Lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence enables long-term secretion of functional chondroitinase ABC in human bone marrow stromal cells

Biotechnol Lett. 2016 May;38(5):893-900. doi: 10.1007/s10529-016-2046-y. Epub 2016 Feb 24.

Abstract

Objective: To test the feasibility of secretion of functional chondroitinase ABC (ChABC), a bacterial enzyme that promotes axonal regeneration after spinal cord injury, from human bone marrow stromal cells (hBMSCs).

Results: A lentiviral-expression vector, Lenti6.3-ChABC-3F, carrying the ChABC-3F gene without the bacterial leader sequence (aa 1-24) was constructed. Transfection of these Lenti6.3-ChABC-3F lentivirus led to stable expression in and secrection of ChABC proteins from hBMSCs for at least ten passages in culture in vitro, which was demonstrated by QRT-PCR, immunostaining, western blotting and ELISA. Moreover, the secreted ChABC proteins exhibited similar functional activity as the commercially-available ChABC.

Conclusions: The lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence induced substantial long-term secretion of functional ChABC in hBMSCs.

Keywords: Bone marrow stromal cells; Chondroitin sulfate proteoglycans; Chondroitinase ABC; Lentivirus; Secretion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Chondroitin ABC Lyase / genetics
  • Chondroitin ABC Lyase / metabolism*
  • Gene Expression
  • Gene Expression Profiling
  • Genomic Instability
  • Humans
  • Immunoassay
  • Lentivirus / genetics
  • Mesenchymal Stem Cells / metabolism*
  • Protein Sorting Signals / genetics
  • Real-Time Polymerase Chain Reaction
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Transfection

Substances

  • Protein Sorting Signals
  • Recombinant Proteins
  • Chondroitin ABC Lyase