International Journal of Industrial Entomology and Biomaterials

Korean Society of Sericultural Science (한국잠사학회)
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Expression of Human Stem Cell Factor with Recombinant Baculovirus in BmN Cell Line and Silkworm

  • Xijie, Guo (Sericultural Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yongfeng, Jin (Institute of Biochemistry, Chinese Academy of Agricultural Sciences) ;
  • Mingguan, Yang (College of Animal Sciences, Zhejiang University) ;
  • Yaozhou, Zhang (Institute of Biochemistry, Chinese Academy of Agricultural Sciences)
  • Published : 2002.03.01
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Abstract

A recombinant transfer vector pBacSCF was constructed by inserting huamn stem cell factor (hSCF) cDNA into plasmid pBacPAK8. BmN cells were co-transfected with modified Bombyx mori, nuclear polyhedrosis virus (BmBacPAK) DNA and the recmbinant transfer vector to construct a recombinant baculovirus containing hSCE gene. DNA dot blotting and RNA dot blotting demonstrated that the hSCE gene was contained in the recombinant virus and transcribed. The recombinant baculovirus was infectious to BmN cells and to silkworm. SDS-PAGE analysis showed a specific band of expressed product in the extract of infected cells and in the heamolymph of infected larvae. Bioactivity of the recombinant hSCE was determined with W-1 cell line and MTT colorimetric method in synergy with interlukin-3 (IL-3). These results revealed that the hSCF gene was over-expressed in cultured cells and lavae of silkworm.

Keywords

References

  1. Arakawa, T., K. E. Langley, K. Kameyama and T. Takagi (1992) Molecular weights of glycosylated and nonglycosylated forms of recombinant human stem cell factor determined by low-angle laser light scattering. Anal. Biochem. 203, 53-57 https://doi.org/10.1016/0003-2697(92)90042-6
  2. Broudy, V. C. (1997) Stem cell factor and hematopoiesis. Blood 90,1345-1364
  3. Kitts, P. A. and R. D. Possee (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810-817
  4. Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  5. Martin, F. H., S. V. Suggs, K. E. Langley, H. S. Lu, T. Ting, K. J. Okino, C. F. Morris, I. K. McNiece, F. W. Jacobsen and E. A. Mendiaz (1990) Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63, 203-211 https://doi.org/10.1016/0092-8674(90)90301-T
  6. Sambrook, J., E. F. Fritsch and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York
  7. Smith, G. E., M. D. Summers and M. Fraser (1983) Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell Biol. 13, 183-192
  8. Summers, M. D. and G. E. Smith (1987) A manual of method for baculovirus vector and insect cell culture procedures. Texas Agncultural Experiment Station Bulletin No. 1555, 33-35
  9. Zhang Dongmei, Qin Junchuan, Zang Yuhui and Zhu Jie (2000) Expression of recombinant human stem cell factor in insect cells. Acta Microbiologica Sinica 40, 44-49
  10. Zhu Yuanxiao, Lai Chunning, Wang Jianan and Shen Beifen (1993a) Cloning and expression in COS7 cells of cDNA for human stem cell factor. Prog. Biochen. Biophys. 20, 127-128
  11. Zhu Yuanxiao, Lai Chunning, Shen Beifen and Li Huiyun(1993b) High expression of soluble form of human SCF inEscherichia coti. Prog. Biochem. Biophys. 20, 373-374