University of Technology,Chongqing 400054,China;Department of Blood Transfusion,Laboratory Medicine Center,The Second Affiliated Hospital,Army Medical University,Chongging 400037,China;Chongqing City Animal Disease Prevention and Control Center,Chongqing 401120,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2022年第16期66-70,75,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
重庆市技术创新与应用示范项目(cstc2019jscx-msxmX0370,cstc2018jscxmszdX0049)
重庆市巴南区科技计划项目(2020TJZ008)
重庆理工大学研究生创新基金项目(lgycx 20202128)
大学生创新创业训练计划项目(2020CX059,2020CX064)
重庆市生猪产业技术体系项目(20211105)。
关键词
禽流感病毒
单克隆抗体
血凝素蛋白
特异性
检测试剂
细胞融合
avian Influenza A virus
monoclonal antibody
hemagglutinin protein
specificity
detection reagent
cell fusion
