牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定  

Preparation and Preliminary Characterization of Monoclonal Antibodies against E2 Protein of Bovine Viral Diarrhea Virus

作　　者：王姝[1,2] 朱远茂[2] 蔡红[2] 马磊[2] 史鸿飞[2] 吕闯[2] 董秀梅[2] 高欲燃[2] 薛飞[2] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150001 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物传染病研究室,哈尔滨150001

出　　处：《中国生物工程杂志》2013年第4期40-45,共6页China Biotechnology

基　　金：国家科技支撑计划子课题(2012BAD12B03-3);中央级公益性科研院所基本科研业务费项目(0302012002);哈尔滨市科技攻关计划项目(2010AA6AN019;AA6BN020)资助项目

摘　　要：为制备牛病毒性腹泻病毒(BVDV)糖蛋白E2单克隆抗体(MAb),利用原核表达并且纯化的重组糖蛋白E2(rE2)免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合。采用以BVDV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E2特异性MAb的杂交瘤细胞株,分别命名为4E3与1G11。用4E3与1G11杂交瘤细胞株接种BALB/c小鼠制备腹水,采用rE2及BVDV包被的ELISA测得的效价分别是6.21×106和6.83×105及6.83×105和7.5×104。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4E3与1G11均为IgM/κ。特异性试验表明4E3与1G11这2株MAb均不与牛传染性鼻气管炎病毒、牛副流感病毒3型、牛腺病毒3型反应;其中4E3不与猪瘟病毒反应,而1G11则可与猪瘟病毒发生交叉反应,这种反应特性可试用于BVDV与猪瘟病毒的鉴别诊断。所制备的4E3与1G11 MAb可以用于BVDV抗原的检测,为建立检测BVDV E2蛋白血清抗体的ELISA奠定了基础。Abstract To prepare monoelonal antibody （MAb） against glycoprotein E2 of bovine viral diarrhea virus （BVDV）, BALB/c mice were immunized with purified recombinant E2 （rE2） expressed in E. coli and two hybridomas secreting MAb were screened from fusing the SP2/0 cells with the spleen cells of the immunized BALB/c mice by indirect ELISA coated with BVDV. The titers of MAb 4E3 and 1Gll in ascites were 6.21 × 10^6 ,6.83 × 10^5, 6.83 × 10^5, and 7.5 × 10^4 as detected by rE2 and BVDV , respectively. The MAbs secreted by hybridomas 4E3 and 1Gll had highly reactivity and specificity in indirect ELISA, Western blotting, IFA and identified as IgM with a light chain of K by indirect ELISA. The specific tests indicated that the MAbs 4E3 and 1G11 had no reaction with bovine infectious rhinotraeheitis virus, bovine parainfluenza virus type 3 and bovine adenovirus type 3. Meanwhile the MAb 4E3 had no reaction with classical swine fever virus（CSFV） and the MAb 1 G11 had reaction with CSFV. Therefore the MAbs 4E3 and 1 G11 might be used for differentiating BVDV and CSFV. On the other hand, the MAbs 4E3 and 1 G11 could be used for establish diagnosis method for BVDV.

关 键 词：牛病毒性腹泻病毒 E2 单克隆抗体 IGM 

分 类 号：Q819[生物学—生物工程]