Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Transgenic tobacco culture cells expressing spike protein gene of porcine epidemic diarrhea virus

돼지 유행성 설사병 바이러스 스파크 단백질 유전자 발현 형질전환 담배 배양세포

  • Yang, Kyoung-Sil (Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Hyeon-Soo (Department of Verternarian, Chungnam National University) ;
  • Kwon, Suk-Yoon (Plant Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kwak, Sang-Soo (Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Haeng-Soon (Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • 양경실 (한국생명공학연구원 환경생명공학연구센터) ;
  • 김현수 (충남대학교 수의과대학) ;
  • 권석윤 (한국생명공학연구원 식물유전체연구센터) ;
  • 곽상수 (한국생명공학연구원 환경생명공학연구센터) ;
  • 이행순 (한국생명공학연구원 환경생명공학연구센터)
  • Published : 2008.03.31
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Abstract

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

Porcine epidemic diarrhea virus (PEDV)는 돼지의 급성장염을 유발하여 설사 등의 증상을 일으키는 바이러스이다. 본 연구에서는 PEDV 항원단백질을 생산하는 담배 배양세포주를 개발하고자 하였다. PEDV에서 항원성이 알려진 스파크 단백질의 일부분을 암호화하는 유전자를 PCR로 합성하여 4종류의 형질전환 벡터를 제작하였다. 담배 배양세포 BY-2를 재료로 하여 Agrobacterium tumefaciens을 매개로 형질전환하였다. 선발배지 (MS salt, $KH_2PO_4$ 370 mg/L, 2,4-D 0.18 mg/L, Thiamin HCl 1 mg/L, kanamycin 100 mg/L, 침랙무 400 mg/L)에서 캘러스를 3주 간격으로 3개월 동안 계대배양하여 카나마이신 저항성 캘러스를 선발하였다. 선발된 캘러스를 대상으로 PCR 분석한 결과 형질전환 효율은 75% 이상이었으며 벡터당 40 여개 이상의 형질전환 배양세포주를 얻었다. 형질전환 배양세포주를 대상으로 Southern blot 분석하여 PEDV 유전자가 고구마 식물체의 게놈으로 안정적으로 도입되었음을 확인하였다. Northern blot 분석 결과 PEDV 스파크 단백질 유전자가 높은 수준으로 발현함을 확인하였으며 dot blot으로 PEDV 스파크 단백질 고생산 배양세포주를 선발하였다. 형질전환 담배 배양세포로부터 생산된 PEDV 항원단백질을 BALB/c 마우스에 경구투여 하여 면역활성을 조사한 결과 형질전환 세포주인 35S::SP1-M, 35S::SP2-M, 35S::SP4-M 세포주에서 1:10의 희석배수까지 바이러스 억제효과가 관찰되었다. 제작된 형질전환 벡터는 고구마와 같은 경구용 사료작물에 활용할 수 있을 것이다.

Keywords

References

  1. Bae JL, Lee JG, Kang TJ, Jang HS, Lang YS, Yang MS (2003) Induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen. Vaccine 21: 4052-4058 https://doi.org/10.1016/S0264-410X(03)00360-8
  2. Chang SH, Bae JL, Kan TJ, Kim J, Chung GH, Lim CW, Laude H, Yang MS, Jang YS (2002) Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus. Mol Cells 14: 295-299
  3. de Bouck P, Pensaert M (1980) Experimental infection of pigs with a new porcine enteric coronavirus, CV 777. Am J Vet Res 41: 219-223
  4. Hellwig S, Drossard J, Twyman R, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotech 22: 1415-1422 https://doi.org/10.1038/nbt1027
  5. Kang TJ, Kang KH, Kin JA, Kwon TH, Jang YS, Yang MS (2004) High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus-based vector. Protein Expr Purif 38: 129-135 https://doi.org/10.1016/j.pep.2004.07.014
  6. Kang TJ, Kim YS, Jang YS, Yang MS (2005a) Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine. Vaccine 23: 2294-22975 https://doi.org/10.1016/j.vaccine.2005.01.027
  7. Kang TJ, Seo JE, Kim DH, Kim TG, Jang YS, Yang MS (2005b) Cloning and sequence analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants. Protein Expr Purif 41: 378-383 https://doi.org/10.1016/j.pep.2005.02.018
  8. Kang TJ, Han SC, Yang MS, Jang YS (2006) Expression of synthetic neutralizating epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants. Protein Expr Purif 46: 16-22 https://doi.org/10.1016/j.pep.2005.07.026
  9. Mason HS, Lam DM, Arntzen CJ (1992) Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA 89: 11745-11749
  10. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  11. Pensaert MB, de Bouck P (1978) A new coronavirus-like particle associated with diarrhea in swine. Arch Viol 58: 243-247
  12. Spaan W, Cavanagh D, Horzinek MC (1988) Coronavirus: structure and genome expression. J Gen Virol 69: 2939-2952 https://doi.org/10.1099/0022-1317-69-12-2939
  13. Tuboly T, Yu W, Bailey A, Degrandis S, Du S, Erickson L, Nagy E (2000) Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine 18: 2023-2028 https://doi.org/10.1016/S0264-410X(99)00525-3