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

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.