牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定

中国生物工程杂志

2013, Vol. 33

Issue (4): 40-45

研究报告

牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定

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

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

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

WANG Shu^1,2, ZHU Yuan-mao², CAI Hong², MA Lei², SHI Hong-fei², LÜ Chuang², DONG Xiu-mei², GAO Yu-ran², XUE Fei²

1. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150001, China;
2. Division of Livestock Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China

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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。

关键词： 牛病毒性腹泻病毒; E2; 单克隆抗体; IgM

Abstract: To prepare monoclonal 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 1G11 in ascites were 6.21×10⁶,6.83×10⁵, 6.83×10⁵, and 7.5×10⁴ as detected by rE2 and BVDV, respectively. The MAbs secreted by hybridomas 4E3 and 1G11 had highly reactivity and specificity in indirect ELISA, Western blotting, IFA and identified as IgM with a light chain of κ by indirect ELISA.The specific tests indicated that the MAbs 4E3 and 1G11 had no reaction with bovine infectious rhinotracheitis 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 1G11 had reaction with CSFV.Therefore the MAbs 4E3 and 1G11 might be used for differentiating BVDV and CSFV.On the other hand, the MAbs 4E3 and 1G11 could be used for establish diagnosis method for BVDV.

Key words: Bovine viral diarrhea virus E2 protein Monoclonal antibody IgM

收稿日期: 2012-12-03 出版日期: 2013-04-25

ZTFLH:

Q819

基金资助: 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;2012AA6BN020)资助项目

通讯作者: 薛飞 E-mail: xuefeicnh@yahoo.com.cn

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引用本文:

王姝, 朱远茂, 蔡红, 马磊, 史鸿飞, 吕闯, 董秀梅, 高欲燃, 薛飞. 牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定[J]. 中国生物工程杂志, 2013, 33(4): 40-45.

WANG Shu, ZHU Yuan-mao, CAI Hong, MA Lei, SHI Hong-fei, LÜ Chuang, DONG Xiu-mei, GAO Yu-ran, XUE Fei. Preparation and Preliminary Characterization of Monoclonal Antibodies against E2 Protein of Bovine Viral Diarrhea Virus. China Biotechnology, 2013, 33(4): 40-45.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/40

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