Article | 08. 2014 Vol. 32, Issue. 4
Expression of Lily mottle virus Coat Protein and Preparation of IgY Antibody against the Recombinant Coat Protein



Department of Microbiology, Dankook University1




2014.08. 544:549


PDF XML




Lily symptomless virus (LSV), Lily mottle virus (LMoV), and Cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf and bulb samples showing characteristic symptoms of virus infection were collected in 2012, and 80 field samples were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The infection frequencies were 79% for LMoV, 5% for LSV, and 3% for CMV. The LMoV coat protein gene was amplified and cloned into the pET21d(+) expression vector to develop serological diagnostic tools to detect LMoV. The resulting carboxy-terminal His-tagged coat proteins were expressed in Escherichia coli strain BL21 (DE3) by induction with IPTG. The recombinant proteins were purified using Ni-NTA agarose beads and used as an antigen to produce polyclonal antibodies in laying hens. The resulting egg yolk immunoglobulin (IgY) specifically recognized LMoV from infected plant tissues in immunoblotting assays and had comparable sensitivity to that of a mammalian antibody. In addition, method of immunocapture RT-PCR using this IgY was developed for sensitive, efficient, and rapid detection of LMoV. Based on these results, large-scale bulb tests and detection of LMoV in epidemiological studies can be performed routinely using this IgY. This is the first report of production of a polyclonal IgY against a plant virus and its use for diagnosis.



1. Asjes, C.J. 2000. Control of aphid-borne Lily symptomless virus and Lily mottle virus in Lilium in the Netherlands. Virus Res. 71:23-32.  

2. Berniak, H., T. Malinowski, and M. Kaminska. 2010. Characterization of polyclonal antibodies raised against two isolates of Cucumber mosaic virus. J. Plant Pathol. 92:231-234.  

3. Beuve, M., L. Sempé, and O. Lemaire. 2007. A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll- associated virus 2 variants in grapevine. J. Virol. Methods 141:117-124.  

4. Boonham, N., L. Laurenson, R. Weekes, and R. Mumford. 2009. Direct detection of plant viruses in potato tubers using real-time PCR. Methods Mol. Biol. 508:249-258.  

5. Dai, Y.C., X.F. Zhang, M. Tan, P. Huang, W. Lei, H. Fang, W. Zhong, and X. Jiang. 2013. A dual chicken IgY against rotavirus and norovirus. Antiviral Res. 97:293-300.  

6. Dias da Silva, W. and D.V. Tambourgi. 2010. IgY: A promising antibody for use in immunodiagnostic and in immunotherapy. Vet. Immunol. Immunopathol. 135:173-180.  

7. Edwards, M.L. and J.I. Cooper. 1985. Plant virus detection using a new form of indirect ELISA. J. Virol. Methods 11:309-319.  

8. Ganesh Selvaraj, D., R. Pokorný, and L. Holková. 2009. Comparative analysis of ELISA, one step RT-PCR and IC-RT-PCR for the detection of bean yellow Mosaic virus in gladiolus. Commun. Agric. Appl. Biol. Sci. 74:853-859.  

9. Gulati-Sakhuja, A., J.L. Sears, A. Nuñez, and H.Y. Liu. 2009. Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis. J. Virol. Methods 160:29-37.  

10. Ha, C., S. Coombs, P.A. Revill, R.M. Harding, M. Vu, and J.L. Dale. 2008. Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Arch. Virol. 153:25-36.  

11. Kim, J.H., H.N. Yoo, E.H. Bae, and Y.T. Jung. 2012. Development of an indirect ELISA and immunocapture RT-PCR for Lily virus detection. J. Microbiol. Biotechnol. 22:1776-1781.  

12. Kim, J.Y., S.T. Choi, and H.T. Hsu. 1995. Comparision of detection efficiency of lily symptomless virus in lily bulbs by ELISA, DBIA and tissue blotting method. J. Kor. Soc. Hort. Sci. 36:560-566.  

13. Kong, B., J. Yu, H. Chen, D. Li, C. Han, and J. Wang. 2009. Production of antiserum to recombinant coat protein for detecting Lily mottle virus in Yunnan, China. J. Phytopathol. 157:362-369.   

