Method for preparing human thrombopoietin polytides by mammalian cell culturesProcedures to produce a biologically active human TPO by mammalian cells in in vitro cultures are disclosed. Murine myeloma cells were genetically modified by introducing the gene of human thrombopoietin through a DNA construction which includes an immunoglobulin promoter associated to an immunoglobulin enhancer. The hTPO obtained is useful with  methods of stimulating proliferation or development of hematopoietic cells of the megakaryocytic leneage in vitro and in vivo.Reproducible expression vector in mammalian cell, it contains to guarantee accurately and the effective following elements formed of the mode of transcribing:a) corresponding to the transcripting promoter of the variable gene of human normal immunoglobulinb) coding comprises the dna section of complete htpo polypeptide of its signal peptide (secretion property lead)c) the immunoglobulin (ig) transcriptional enhancer dna section of people's kappa gened) transcription terminatore) polyadenylation signalf) coding has the gene of resistance to the antibiotic neomycin types of drugg) coding has the gene of resistance to the microbiotic penbritinh) colibacillary replication orgin (cole1 ori)
포유동물 세포 배양물을 이용하여 인간의 트롬보포에틴폴리펩타이드를 제조하는 방법시험관내 배양물에서 포유동물 세포에 의해 생물학적 활성인 인간 TPO를 생산하는 방법을 개시한다. 면역글로블린 인헨서에 결합된 면역글로불린 프로모터를 포함하는 DNA 작제물을 통해 인간 트롬보포에틴 유전자를 도입시켜 뮤린의 골수종 세포를 유전자 변형시킨다. 그 결과 수득되는 hTPO는 시험관내 및 생체내에서 거대핵 계열의 조혈 세포의 증식이나 발생을 자극하는 방법에 유용하다. [참고문헌] 1. Kuter, D. J. 1996, Thrombopoietin: Biology and Clinical Applications. The Oncologist 1:98. 2. Kelemen, E., I. Cserhati, and B. Tanos. 1958. Demonstration and some properties of human thrombopoietin in thrombocythemic sera. Acta Haematol. (Basel) 20:350. 3. Souyri, M., I. Vigon, J. F. Penciolelli, J. M. Heard, P. Tambourin, and F. Wendling. 1990. A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopieitc progenitors. Cell 63:1137. 4. Bartley, T. D., J. Bogenberger, P. Hunt, Y. L. Li, H. S. Lu, f. Martin, M. S. Chang, B. Samai, J. L. Nichol, S. Swift, M. J. Johnson, R. Y. Hsu, V. P. Parker, S. Suggs, J. D. Skrine, L. A. Merewether, C. Clogston, E. Hau, M. M. Hokom, A. Hornkohl, E. Choi, M. Pangelinan, Y. Sun, V. Mar, J. McNinch, L. Simonet, F. Jacobsen, C. Xie, J. Shutter, H. Chute, R. Basu, L. Selander, D. Trollinger, L. Sieu, D. Padilla, G. Trail, G. Elliott, R. Izumi, T. Covey, J. Crouse, A. Garcia, W. Xu, J. Del Castillo, J. Biron, S. Cole, H. MC.T., R. Pacifici, I. Ponting, C. Saris, D. Wen, Y. P. Yung, H. Lin, and R. A. Bosselman. 1994, Identification and cloning of a megakaryiocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77:1117. 5. de Sauvage, F. J., P. E. Hass, S. D. Spencer, B. E. Malloy, A. L. Gurney, S. A. Spencer, W. C. Darbonne, W. J. Henxel, S. C. Womg, W. J. Kuang, K. J. Oles, B. Hultgren, L. A. Solberg Jr, D. V. Goeddel, and D. L. Eaton. 1994. Stimulation of megacaryocytopoiesis and thrombopoiesis by the c-mpl ligand. Nature 369:533. 6. Kaushansky, K., S. Lok, R. D. Holly, V. C. Broudy, N. Lin, M. C. Bailey, J. W. Forstrom, M. M. Buddle, P. J. Oort, F. S. Hagen, G. J. Roth, T. Papayannopoulou, and D. C. Foster. 1994. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 369:568. 7. Lok, S., K. Kaushansky, R. D. Holly, J. L. Kuijper, C. E. Lofton-Day, P. J. Oort, F. J. Grant, M. D. Heipel, S. K. Burkhead, J. M. Kramer, L. A. Bell, C. A. Sprecher, H. C. Bailey, J. W. Forstrom, M. M. Buddle, S. G. Osborn, S. J. Evans, P. O. Sheppard, S. R. Presnell, P. J. O'Hara, F. S. Hagen, G. J. Roth, and D. C. Foster. 1994. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369:565. 8. Wdndling, F., E. Maraskovsky, N. Debill, C. Florindo, M. Teepe, M. Titeux, N. Methia, J. Breton-Gorius, D. Cosman, and W. Vainchenker. 1994. c-Mpl ligand is a humoral regulator of megakaryocytopiesis. Nature 369:571. 9. Kaushansky, K. 1995. Thrombopoietin: The primary regulator of platelet production. Blood 86:419. 10. Leonard, J. P., C. M. Quinto, M. K. Kozitza, T. Y. Neben, and S. J. Goldman. 1994. Recombinant human interleukin-11 stimulates multilineage hematopoietic recovery in mice after a myelosuppressive regimen of sublethal irradiation and carboplatin. Blood 83:1499. 11. Foster, D. C., C. A. Sprecher, and F. J. Grant. 1994. Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization. Proc. Natl. Acad. Sci. USA 91:13023. 12. Gurney, A.L., W. J. Kuang, M. H. Xie, B. E. Malloy, D. L. Eaton, and F. J. de Sauvage. 1995. Genomic structure, chromosomal localization and conserved alternative splice forms of thrombopoietin. Blood 85:981. 13. Wigler, M., A. Pellicer, S. Silverstein, and R. Axel. 1978. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725. 14. Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1:841. 15. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156. 16. Vogelstein, B., and D. Gillespie. 1979. Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76:615. 17. Kuter, D. J., P. Hunt, W. Sheridan, and D. Zucker-Franklin. 1997. Thrombopoiesis and Thrombopoietins: Molecular, Cellular, Preclinical, and Clinical Biology. Humana Press Inc. 18. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. (USA) 74:5463.다음과 같은 구성인자들이 정확하고 효율적으로 전사를 일으킬 수 있는 방식으로 구성되어 있는, 포유동물 세포에서 복제할 수 있는 발현 벡터: a) 인간 면역글로불린의 가변 유전자에 상응하는 전사 프로모터 b) 시그널 펩타이드(분비 리더)를 비롯하여 전체 hTPO 폴리펩타이드를 암호화하는 DNA 분절 c) 인간 카파 유전자의 면역글로불린 전사 인헨서 DNA 분절 d) 전사 터이네이터 e) 폴리아데닐화 시그널 f) 항생제 네오마이신형 약물에 내성을 부여하는 유전자 g) 항생제 앰피실린에 내성을 부여하는 유전자 h) 대장균의 복제 오리진(ColEI ori).