생명과학회지 (Journal of Life Science)

  • 제17권11호
  • /
  • Pages.1497-1504
  • /
  • 2007
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

유전자 재조합 인간의 Thrombopoietin의 생리활성

Biological Activity of Recombinant Human Thrombopoietin

  • 김봉순 (양평농업기술센터) ;
  • ;
  • 민관식 (한경대학교 생물환경정보통신전문대학원 동물생명공학)
  • Kim, Boing-Soon (YangPyeong Agricultural Development & Technology Center) ;
  • Naidansuren, Purevjargal (Animal Biotechnology, The Graduate School of Bio. & Information Technology, Hankyong National University) ;
  • Min, Kwan-Sik (Animal Biotechnology, The Graduate School of Bio. & Information Technology, Hankyong National University)
  • 발행 : 2007.11.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

사람의 혈소판조절인자 (TPO)의 분비와 기능을 분석하기 위하여 사람 간 cDNA로부터 TPO cDNA를 분리하여 동물세포에서 재조합체를 생산하였다. 또한, 당쇄의 기능분석을 위하여 6개의 당쇄첨가부위를 Ala으로 치환하여 각각의 돌연변이체 재조합체도 생산하여 이들의 생리활성분석을 위하여 피하주사하여 혈소판의 증가여부를 분석하였으며, 체내 약동학검사를 위하여 꼬리 정맥에 재조합체를 주사하여 24시간까지 혈액을 채취하였다. Wild-type TPO는 효과적으로 분비하였으나, 크로닝에서 분석되어 진 116개 아미노산이 삭제된 돌연변이체는 배양상층으로 분비되지 않았다. N-linked 당쇄첨가 부위는 3번과 4번을 제외하고는 거의 비슷한 발현양상을 나타내었다. 특히 4번당쇄부위는 TPO의 분비에 중요한 역할을 하는 것으로 나타났다. 재조합체 10ng을 피하주사에 의하여 체내 혈소판이 유의적으로 증가하였으며, 5ng을 이용한 약동학 분석결과 1시간에 최대로 증가하였으며 그 이후 급격하게 감소하여 10시간에는 거의 존재하지 않았다. 따라서, 이러한 연구는 고 활성을 가지는 유전자 재조합체 TPO의 생산을 가능하게 하고, 또한 새로운 분자의 TPO를 가능하게 할 것으로 사료된다.

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.