14. Liebenberg, A., M.J. Freeborough, C.J. Visser, D.U. Bellstedt, and J.T. Burger. 2009. Genetic variability within the coat protein gene of Grapevine fanleaf virus isolates from South Africa and the evaluation of RT-PCR, DAS-ELISA and ImmunoStrips as virus diagnostic assays. Virus Res. 142:28-35.  

15. Lim, J.H., E.H. Bae, Y.J. Lee, S.H. Park, K.J. Lee, S.R.M. Kim, and Y.T. Jung. 2009. Detection of Lily symptomless virus, Lily mottle virus and Cucumber mosaic virus from Lilium grown in Korea by RT-PCR. Kor. J. Microbiol. 45:251-256.  

16. Lim, J.H., E.H. Bae, S.H. Park, K.J. Lee, S.R.M. Kim, E.Y. Kwon, Y.J. Lee, and Y.T. Jung. 2010. Development of a real time RT-PCR with SYBR Green dye for the detection of several viruses from the bulbs and leaves of lily. Acta Virol. 54:319-321.  

17. Mortimer-Jones, S.M., M.G. Jones, R.A. Jones, G. Thomson, and G.I. Dwyer. 2009. A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously. J. Virol. Methods 161:289-296.  

18. Niimi, Y., T. Gondaira, Y. Kutsuwada, and H. Tsuji. 1999. Detection by ELISA and DIBa tests of Lily symptomless virus (LSV), Tulip breaking virus-lily (TBV-L) and Cucumber mosaic virus (CMV) in Lilium spp. grown in the field. J. Japan. Soc. Hort. Sci. 68:176-183.  

19. Schade, R., E.G. Calzado, R. Sarmiento, P.A. Chacana, J. Porankiewicz- Asplund, and H.R. Terzolo. 2005. Chicken egg yolk antibodies (IgY-technology): A review of progress in production and use in research and human and veterinary medicine. Altern. Lab. Anim. 33:129-154.  

20. Spillner, E., I. Braren, K. Greunke, H. Seismann, S. Blank, and D. du Plessis. 2012. Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy. Biologicals. 40:313-322.  

21. Tatineni, S., G. Sarath, D. Seifers, and R. French. 2013. Immuno-detection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: Potential for high-throughput diagnostic methods. J. Virol. Methods. 189:196-203.  

22. Wang, R., G. Wang, Q. Zhao, Y. Zhang, L. An, and Y. Wang. 2010. Expression, purification and characterization of the Lily symptomless virus coat protein from Lanzhou Isolate. Virol. J. 34:1-7.  

23. Wen, J., S. Zhao, D. He, Y. Yang, Y. Li, and S. Zhu. 2012. Preparation and characterization of egg yolk immunoglobulin Y specific to influenza B virus. Antiviral Res. 93:154-159.  

24. Wu, J., C. Meng, H. Shang, S. Rong, C. Zhang, J. Hong, and X. Zhou. 2011. Monoclonal antibody-based triple antibody sandwich-enzyme-linked immunosorbent assay and immunocapture reverse transcription-polymerase chain reaction for Odontoglossum ringspot virus detection. J. Virol. Methods 171:40-45.  

25. Wu, J., Y. Ni, H. Liu, L. Rao, Y. Zhou, and X. Zhou. 2013. Development and use of three monoclonal antibodies for the detection of rice black-streaked dwarf virus in field plants and planthopper vectors. Virol. J. 10:114.  

26. Zein, H.S., M. Nakazawa, M. Ueda, and K. Miyatake. 2007. Development of serological procedures for rapid and reliable detection of Cucumber mosaic virus with dot-immunobinding assay. World J. Agric. Sci. 3:430-439.   

27. Zheng, H.Y., J. Chen, M.F. Zhao, L. Lin, J.P. Chen, J.F. Antoniw, and M.J. Adams. 2003. Occurrence and sequences of Lily mottle virus and Lily symptomless virus in plants grown from imported bulbs in Zhejiang province, China. Arch. Virol. 148:2419-2428.