摘要
目的制备抗猪呼吸与生殖综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)Nsp9蛋白单克隆抗体,并进行初步鉴定。方法用纯化的重组Nsp9蛋白免疫BALB/c小鼠,采用杂交瘤技术制备抗PRRSV Nsp9蛋白单克隆抗体,采用Western blot和间接免疫荧光试验分析单抗的特异性;使用免疫球蛋白标准亚类鉴定试剂盒鉴定单抗的亚类;将杂交瘤细胞分别保存1、2、3、4个月后复苏,采用间接ELISA法检测细胞分泌抗体的能力。结果共获得3株可稳定分泌特异性单抗的杂交瘤细胞株,分别命名为2D6、3C11和4E6,其细胞培养上清的ELISA效价分别为1∶6 400、1∶3 200和1∶1 600,腹水的ELISA效价分别为1∶640 000、1∶512 000和1∶256 000。3株单抗均可与Nsp9蛋白和PRRSV特异性结合;2D6和3C11株单抗的Ig亚型为IgG1,4E6株单抗为IgM亚类抗体;3株杂交瘤细胞保存4个月均能稳定分泌单抗。结论已制备抗PRRSVNsp9蛋白单克隆抗体,其特异性高,稳定性良好,为建立鉴别自然感染与注射疫苗感染PRRSV的诊断方法提供了材料。
Objective To prepare and preliminarily identify the monoclonal antibodies（McAbs） against Nsp9 protein of porcine reproductive and respiratory syndrome（PRRSV）.Methods BALB / c mice were immunized with purified recombinant Nsp9 protein,based on which McAbs were prepared by hybridoma technique,then analyzed for specificity by Western blot and indirect IFA,and identified for subclass by IgG standard subclass identification kit.The hybridoma cells were resuscitated after storage for 1,2,3 and 4 months respectively and determined for antibody-secreting ability by ELISA.Results Three hybridoma cell strains stably secreting specific McAbs,named as 2D6,3C11 and 4E6,were obtained,by which the secreted McAbs reached ELISA titers of 1 ∶ 6 400,1 ∶ 3 200 and 1 ∶ 1 600 in culture supernatant,and 1 ∶ 640 000,1 ∶ 512 000 and 1 ∶ 256 000 in ascites,respectively.All the McAbs secreted by three strains showed specific binding to Nsp9 and PRRSV.The McAbs secreted by 2D6 and 3C11 strains were IgG1,while that by 4E6 strain were IgM.All the three strains secreted McAbs stably after storage for 4 months.Conclusion The McAbs against PRRSV Nsp9 were prepared,and showed high specificity and stability,which provided a material for development of method for identification of natural infection and vaccine-associated infection of PRRSV.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第9期1180-1184,共5页
Chinese Journal of Biologicals
基金
吉林大学大学生创新性实验项目(2011C81326)
关键词
猪呼吸与生殖综合征病毒
Nsp9蛋白
单克隆抗体
Porcine reproductive and respiratory syndrome virus（PRRSV）
Nsp9 protein
Monoclonal antibody