키워드

참고문헌

  1. Barr, P. J. 1994. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66, 1-3. https://doi.org/10.1016/S0006-3495(94)80746-5
  2. Bartley, I. D., J. Bogenberger and P. Hunt. 1994. Identification and cloning of a megakaryocyte growth and development factor that is ligand for the cytokine receptor MPl. Cell 77, 1117-1124. https://doi.org/10.1016/0092-8674(94)90450-2
  3. Bazan, J. F. 1990. Haematopoietic receptors and helical cytokines. Immunol. Today 11, 350-354. https://doi.org/10.1016/0167-5699(90)90139-Z
  4. de Sauvage, F. J., P. E. Hass and S. D. Spencer. 1994. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-MPl ligand. Nature 369, 533-538. https://doi.org/10.1038/369533a0
  5. Eaton, D. L., A.Gurney and W. J. Malloy. 1994. Biological activity of human thrombopoietin (TPO), the c-MP1 ligand, and TPO variants and the chromosomal location of TPO. Blood 84, 241a (abstrat no 948).
  6. Foster, D. and H. Pamela. 1997. Thrombopoiesis and thrombopietins: The biological significance of truncated and full-length forms of MPI ligand. pp. 203-214, Humana Press.
  7. Ghilardi, N., A. Wiestner and R. C. Skoda. 1998. Thrombopoietin production is inhibited by a translational mechanism. Blood 92, 4023-4027.
  8. Hill, R. J., J. Levin and F. C. Levin. 1992. Correlation of in vitro and in vivo biological activities during the partial purification of thrmbopoietin. Exp. Hematol. 20, 354-907.
  9. Hokom, M., D. Lacey and O. Kinstler. 1995. Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice. Blood 86, 971-976.
  10. Hunt, P., Y. S. Li, and J. L. Nichol. 1995. Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor. Blood 86, 540-547.
  11. Jackson, C. W., L. K. Brown, B. C. Somerville, S. A. Lyles and A. T. Look. 1984. Two color cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions. Blood 63, 768-778
  12. Kato, T., T. Ozawa and T. Muto T. 1995. Essential structure for the biological activity of thrombopoietin. Blood 86, 365a (abstract no 1448).
  13. Kondo, T., M. Okabe, M. Sanada, M. Kurosawa, S. Suzuki, M. Kobayashi, M. Hosokawa M and M. Asaka. 1998. Familial essential thrombocythemia associated with one-base deletion in the 5'-untranslated region of the thrombopoietin gene. Blood 92, 1091-1099.
  14. Lee, H. G., P. Y. Lee, Y. K. Lee, S. J. Kim, H. K. Chung, M. K. Seo, J. K. Park, M. S. Min and W. K. Chang. 2003. Effects of changes in glycosylation sites on secretion of recombinant erythropoietin in cultured CHO cells. Korean J. Animal Reprod. 27, 299-307.
  15. Lok, S., K. Kaushanky, R. D. Holly. 1994. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369, 565-568. https://doi.org/10.1038/369565a0
  16. Min, K. S. 2000. Biological functions of N- and O-linked Oligosaccharides of equine chorionic gonadotropin and lutropin/ chorionic gonadotropin receptor. Korean J. Animal Reprod. 24, 357-364.
  17. Min, K. S. 2001. Biosynthesis of a biological active single chain equine chorionic gonadotropin. J. Life Sci. 11, 103-107. https://doi.org/10.1016/0024-3205(72)90223-8
  18. Min, K. S., N. Hattori, J. I.. Aikawa, K. Shiota and T. Ogawa. 1996. Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormoneand follicle-stimulaiting hormone-like activities. Endocrine J. 43, 585-593. https://doi.org/10.1507/endocrj.43.585
  19. Min, K. S., T. Hiyama, H. H. Seong, N. Hattori, S. Tanaka and K. Shiota. 2004. Biological activities of tethered chorionic gonadotropin (eCG) and its deglycosylated mutants. J. Reprod. Dev. 50, 297-304. https://doi.org/10.1262/jrd.50.297
  20. Min, K. S., M. H. Kang, J. T. Yoon, H. J. Jin, H. H. Seong, Y. M. Chang, H. J. Chung, S. J. Oh, S. G. Yun and W. K. Chang. 2003. Production of biological active single chain bovine LH and FSH. Asian-Aust J. Anim. Sci. 16, 498-503. https://doi.org/10.5713/ajas.2003.498
  21. Min, K. S., K. Shiota, T. Saneyoshi, M. Hirosawa and T. Ogawa. 1997. Differential role of oligosacchairdes in equine chorionic gonadotropin (eCG)/luteinizing hormone (LH) to express follicle stimulating hormone (FSH) -like and LH-like activities. J. Reprod. Dev. 43, 177-179.
  22. Miyake, T., M. Kawakita, K. Enomoto and M. J. Murphy. 1982. Partial purification and biological properties of thrombopoietin extracted from the urine of aplastic anemia patients. Stem Cells 2, 129-144.
  23. Narhi, L. O., T. Arakawa and K. H. Aoki. 1991. The effect of carbohydrate on the structure and stablility of erythropoietin. J. Biol. Chem. 266, 23022-23026.
  24. Park, J. J., H. G. Lee, I. S. Nam, H. J. Park, M. S. Kim, Y. H. Chung, P. J. Naidansuren, H. Y. Kang, P. Y. Lee, J. G. Park, H. H. Seong, W. K. Chang, M. H. Kang, Y. S. Park, S. S. Hwang, S. Y. Hwang, J. T. Yun and K. S. Min. 2005. Biological activity of recombinant human erythropoietin (EPO) in vivo and in vitro. Reprod. Dev. Biol. 29, 69-73.
  25. Penington, D. G. and T. E. Olsen. 1970. Megakaryocytes in states of altered platelet production: cell numbers, sizes and DNA count. Br. J. Haematol. 18, 447-463. https://doi.org/10.1111/j.1365-2141.1970.tb01458.x
  26. Saneyoshi, T., K. S. Min, X. J. Ma, Y. Nambo, T. Hiyama, S. Tanaka and K. Shiota. 2001. Equine follicle-stimulating hormone: Molecular cloning of b-subunit and biological role of the asparagine-linked oligosaccharide at asparagine56 of a-subunit. Biol. Reproduc. 65, 1686-1690. https://doi.org/10.1095/biolreprod65.6.1686
  27. Shimada, Y., T. Kato and K. Ogami K. 1995. Production of thrombopoietin by rat hepatocytes and hepatoma cell lines. Exp. Hematol. 23, 1388-1396.
  28. Stenberg, P. E., J. Levin, G. Baker, Y. Mok and L. Corash. 1991. Neuraminidase-induced thrombocytopenia in mice: effects on thrombopoiesis. J. Cell Physiol. 147, 7-16. https://doi.org/10.1002/jcp.1041470103
  29. Takeuchi, M., N. Inoue and T. W. Strickland. 1989. Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese Hamster Ovary cells. Proc. Natl. Acad. Sci. USA 86, 7819-7822. https://doi.org/10.1073/pnas.86.20.7819
  30. Wiestner, A., R. J. Schlemper, A. P. van der Mass and R. C. Skoda. 1998. An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia. Nat. Genet. 18, 49-52. https://doi.org/10.1038/ng0198-49

피인용 문헌

  1. Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica vol.233, 2016, https://doi.org/10.1016/j.ygcen.2016.04.030