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Monoclonal antibody production for CP4 EPSPS detection assays

CP4 EPSPS 검출을 위한 단클론 항체 생산

  • A-Mi Yoon (Division of Ecological Safety, National Institute of Ecology(NIE)) ;
  • Il Ryong Kim (Division of Ecological Safety, National Institute of Ecology(NIE)) ;
  • Wonkyun Choi (Division of Ecological Safety, National Institute of Ecology(NIE))
  • 윤아미 (국립생태원 생태안전연구실) ;
  • 김일룡 (국립생태원 생태안전연구실) ;
  • 최원균 (국립생태원 생태안전연구실)
  • Received : 2021.10.26
  • Accepted : 2021.11.16
  • Published : 2021.12.31

Abstract

In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.

Agrobacterium tumefaciens strain CP4 유래 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) 유전자를 포함하는 유전자변형생물체(Living modified organism, LMO)가 개발되었다. 이 같은 LMO는 국내 승인되어 사료용, 식품용, 가공용으로 이용 중이다. 간이면역 검사키트 개발을 위해서는 고효율의 단클론 항체 개발이 필수적이다. 본 연구에서는 대장균 BL21 (DE3)에서 재조합 CP4 EPSPS 단백질을 정제하였으며 SDS-PAGE와 MALDI-TOF MS 분석으로 단백질 특성을 분석하였다. 단클론 항체 제작은 (주)앱클론의 SOP 매뉴얼에 따라 진행하였다. 본 연구 결과 5개의 단클론 항체 클론(2F2, 4B9, 6C11, 10A9, 10G9)를 확보하였다. 5종의 단클론 항체의 효율과 특이도 검정을 위해서 LM 면화 추출액을 이용한 western blotting 분석을 실시하였다. 모든 단클론 항체는 CP4 EPSPS를 함유하는 MON1445와 MON88913을 특이적으로 검출하였으며 비변형 면화 및 타종의 LM 면화에서는 검출되지 않았다. 이러한 결과들을 바탕으로 CP4 EPSPS 단클론 항체는 LMO에 함유된 CP4 EPSPS 단백질을 타겟으로 항체 기반 검출법 개발에 활용될 것으로 사료된다.

Keywords

Acknowledgement

본 연구는 환경부 재원으로 NIE-법정연구-2021-06 지원을 받아 수행하였습니다.

References

  1. Apiratmateekul N, P Phunpae and W Kasinrerk. 2009. A modified hybridoma technique for production of monoclonal antibodies having desired isotypes. Cytotechnology 60:45-51. https://doi.org/10.1007/s10616-009-9213-0
  2. Balasubramani G, KP Raghavendra, J Das, R Kumar, HB Santosh, J Amudha, S Kranthi and KR Kranthi. 2021. Critical evaluation of GM cotton. pp. 351-410. In: Cotton Precision Breeding (Rahman M, Y Zafar and T Zhang eds.). Springer. Cham, Germany.
  3. Center for Environmental Risk Assessment and ILSI Research Foundation. 2011. A review of the environmental safety of the CP4 EPSPS protein. Environ. Biosafety Res. 10:5-25. https://doi.org/10.1051/ebr/2012001
  4. Chinnadurai P, D Stojsin, K Liu, GE Frierdich, KC Glenn, T Geng, A Schapaugh, K Huang, AE Deffenbaugh, ZL Liu and LA Burzio. 2018. Variability of CP4 EPSPS expression in genetically engineered soybean (Glycine max L. Merrill). Transgenic Res. 27:511-524. https://doi.org/10.1007/s11248-018-0092-z
  5. Della-Cioppa G, SC Bauer, BK Klein, DM Shah, RT Fraley and GM Kishore. 1986. Translocation of the precursor of the 5-enolpyruvylshikimate-3-phosphate into chloroplasts of higher plants in vitro. Proc. Natl. Acad. Sci. U.S.A. 83:6873-6877. https://doi.org/10.1073/pnas.83.18.6873
  6. Edholm ES, M Wilson and E Bengten. 2011. Immunoglobulin light (IgL) chains in ectothermic vertebrates. Dev. Comp. Immunol. 35:906-915. https://doi.org/10.1016/j.dci.2011.01.012
  7. Funke T, Y Yang, H Han, M Healy-Fried, S Olesen, A Becker and E Schonbrunn. 2009. Structural basis of glyphosate resistance resulting from the double mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli. J. Biol. Chem. 284:9854-9860. https://doi.org/10.1074/jbc.M809771200
  8. KBCH. 2021. Korea Biosafety Clearing House. Korea Research Institute of Bioscience and Biotechnology. Daejeon, Korea. Retrived from http://biosafety.or.kr/(accessed on 1 Oct 2021).
  9. Kwak BY, SH Ko, CW Park, DY Son and DH Shon. 2003. Development of enzyme-linked immunosorbent assay for glyphosatetolerant soybeans. Korean J. Food Sci. Technol. 35:366-372.
  10. Li Z, H Yu, Y Hu, S Shi, Y Liu, Y Duan, X Li, Y Wang and Q Li. 2013. Preparation and analysis on biological characteristics of monoclonal antibodies against CP4-EPSPS for sandwich ELISA. Biotechnol. Bull. 4:206-209.
  11. Morisset D, D Stebih, M Milavec, K Gruden and J Zel. 2013. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One 8:e62583.
  12. Nelson PN, GM Reynolds, EE Waldron, E Ward, K Giannopoulos and PG Murray. 2000. Monoclonal antibodies. Mol. Pathol. 53:111-117. https://doi.org/10.1136/mp.53.3.111
  13. Nida DL, KH Kolacz, RE Buehler, WR Deaton, WR Schuler, TA Armstrong, ML Taylor, CC Ebert, GJ Rogan, SR Padgette and RL Fuchs. 1996. Glyphosate-tolerant cotton: Genetic characterization and protein expression. J. Agric. Food Chem. 44:1960-1966. https://doi.org/10.1021/jf9505640
  14. NIE. 2020a. Study on Environmental Monitoring and Post-management of LMO. National Institute of Ecology. Seocheon, Korea.
  15. NIE. 2020b. Establishment of Detection Method for LMO. National Institute of Ecology. Seocheon, Korea.
  16. Padgette SR, KH Kolacz, X Delannay, DB Re, BJ LaVallee, CN Tinius, WK Rhodes, YI Otero, GF Barry, DA Eichholtz, VM Peschke, DL Nida, NB Taylor and GM Kishore. 1995. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci. 35:1451-1461. https://doi.org/10.2135/cropsci1995.0011183X003500050032x
  17. Renz B, JK Davies, D Carling, H Watkins and C Redwood. 2009. Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale. Protein Expr. Purif. 66:181-184. https://doi.org/10.1016/j.pep.2009.02.016
  18. Sharon E, J Juye and M Jack. 2019. The effects of acculturative stress, intergenerational conflict, and negative mood regulation expectancies of the mental health of Korean immigrants. Asian J. Fam. Ther. 3:1-27.
  19. Webster J and D Oxley. 2012. Protein identification by MALDI-TOF mass spectrometry. pp. 227-240. In: Chemical Genomics and Proteomics(Zanders ED, ed.). Springer, London.
  20. Zeng H, Q Yang, H Liu, G Wu, W Jiang, X Liu, J Wang and X Tang. 2021. A sensitive immunosensor based on graphene-PAMAM composites for rapid detection of the CP4-EPSPS protein in genetically modified crops. Food Chem. 361:129901.