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소나무재선충[Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle]의 GaLectin에 대한 특이적인 단클론 항체 제작과 진단에의 활용

Development of Monoclonal Antibodies Specific to Galectin of Pine Wood Nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle and Their Utilization for Detection of Pine Wood Nematodes

  • 김아영 (한림대학교 일송생명과학연구소) ;
  • 김영하 (한림대학교 일송생명과학연구소) ;
  • 최보혜 (한림대학교 일송생명과학연구소) ;
  • 트랑뉴겐 (한림대학교 일송생명과학연구소) ;
  • 윤경재 (서울대학교 농생물학과) ;
  • 이시혁 (서울대학교 농생물학과) ;
  • 한혜림 (국립산림과학원) ;
  • 고영호 (한림대학교 일송생명과학연구소)
  • Kim, A-Young (Ilsong Institute of Life Science, Hallym University) ;
  • Kim, Young Ha (Ilsong Institute of Life Science, Hallym University) ;
  • Choi, Bo-Hye (Ilsong Institute of Life Science, Hallym University) ;
  • Nguyen, Trang (Ilsong Institute of Life Science, Hallym University) ;
  • Yoon, Kyungjae Andrew (Department of Agricultural Biology, Seoul National University) ;
  • Lee, Si Hyeock (Department of Agricultural Biology, Seoul National University) ;
  • Han, Hye-Rim (Laboratory of Pine Wilt Disease, Division of Forest Insect Pests & Disease, National Institute of Forest Science) ;
  • Koh, Young Ho (Ilsong Institute of Life Science, Hallym University)
  • 투고 : 2017.10.24
  • 심사 : 2018.01.16
  • 발행 : 2018.03.01

초록

소나무재선충(pine wood nematode, PWN) 감염 소나무를 현장에서 신속하게 진단을 할 수 있는 방법은 현재 없다. 본 연구에서는 PWN특이적인 항원으로 알려진 PWN-GaLectin을 baculovirus발현체계로 발현시켜서 총 1,464개의 fusion hybridoma 세포 주 라이브러리를 제작 하는 항원으로 이용하였다. 총 1,464개의 fusion hybridoma 세포 주 중, PWN-GaLectin에 대한 높은 반응을 보이는 62개의 fusion hybridoma 세포 주를 선별했다. 이들 중, 표준 PWN 감염소나무 PBS추출물과 PWN 단백질 추출물에 강한 반응을 보이는 세포 주 12개를 선별하여 단클론 항체(monoclonal antibody, Mab) 분비세포 주 확립을 위한 limited dilution을 실시하였다. Mab분비세포 주 확립을 위해서 표준 PWN 감염소나무 추출물에 대한 반응이 표준 정상 소나무 추출물 보다 높은 세포 주들을 선별했다. 그리고 추가로 PWN 단백질 추출물에 대해서 3종의 비 병원성 선충 단백질 추출물 보다 높은 반응을 보이는 세포 주들도 선별, 확립했다. 본 연구에서 확립된 Mab들을 우리는 현장과 실험실에서 사용할 수 있는 신속진단키트의 개발에 이용할 수 있을 것이다.

Currently, there is no available tool that rapidly diagnoses pine wood nematode (PWN)-infected pine trees in the field. In this study, we synthesized and purified PWN Galectin, which might be an antigen specific to PWN, using the Baculovirus expression system. We used PWN Galectin as an antigen for generating 1,464 fusion hybridoma cell lines secreting monoclonal antibodies (Mabs). Among them, we selected 62 fusion hybridoma cell lines showing high reactivity to PWN Galectin. We further selected 12 fusion hybridoma cell lines showing high reactivity to the standard PWN-infected pine tree phosphate buffered saline (PBS) extract. Additionally, two fusion hybridoma cell lines showing no or extremely low reactivity were used as controls. The selected fusion hybridoma cell lines were subjected to limiting dilutions for selecting and establishing Mab-secreting cell lines showing higher reactivity to the standard PWN-infected pine tree extract than to the standard normal pine tree PBS extract. Moreover, the selected fusion hybridoma cell lines were further selected based on their higher reactivity to PWN protein extracts than to three non-pathogenic nematode protein extracts. The Mab-secreting cell lines established in this study could be used to develop rapid diagnostic tools that can be used in the field or in laboratories for detecting PWN-infected pine trees or PWN.

