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Vaccination of Shrimp (Litopenaeus vannamei) against White Spot Syndrome Virus (WSSV) by Oral Vaccination of Recombinant Fusion Protein, rVP19+28

사료급이(oral feeding)에 의한 vaccination을 통한 흰반점바이러스(WSSV)에 대한 재조합단백질 rVP19+28의 백신효능의 확인

  • Nguyen, Thi-Hoai (Department of Biotechnology, Pukyong National University) ;
  • Kim, Yeong-Jin (Department of Biotechnology, Pukyong National University) ;
  • Choi, Mi-Ran (Department of Biotechnology, Pukyong National University) ;
  • Kim, Sung-Koo (Department of Biotechnology, Pukyong National University)
  • Received : 2010.06.14
  • Accepted : 2010.07.26
  • Published : 2010.08.30

Abstract

This study was carried out to evaluate the vaccination effects of recombinant fusion protein rVP19+28 against WSSV in shrimp, Litopenaeus vannamei. The VP19+28 gene fused with VP19 and VP28 genes was inserted into pET-28a(+) expression vector and cloned in E. coli BL21 (DE3) to produce fused gene product recombinant VP19+VP28 as a single protein. For the vaccination, the shrimps were fed with pellets coated with purified recombinant protein, rVP19+28, for 2 weeks. Then, constant amounts of WSSV at $1{\times}10^2$ diluted stocks were injected to the muscle of the shrimp for the in vivo challenge tests. Non-vaccinated shrimps showed a cumulative mortality of 100% at 11 days post-challenge. The shrimps vaccinated with the inactivated E. coli BL21 as a host cell control showed cumulative mortality of 100% at 17 days post-challenge. The shrimps vaccinated with rVP19, rVP28 and rVP19+28 showed mortalities of 66.7%, 41.7% and 41.7% at 21 days post-challenge, respectively. These results indicated that the rVP28 and rVP19+28 had relatively high vaccination effects against WSSV infection. However, this study suggests that the fusion protein rVP19+28 was more effective for the protection of shrimp against WSSV than rVP28, even though the cumulative mortalities were the same 21 days post-challenge.

본 연구는 WSSV의 주요 구조단백질인 VP19와 VP28을 모두 포함하는 VP19+28 fusion protein을 제조하여, Litopenaeus vannamei에서 WSSV에 대한 백신으로서의 효능을 평가하고자 수행하였다. VP19와 VP28 유전자를 fusion하여 제작한 VP19+28 유전자를 pET-28a(+) vector에 삽입하고 단일단백질로서 제작된 VP19+28 유전자를 E. coli BL21 (DE3)에서 발현시켰다. 백신실험을 위해 새우에게 2주 동안 실험용 사료를 급이하였으며, 그 후 바이러스액($1{\times}10^2$배로 희석한 WSSV)을 이용하여 새우에게 주사 감염에 의해 in vivo 공격실험(challenge test)을 수행하였다. 실험결과, vaccination을 하지 않은 새우들은 감염 후 11일째에 100%의 누적폐사율을 보였으며, host control로써 E. coli BL21을 사용하여 vaccination한 새우들은 감염 후 17일째에 100%의 누적폐사율을 보였다. rVP19, rVP28, rVP19+28을 이용하여 vaccination한 새우들의 경우 감염 후 21일째에 각각 66.7%, 41.7%, 41.7%의 누적폐사율을 보였다. 이상의 결과를 통해 rVP28과 rVP19+28이 WSSV에 대해 높은 백신효능을 가짐을 확인하였다. 또한 감염 후 21일째에 fusion protein rVP19+28과 rVP28의 누적폐사율은 동일하였지만 공격실험기간 동안 폐사율이 rVP19+28을 투여 한 실험군이 낮게 나타나는 것을 보아 WSSV에 대한 새우의 방어효능은 rVP19+28이 더 높음을 나타내는 것이다.

Keywords

References

  1. Escobedo-Bonilla, C. M., V. Alday-Sanz, M. Wille, P. Sorgeloos, M. B. Pensaert, and H. J. Nauwynck. 2008. A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus. J. Fish Dis. 31, 1-18. https://doi.org/10.1111/j.1365-2761.2007.00877.x
  2. Kurtz, J. and K. Franz. 2003. Innate defence: evidence for memory in invertebrate immunity. Nature 425, 37-38. https://doi.org/10.1038/425037a
  3. Kurtz, J. and K. Franz. 2003. Innate defence: evidence for memory in invertebrate immunity. Nature 425, 37-38. https://doi.org/10.1038/425037a
  4. Rojtinnakorn, J., I. Hirono, T. Itami, Y. Takahashi, and T. Aoki. 2002. Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach. Fish Shellfish Immunol. 13, 69-83. https://doi.org/10.1006/fsim.2001.0382
  5. Roux, M. M., A. Pain, K. R. Klimpel, and A. K. Dhar. 2002. The lipopolysaccharide and $\beta$-1, 3-glucan binding protein gene is upregulated in white spot virus-infected shrimp (Penaeus stylirostris). J. Virol. 76, 7140-7149. https://doi.org/10.1128/JVI.76.14.7140-7149.2002
  6. Van Hulten, M. C. W., J. Witteveldt, M. Snippe, and J. M. Vlak. 2001. White spot syndrome virus envelop protein VP28 is involved in the systemic infection of shrimp. Virology 285, 228-233. https://doi.org/10.1006/viro.2001.0928
  7. Venegas, C. A., L. Nonaka, K. Mushiake, T. Nishizawa, and K. Muroga. 2000. Quasi-immune response of Penaeus japonicus to penaeid rod-shaped DNA virus (PRDV). Dis. Aquat. Org. 42, 83-89. https://doi.org/10.3354/dao042083
  8. Vlak, J. M., J. R. Bonami, T. W. Flegel, G. H. Kou, D. V. Lightner, C. F. Loh, P. C. Loh, and P. W. Walker. 2005. Nimaviridae, pp. 1162, Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, Academic Press, London, UK.
  9. Witteveldt, J., C. C. Cifuentes, J. M. Vlak, and M. C. W. Van Hulten. 2004. Protection of Penaeus monodon against white spot syndrome virus by oral vaccination. J. Virol. 78, 2057-2061. https://doi.org/10.1128/JVI.78.4.2057-2061.2004
  10. Wu, J. L., T. Nishioka, K. Mori, T. Nishizawa, and K. A. Muroga. 2002. A time-course study on the resistance of Penaeus japonicus induced by artificial infection with white spot syndrome virus. Fish Shellfish Immunol. 13, 391-403. https://doi.org/10.1006/fsim.2002.0414