Preparation and Primary Mapping the Epitopes of Monoclonal Antibodies Against RHDV VLPs

doi:10.7668/hbnxb.2015.05.011

Abstract

Abstract: To select specific mAbs for the capsid protein(VP60)of Rabbit hemorrhagic disease virus(RHDV)and further analysis the distribution of antigenic epitopes recognized by mAbs,the recombinant baculoviruses rAcV-Bac-VP60 infected Sf9 insect cell monolayers to effectively express the recombinant VP60 protein,which could self-assemble into VLPs.To prepare monoclonal antibody(MAbs)specific to RHDV VLPs,BALB/c mice were immunized with the recombinant VP60 protein emulsified with Freund's adjuvant.Splenocytes from immunized mice were fused with SP2/0 murine myeloma cells on day 3 after the last injection.After three times subcloning,eleven monoclonal antibodies were screened,which could recognize RHDV VLPs and native RHDV.The subtypes of 1D4 and 3F7 belonged to IgG1-λ.And other MAbs belonged to IgG1-κ.Western Blot results indicated that 5A3 recognized the NTA domain of VP60 protein,5F3 recognized the S domain of VP60 protein and 9 MAbs(1B8,1D4,3D11,3F7,4C2,4G2,5G2,5H3 and 6B2)recognized the P domain of VP60 protein.These MAbs will be useful for mapping the epitopes of RHDV VLPs and the research of the structure and function of RHDV in the future,which will also contribute to the detection of RHDV and its novel vaccine development.

Key words: RHDV VLPs, MAbs, Epitope, Primary mapping

CLC Number:

S858.2912.65

SONG Yan-hua, HU Bo, FAN Zhi-yu, PENG Wei, WEI Hou-jun, XUE Jia-bin, XU Wei-zhong, WANG Fang. Preparation and Primary Mapping the Epitopes of Monoclonal Antibodies Against RHDV VLPs[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(5): 65-70. doi: 10.7668/hbnxb.2015.05.011.

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References

[1] Abrantes J,Van Der Loo W,Le Pendu J A.Rabbit haemorrhagic disease(RHD)and rabbit haemorrhagic disease virus(RHDV):a review[J].Veterinary Research,2012,43:12.
[2] Meyers G,Wirblich C,Thiel H J.Genomic and subgenomic RNAs of rabbit hemorrhagic disease virus are both protein-linked and packaged into particles[J].Virology,1991,184(2):677-686.
[3] Meyers G,Wirblich C,Thiel H J.Rabbit hemorrhagic disease virus-molecular cloning and nucleotide sequencing of a calicivirus genome[J].Virology,1991,184(2):664-676.
[4] Meyers G,Wirblich C,Thiel H J,et al.Rabbit hemorrhagic disease virus:genome organization and polyprotein processing of a calicivirus studied after transient expression of cDNA constructs[J].Virology,2000,276(2):349-363.
[5] König M,Thiel H J,Meyers G.Detection of viral proteins after infection of cultured hepatocytes with rabbit hemorrhagic disease virus[J].Journal of Virology,1998,72(5):4492-4497.
[6] Wang X E,Xu F T,Liu J S,et al.Atomic model of rabbit hemorrhagic disease virus by Cryo-Electron microscopy and crystallography[J].PLOS Pathogens,2013,9(1):e1003132.
[7] Esteves P J,Abrantes J,Carneiro M,et al.Detection of positive selection in the major capsid protein VP60 of the rabbit haemorrhagic disease virus(RHDV)[J].Virus Research,2008,137(2):253-256.
[8] 王芳,胡波,任雪枫,等.兔出血症病毒衣壳蛋白在昆虫细胞中的表达及对家兔的免疫保护效果[J].畜牧兽医学报,2008,39(10):1382-1387.
[9] Song Y,Zhou Y,Li Y,et al.Identification of B-cell epitopes in the NSP1 protein of porcine reproductive and respiratory syndrome virus[J].Veterinary Microbiology,2012,155(2/4):220-229.
[10] 刘秀梵.单克隆抗体在农业上的应用[M].合肥:安徽科学技术出版社,1994:8-36.
[11] Viaplana E,Plana J,Villaverde A.Antigenicity of VP60 structural protein of rabbit haemorrhagic disease virus[J].Archives of Virology,1997,142(9):1843-1848.
[12] Martínez-Torrecuadrada J L,Cortés E,Vela C,et al.Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus[J].The Journal of General Virology,1998,79(Pt 8):1901-1909.
[13] Barcena J,Verdaguer N,Roca R,et al.The coat protein of rabbit hemorrhagic disease virus contains a molecular Switch at the N-terminal region facing the inner surface of the capsid[J].Virology,2004,322(1):118-134.
[14] Laurent S,Vautherot J F,Madelaine M F,et al.Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection[J].Journal of Virology,1994,68(10):6794-6798.
[15] Parra F,Prieto M.Purification and characterization of a calicivirus as the causative agent of a lethal hemorrhagic disease in rabbits[J].J Virol,1990,64:4013-4015.

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