키워드

참고문헌

  1. Barondes, S.H., Cooper, D.N., Gitt, M.A., Leffler, H., 1994. Galectins: Structure and function of a large family of animal lectins. J. Biol. Chem. 269, 20807-20810.
  2. Cao, A.X., Liu, X.Z., Zhu, S.F., B.S., L., 2005. Detection of the pinewood nematode, Bursaphelenchus xylophilus, using a real-time polymerase chain reaction assay. Phytophathology 95, 566-571. https://doi.org/10.1094/PHYTO-95-0566
  3. Futai, K., 2013. Pine wood nematode, Bursaphelenchus xylophilus. Annu. Rev. Phytopathol. 51, 61-83. https://doi.org/10.1146/annurev-phyto-081211-172910
  4. Kang, J.S., Choi, K.S., Shin, S.C., Moon, I.S., Lee, S.G., Lee, S.H., 2004. Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae). Nematology 6, 279-285. https://doi.org/10.1163/1568541041217915
  5. Kang, J.S., Kim, A.-Y., Han, H.R., Moon, Y.S., Koh, Y.H., 2015. Development of two alternative loop-mediated isothermal amplification tools for detecting pathogenic pine wood nematodes. For. Pathol. 45, 127-133. https://doi.org/10.1111/efp.12147
  6. Kikuchi, T., Aikawa, T., Oeda, Y., Karim, N., Kanzaki, N., 2009. A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology 99, 1365-1369. https://doi.org/10.1094/PHYTO-99-12-1365
  7. Kim, D.S., Lee, S.M., Hur, H.S., Park, C.N., Park, J.G., 2009. Escape of pine wood nematode, Bursaphelenchus xylophilus, through feeding and oviposition behavior of Monochamus alternatus and M. saltuarius (coleoptera: cerambycidae) adults. Korean J. Appl. Entomol. 48, 527-533. https://doi.org/10.5656/KSAE.2009.48.4.527
  8. Kim, Y.H., Kim, A.-Y., Choi, B.-H., Han, H.-R., Koh, Y.H., 2017. ExpansinB3 as a marker for detecting pine wood nematodeinfected pine trees. J. Asia-Pacific Entomol. 20, 1228-1233. https://doi.org/10.1016/j.aspen.2017.08.029
  9. Koczula, K.M., Gallotta, A., 2016. Lateral flow assays. Essays Biochem. 60, 111-120. https://doi.org/10.1042/EBC20150012
  10. Lee, D.-W., Seo, J.B., Nam, M.H., Kang, J.S., Kim, S.Y., Kim, A.-Y., Kim, W.T., Choi, J.K., Um, Y., Lee, Y., Moon, I.-S., Han, H.R., Koh, S.-H., Je, Y.H., Lim, K.J., Lee, S.H., Koh, Y.H., 2011. A combination of biochemical and proteomic analyses reveals Bx-LEC-1 as an antigenic target for the monoclonal antibody 3-2A7-2H5-D9-F10 specific to the pine wood nematode. Mol. Cell Proteomics 10, M900521-MCP900200-900521.
  11. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63. https://doi.org/10.1093/nar/28.12.e63
  12. Shinya, R., Morisaka, H., Kikuchi, T., Takeuchi, Y., Ueda, M., Futai, K., 2013. Secretome analysis of the pine wood nematode Bursaphelenchus xylophilus reveals the tangled roots of parasitism and its potential for molecular mimicry. PLoS One 8, e67377. https://doi.org/10.1371/journal.pone.0067377
  13. Zhao, L., Mota, M., Vieira, P., Butcher, R.A., Sun, J., 2014. Interspecific communication between pinewood nematode, its insect vector, and associated microbes. Trends Parasitol. 30, 299-308. https://doi.org/10.1016/j.pt.2014.04.007