|
|
|
| Prokaryotic Expression and Preparation of Monoclonal Antibody of the Nucleoprotein of Bovine Respiratory Syncytial Virus (BRSV) |
| MA Lei, ZHU Yuan-mao, CAI Hong, WANG Shu, SHI Hong-fei, LÜ Chuang, DONG XIU-mei, GAO Yu-ran, XUE Fei |
| Division of Livestock Infectious Diseases National Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences, Harbin 150001, China |
|
|
|
Abstract 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 1×105, 1×106 and 1×102, 1×103 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 κ 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.
|
|
Received: 04 September 2012
Published: 25 December 2012
|
|
|
|
Cite this article:
MA Lei, ZHU Yuan-mao, CAI Hong, WANG Shu, SHI Hong-fei, LÜ Chuang, DONG XIU-mei, GAO Yu-ran, XUE Fei. Prokaryotic Expression and Preparation of Monoclonal Antibody of the Nucleoprotein of Bovine Respiratory Syncytial Virus (BRSV). China Biotechnology, 2012, 32(12): 20-24.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2012/V32/I12/20
|
|
|
[1] Stott E J, Taylor G. Respiratory syncytial virus. Arch Virol,1985,84:1-52.
[2] Paccaud M F, Jacquier C. A respiratory syneytial virus of bovine origin. Archives of Virology,1970,30:327-342.
[3] 史鸿飞,朱远茂,高欲燃,等.套式RT-PCR检测牛呼吸道合胞体病毒的研究.中国预防兽医学报,2010,32(3):238-240. Shi H F, Zhu Y M, Gao Y R,et al. Detection of bovine respiratory syncytial virus by a reverse transcriptase-nested-polymerase chain reaction in bovine clinical samples.Chinese Journal of Preventive Veterinary Medicine, 2010,32(3):238-240.
[4] Bachi T, Howe C. Morphogenesis and ultrastructure of respiratory syncytial virus. J Virol, 1973, 12(5):1173-1180.
[5] 薛飞,相文华,沈荣显.绵羊进行性肺炎病毒与山羊关节炎-脑炎病毒的血清学交叉反应的研究. 畜牧兽医学报,1999,30(4):365-369. Xue F, Xiang W H, Shen R X. Serological cross-reactivity between ovine progressive pneumonia virus and caprine arthritis-encephalitis virus. Acta Veterinaria et Zootechnica Sinica,1999, 30(4):365-369.
[6] Ward P A, Adams J, Gebhart G F, et al. Monoclonal antibody production: a report of the committee in methods of producing monoclonal antibodies.Institute for Laboratory Animal Research,National Research Council,1999,9-23.
[7] 冯军科,薛飞,李娇,等.牛呼吸道合胞体病毒G蛋白的截短表达与鉴定.中国生物工程杂志,2008,28(12): 24-29. Feng J K, Xue F, Li J,et al. Expression and identification truncated glycoprotein G of bovine respiratory syncytial virus in Escherichia coli. China Biotechnology, 2008,28(12): 24-29.
[8] Lerch R A,Stott E J,Wertz G W. Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs. J Virol,1989,63:833-840.
[9] Westenbrink F, Kimman T G, Brinkhof J M. Analysis of the antibody response to bovine respiratory syncytial virus proteins in calves. J Gen Virol,1989,70:591-601.
[10] Valarcher J L, Taylor G. Bovine respiratory syncytial virus infection. Vet Res, 2007,38:153-180. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
