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Related Experiment Videos

[Expression of recombinant human stem cell factor in insect cells].

D Zhang1, J Qin, Y Zang

  • 1Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093.

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica
|January 29, 2003
PubMed
Summary

Researchers developed a recombinant baculovirus, AcNPV-SCF, to express biologically active human stem cell factor (hSCF) in Sf9 cells. This method successfully produced secreted hSCF, demonstrating its potential for therapeutic applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Cell Biology

Background:

  • Human stem cell factor (hSCF) plays a crucial role in hematopoiesis and stem cell development.
  • Efficient and scalable production of biologically active hSCF is essential for research and potential therapeutic use.

Purpose of the Study:

  • To construct a recombinant baculovirus for the expression of human stem cell factor (hSCF).
  • To evaluate the biological activity and secretion of recombinant hSCF (rhSCF) produced in Sf9 insect cells.
  • To assess the synergistic effects of rhSCF with human interleukin-3 (hIL-3) on cell proliferation.

Main Methods:

  • Cloning of hSCF cDNA into the AcNPV transfer vector pVL941 to create pVL941-SCF.
  • Generation of recombinant baculovirus AcNPV-SCF via homologous recombination in Sf9 cells.

Related Experiment Videos

  • Confirmation of hSCF cDNA integration using Southern hybridization.
  • Expression and secretion analysis of rhSCF in infected Sf9 cells.
  • Assessment of synergistic activity with hIL-3 using MTT assay and TF-1 cell line.
  • Detection of rhSCF protein bands via immunoblotting.

    • Main Results:

    • A recombinant baculovirus, AcNPV-SCF, was successfully constructed and confirmed to contain the hSCF cDNA.
    • Sf9 cells infected with AcNPV-SCF expressed and secreted biologically active rhSCF into the cell culture medium.
    • The highest expression level of rhSCF reached approximately 1970 units/ml on day 3 post-infection.
    • Immunoblotting detected three distinct rhSCF bands with molecular masses of 18, 20, and 22 kDa.
    • rhSCF demonstrated synergistic activity with hIL-3 in promoting TF-1 cell proliferation.

    Conclusions:

    • The baculovirus expression system provides an effective platform for producing biologically active, secreted rhSCF.
    • The recombinant rhSCF exhibits functional activity, including synergistic effects with hIL-3, indicating its potential therapeutic value.
    • This study establishes a foundation for further investigation and application of rhSCF in stem cell research and regenerative medicine.