猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及单克隆抗体制备  

Prokaryotic expression and monoclonal antibodies preparation of PRRSV N protein

作　　者：吴胜昔[1] 邵烈刚[2] 曾政[3] 陈晶[1] 刘人菊[1] 袁丽萍[1] 熊仲良[3] 蔡家利[1] 

机构地区：[1]重庆理工大学药学与生物工程学院,400054 [2]西南大学动物科技学院,重庆400700 [3]重庆市动物疫病预防控制中心,401120

出　　处：《免疫学杂志》2013年第10期894-897,共4页Immunological Journal

基　　金：重庆市科技攻关计划项目(CSTC;2011AB1078;2012GG-YYJS80014);重庆理工大学博士启动基金项目(2011ZD23);重庆理工大学实验创新基金(SC201228)

摘　　要：目的获得原核表达的PRRSV N蛋白,并制备N蛋白的单克隆抗体。方法将已构建的含有PRRSV N蛋白基因的重组表达载体阳性菌,在37℃条件下,用终浓度1 mmol/L的IPTG诱导表达4 h。经SDS-PAGE分析,表明N基因在大肠杆菌中得到了高效表达。经镍柱亲和层析纯化,以此蛋白作为抗原采用长程免疫法免疫Balb/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合。结果获得了纯度较高、免疫学活性较好的N蛋白,细胞融合率为81.3%。建立间接ELISA,用于检测杂交瘤细胞上清中的单克隆抗体。初检阳性率为17.3%,经2次亚克隆反复筛选获得了2株分泌抗PRRSV单抗的杂交瘤细胞株,命名为4G10和2F9。ELISA证实所获得的单抗是特异性针对PRRSV共同的抗原表位,4G10和2F9经反复冻存、复苏及多次传代,仍能分泌高效价抗体。结论成功制备出高效价的抗PRRSV N蛋白的单克隆抗体,为建立一种快速、特异的PRRSV检测方法奠定了基础。A large body of information shows that porcine reproductive and respiratory syndrome virus（PRRSV） N protein is the most immunogenic protein and an ideal target for the serological detection of infected pigs.This study aimed to prepare monoclonal antibodies（Mabs） against PRRSV N protein,which would be very useful in diagnosis and control of PRRSV.Firstly,N protein gene of PRRSV was cloned into the pRSETA vector to construct a recombinant expression plasmid.After the recombinant expression plasmid was transformed into E.coli,the recombinant protein was induced by 1 mmol/L IPTG at 37 ℃ for 4 hours and analyzed by SDS-PAGE.The result showed that N protein was highly expressed in E.coli.The expressed N protein of PRRSV was denatured and purified by Ni affinity chromatograph,then analyzed by SDS-PAGE and Western blot.Balb/c mice were immunized four times with the purified N protein,then the spleen cells of the hyperimmunized mouse and SP2/0 cells were fused with PEG-1500 at fusion rate of 81.3%.An indirect enzyme-linked immunosorbent assay（ELISA） was developed to detect Mabs secreted by hybridoma cell lines,and found the positive rate was 17.3%.Two hybridoma cell lines secreting Mabs,named 4G10 and 2F9,were developed by two subclones.The specificity of Mab was identified by means of indirect ELISA.After repeat frozen storage,thawed revival,and generation,the two cell lines are still steadily producing high titer of Mabs.In this study,one of high titer specific Mab against PRRSV N protein has been successfully prepared,which may be useful in the development of a quick and specific method to detect PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 核衣壳蛋白 单克隆抗体 

分 类 号：S858.28[农业科学—临床兽医学]