猪细小病毒NS1蛋白单克隆抗体的制备与鉴定
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摘要
为制备针对猪细小病毒(PPV)NS1蛋白的单克隆抗体,本研究以原核表达的NS1重组蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0细胞进行融合,经间接ELISA筛选阳性杂交瘤细胞株,获得了3株能稳定分泌抗PPV NS1蛋白的杂交瘤细胞株,间接ELISA检测3株单克隆抗体腹水的抗体效价分别达到1∶2.048×106、1∶4.096×106和1∶2.048×106,染色体数分别为105、104和106,均能与PPV NS1重组蛋白及PRV毒株感染的PK-15细胞发生特异性反应,而与6种常见病毒不发生交叉反应。表明制备的3株NS1蛋白单克隆抗体效价高、特异性强,为NS1蛋白抗原表位分析、结构与功能研究,以及PPV免疫学诊断试剂的开发奠定了基础。
To prepare monoclonal antibodies against porcine parvovirus(PPV)NS1 protein,BALB/c mice were immunized with Prokaryotic Recombinant NS1 protein,and the spleen cells were fused with SP2/0 cells,then the hybridoma cell lines were screened by indirect ELISA,and three hybridoma cell lines stably secreting anti PPV NS1 protein were obtained,the antibody titer of indirect ELISA detection of three strains of monoclonal antibody ascites reached 1:2.048×106,1:4.096×106 and 1:2.048×106,chromosome number were 105,104,106,they all reacted specifically with PPV NS1 recombinant protein and PRV infected PK-15 cells,but did not cross react with six common viruses.The results show that the three monoclonal antibodies against NS1 protein has high titer and strong specificity,which lays a foundation for NS1 protein epitope analysis,structure and function study,and the development of PPV immunological diagnostic reagents.
To prepare monoclonal antibodies against porcine parvovirus(PPV)NS1 protein,BALB/c mice were immunized with Prokaryotic Recombinant NS1 protein,and the spleen cells were fused with SP2/0 cells,then the hybridoma cell lines were screened by indirect ELISA,and three hybridoma cell lines stably secreting anti PPV NS1 protein were obtained,the antibody titer of indirect ELISA detection of three strains of monoclonal antibody ascites reached 1:2.048×106,1:4.096×106 and 1:2.048×106,chromosome number were 105,104,106,they all reacted specifically with PPV NS1 recombinant protein and PRV infected PK-15 cells,but did not cross react with six common viruses.The results show that the three monoclonal antibodies against NS1 protein has high titer and strong specificity,which lays a foundation for NS1 protein epitope analysis,structure and function study,and the development of PPV immunological diagnostic reagents.
引文
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[7]刘建,汤德元,罗险峰,等.猪细小病毒非结构蛋白NS1基因的克隆、序列分析及蛋白质结构预测[J].中国畜牧兽医,2013,40(5):8-13.
[8]李兴玉,李金海,林毅,等.猪细小病毒疫苗株NS1基因克隆及生物信息学分析[J].中国畜牧兽医,2014,41(7):21-26.
[9]丁晨曦,艾乐乐,龚秀芳,等.2型猪链球菌SrtA蛋白单克隆抗体的制备与鉴定[J].中国病原生物学杂志,2017,12(12):1 144-1 147.
[10]郭忠华.猪细小病毒病的流行病学、临床症状、鉴别诊断和防控措施[J].现代畜牧科技,2017(2):78.
[11]谢红玲,崔尚金,崔玉东,等.抗猪细小病毒单克隆抗体的制备[J].中国预防兽医学报,2009,31(10):813-815.
[12]张倩,李晓霞,董昕欣,等.猪细小病毒单克隆抗体的制备和鉴定[J].中国比较医学杂志,2011,21(9):54-57.
[2]刘立兵,王建昌,经美,等.猪细小病毒重组酶聚合酶扩增快速检测方法的建立[J].中国畜牧兽医,2017,44(11):3 320-3 326.
[3]吴佳俊,张硕,刘颖昳,等.我国种猪主要垂直传播性疫病流行态势分析及控制策略探讨[J].中国动物检疫,2017,34(8):14-16.
[4] REN X,TAO Y,CUI J,et al.Phylogeny and evolution of porcine parvovirus[J].Virus Res,2013,178(2):392-397.
[5]陈龙彪,高晓云,吴宇阳,等.猪细小病毒河南分离株全基因组克隆及序列分析[J].安徽农业科学,2015,43(4):26-29.
[6] YANG Y,QIN X,ZHANG W,et al.Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays[J].Mol Cell Probes,2016,30(5):300-305.
[7]刘建,汤德元,罗险峰,等.猪细小病毒非结构蛋白NS1基因的克隆、序列分析及蛋白质结构预测[J].中国畜牧兽医,2013,40(5):8-13.
[8]李兴玉,李金海,林毅,等.猪细小病毒疫苗株NS1基因克隆及生物信息学分析[J].中国畜牧兽医,2014,41(7):21-26.
[9]丁晨曦,艾乐乐,龚秀芳,等.2型猪链球菌SrtA蛋白单克隆抗体的制备与鉴定[J].中国病原生物学杂志,2017,12(12):1 144-1 147.
[10]郭忠华.猪细小病毒病的流行病学、临床症状、鉴别诊断和防控措施[J].现代畜牧科技,2017(2):78.
[11]谢红玲,崔尚金,崔玉东,等.抗猪细小病毒单克隆抗体的制备[J].中国预防兽医学报,2009,31(10):813-815.
[12]张倩,李晓霞,董昕欣,等.猪细小病毒单克隆抗体的制备和鉴定[J].中国比较医学杂志,2011,21(9):54-57.