Cloning of Soluble Human Stem Cell Factor in pET-26b(+) Vector

Area of Science:

• Biotechnology
• Molecular Biology
• Recombinant Protein Expression

Background:

• Stem cell factor (SCF) is vital for hematopoietic stem and progenitor cell functions.
• Therapeutic uses of SCF include stem cell mobilization, ex vivo expansion, gene therapy, and immunotherapy.
• Local production of rhSCF is needed in Iran due to cost and accessibility issues.

Purpose of the Study:

• To construct a soluble rhSCF expression vector with periplasmic localization potential.
• To indigenize the production of recombinant human SCF (rhSCF).

Main Methods:

• Polymerase chain reaction (PCR) amplification of the human SCF open reading frame (ORF).
• Cloning of the SCF ORF into the pET-26b (+) vector using NcoI and XhoI restriction sites.
• Transformation of the recombinant construct into BL21 (DE3) E. coli strains.

Main Results:

• Construction of the recombinant vector was confirmed by colony PCR and sequence analysis.
• Sequence analysis verified the correct insertion and orientation of the human SCF ORF.
• No nucleotide errors were detected in the pET26b-hSCF vector.

Conclusions:

• The SCF ORF was successfully cloned into the pET-26b (+) expression vector.
• The recombinant vector is prepared for the subsequent production of SCF protein.
• This work facilitates the domestic production of rhSCF.