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Volume 26 Issue 4
August 2011
    Citation: Chong-lin Hou, Yu Cao, Rong-hui Xie, Yi-zhen Wang, Hua-hua Du. Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus * .VIROLOGICA SINICA, 2011, 26(4) : 260-266.  http://dx.doi.org/10.1007/s12250-011-3202-0
    shu

    Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus *

    cstr: 32224.14.s12250-011-3202-0
    • 1.

      Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Feed Science Institute, Zhejiang University, Hangzhou 310029, China

    • 2.

      Zhejiang Provincial Center for Disease Prevention and control, Hangzhou 310029, China

    • Corresponding author: Hua-hua Du, huahuadu@zju.edu.cn
    • Received Date: 17 May 2011
      Accepted Date: 29 June 2011
      Available online: 01 August 2011

      Fund Project: NSFC 30901116Zhejiang Provincial Natural Science Foundation of China Y3080212The Planned Science and Technology Project of Zhejiang Province, China 2008C32034

    • The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.
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      1. Chaivisuthangkura P, Longyant S, Rukpratanporn S, et al. 2010. Enhanced white spot syndrome virus (WSSV) detection sensitivity using monoclonal antibody specific to heterologously expressed VP19 envelope protein. Aquaculture, 299: 15-20.
          doi: 10.1016/j.aquaculture.2009.12.005

      2. Chou H Y, Huang C Y, Wang C H, et al. 1995. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured Penaeid shrimp in Taiwan. Dis Aquat Org, 23: 165-173.
          doi: 10.3354/dao023165

      3. Du H H, Fu L L, Xu Y X, et al. 2007. Improvement in a simple method for isolating white spot syndrome virus (WSSV) from the crayfish Procambarus clarkii. Aquaculture, 262: 532-534.
          doi: 10.1016/j.aquaculture.2006.11.002

      4. Du H H, Li W F, Xu Z R, Kil, et al. 2006. Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii. Dis Aquat Org, 71: 175-178.
          doi: 10.3354/dao071175

      5. Du H H, Xu Z R, Wu X F, et al. 2006. Increased resistance to white spot syndrome virus in Procambarus clarkii by injection of envelope protein VP28 expressed using recombinant baculovirus. Aquaculture, 260: 39-43.
          doi: 10.1016/j.aquaculture.2006.06.032

      6. Durand S V, Redman R M, Mohney L L, et al. 2003. Qualitative and quantitative studies on the relative virus load of tails and heads of shrimp acutely infected with WSSV. Aquaculture, 216: 9-18.
          doi: 10.1016/S0044-8486(02)00230-2

      7. Fauquet C M, Mayo M A, Maniloff J, et al. 2005. Virus Taxonomy. Classification and Nomenclature of Viruses. In: Eighth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press.

      8. Inouye K, Yamano K, Ikeda N, et al. 1996. The penaeid rod shaped DNA virus (PRDV), which causes penaeid acute viremia (PAV). Fish Pathol, 31: 39-45.
          doi: 10.3147/jsfp.31.39

      9. Jaroenram W, Kiatpathomchai W, Flegel T W. 2009. Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. Mol Cell Probes, 23: 65-70.
          doi: 10.1016/j.mcp.2008.12.003

      10. Jiang Y S, Zhan W B, Sheng X Z. 2007. Neutralizing assay of monoclonal antibodies against white spot syndrome virus (WSSV). Aquaculture, 272: 216-222.
          doi: 10.1016/j.aquaculture.2007.07.229

      11. Jory D E, Dixon H M. 1999. Shrimp white spot syndrome virus in the western hemisphere. Aquaculture Magazine, 25: 83-91.

      12. Makesh M, Koteeswaran A, Danel Joy, et al. 2006. Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb. Aquaculture, 261: 64-71.
          doi: 10.1016/j.aquaculture.2006.08.034

      13. Otta S K, Karunasagar I, Karunasagar I. 2003. Detection of monodon baculovirus and white spot syndrome virus in apparently healthy Penaeus monodon postlarvae from India by polymerase chain reaction. Aquculture, 220: 59-67.
          doi: 10.1016/S0044-8486(02)00340-X

      14. van Hulten M C, Witteveldt J, Peters S, et al. 2001. The white spot syndrome virus DNA genome sequence. Virology, 286: 7-22.
          doi: 10.1006/viro.2001.1002

      15. Wang Y Z, Zhang X H, Yuan L, et al. 2008. Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp. Biochem Biophy Res Communi, 372: 902-906.
          doi: 10.1016/j.bbrc.2008.05.152

      16. Xie X, Xu L, Yang F. 2006. Proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus. J Virol, 80: 10615-10623.
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      17. Yi G, Wang Z, Qi Y, et al. 2004. Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J Biochem Mol Biol, 37: 726-734.

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      Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus *

        Corresponding author: Hua-hua Du, huahuadu@zju.edu.cn
      • 1. Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Feed Science Institute, Zhejiang University, Hangzhou 310029, China
      • 2. Zhejiang Provincial Center for Disease Prevention and control, Hangzhou 310029, China
      • Received Date: 17 May 2011
      • Accepted Date: 29 June 2011
      Fund Project:  NSFC 30901116Zhejiang Provincial Natural Science Foundation of China Y3080212The Planned Science and Technology Project of Zhejiang Province, China 2008C32034

      Abstract: The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.

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