摘要
利用牛呼吸道合胞体病毒(BRSV)接种牛肺细胞(BL),提取病毒RNA。通过RT-PCR扩增BRSV的核蛋白基因,然后定向克隆到原核表达载体pET30a,获得重组表达质粒pET30a-N。将重组质粒转化表达菌BL21(DE3),经增菌培养和IPTG诱导以及SDS-PAGE和Western blot分析,成功表达出了核蛋白(N),其分子量约为49kDa。为制备BRSV核蛋白的单克隆抗体,用纯化的重组核蛋白免疫BALB/c小鼠,取免疫鼠脾细胞与SP2/0细胞融合。采用以BRSV为检测抗原的间接ELISA筛选阳性细胞克隆,经3次克隆纯化后获得2株稳定分泌抗N特异性MAb的杂交瘤细胞株,分别命名为2D12与4B10。用2D12与4B10杂交瘤细胞株接种BALB/c小鼠制备腹水,采用rN及BRSV包被的ELISA测得的效价分别是1×105和1×106及1×102和1×103。间接ELISA、Western blot、IFA试验表明两株杂交瘤细胞所分泌的MAb具有良好的反应性和特异性。经抗体亚类鉴定D12与4B10均为IgG1/κ。特异性试验表明单抗2D12与4B10均不与牛副流感病毒3型和牛病毒性腹泻病毒反应。所制备的D12与4B10可用于建立检测BRSV病原及抗体的诊断方法。
The nucleoprotein gene was amplified by RT-PCR from RNA of bovine respiratory syncytial virus （BRSV） and cloned into pET30a. The transformed BL21 （DE3） with pET30a-N were cultivated and induced with IPTG. The nucleoprotein （N） was successfully expressed in E. coli BL21 with a molecular weight of approximately 49kDa and analyzed with Western blotting. To prepare monolconal antibody （MAb） to nucleoprotein of BRSV, BALB/c mice were immunized with purified recombinant N （ rN ） expressed in E. coli and two hybridomas secreting MAb were obtained by screening from the SP2/0 cells fused with the spleen cells of the immunized BALB/c mice by indirect ELISA coated with BRSV. The titers of MAbs 2D12 and 4B10 in ascites were 1 x 10^5 , 1 x 10^6 and 1 x 10^2, 1 x 10^3 as detected by rN and BRSV itself, respectively. The MAbs secreted by hybridomas 2D12 and 4B10 had highly reactivity and specificity in indirect ELISA, Western blotting and IFA, and identified as IgG1 with a light chain of K by indirect ELISA. The specific tests indicated that the MAbs 2D12 and 4B10 had no reaction with bovine parainfluenza virus type 3 and bovine viral diarrhea virus. Therefore, the MAbs 2D12 and 4B10 could be used to establish diagnosis method for BRSV.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2012年第12期20-24,共5页
China Biotechnology
基金
哈尔滨市科技攻关计划(2010AA6AN019
2012AA6BN020)
公益性行业(农业)科研专项经费(201003060-04)
中央级公益性科研院所基本科研业务费项目(0302012002)资助项目
关键词
牛呼吸道合胞体病毒核蛋白
单克隆抗体特异性
Bovine respiratory syncytial virus Nucleoprotein Monoclonal antibody Specificity
