猪繁殖与呼吸综合征病毒核蛋白单克隆抗体的制备和初步鉴定  

Preparation and Identification of the Monoclonal Antibodies against the Nucleoprotein of Porcine Reproductive and Respiratory Syndrome Virus

作　　者：米立娟[1,3] 吕宗吉[2] 张守峰[3] 刘晔[3] 王永志[4] 扈荣良[1,3] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]佛山科学技术学院,佛山528200 [3]军事医学科学院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,长春130122 [4]吉林省农业科学院,长春130133

出　　处：《吉林农业大学学报》2012年第6期661-666,共6页Journal of Jilin Agricultural University

基　　金：农业部公益性行业项目(201203056)

摘　　要：克隆了猪繁殖与呼吸综合征病毒(PRRSV)CC-11株的N基因全序列,将其克隆至原核表达载体pET28a,转化E.coliRosetta株后经0.5 mmol/L IPTG诱导,获得了高水平表达。对表达的N蛋白进行纯化,以50μgN蛋白免疫BALB/c小鼠,3次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)进行融合,通过间接ELISA筛选,获得9B12、7C2、7C6共3株稳定分泌抗PRRSV-N蛋白单克隆抗体的杂交瘤细胞,亚型鉴定结果分别为IgG2b、IgG1、IgG1亚型。通过Western blot分析和间接免疫荧光测定表明,3株单抗对PRRSV CC-11株、CH-1R和JXA1-R细胞毒以及4份临床疑似病料均发生特异性反应。The full sequence of the nucleoprotein gene of porcine reproductive and respiratory syndrome virus strain CC-11 was cloned and inserted into the expression vector pET28a.The resultant expression plasmid was transformed into E.coli Rosetta and induced by 0.5 mmol/L IPTG.High level expression of the nucleoprotein was achieved.After purification of the nucleoprotein by Ni affinity chromatography column,it was used to vaccinate BALB/c mice by 50 μg for 3 times.The spleen cells were fused with SP2/0 cells and after rounds of screening by indirect ELISA,3 hybridoma cell lines,named as 9B12,7C2,7C6,secreting monoclonal antibodies against PRRSV nucleoprotein,were obtained.The subtypes of the monoclonal antibodies are IgG2b,IgG2a and IgG1,respectively.These monoclonal antibodies can react with the PRRSV stains CC-11,CH-1R,and JXA1-R maintained in our laboratory and the 4 suspected clinical specimens.

关 键 词：猪繁殖与呼吸综合征病毒 N蛋白 单克隆抗体 

分 类 号：S858.28[农业科学—临床兽医学]