PRODUCTION AND ANALYSIS OF THE BIOLOGICAL PROPERTIES OF RECOMBINANT HUMAN GRANULOCYTE COLONY STIMULATING FACTOR CHIMERIC FORM
Mariya Borisovna PYKHTINA1,2, Vladimir Pavlovich ROMANOV3, Svetlana Mikhaylovna MIROSHNICHENKO1,2, Anatoliy Borisovich BEKLEMISHEV3
1Research Institute for
Biochemistry of Federal Research Center for Fundamental and
Translational Medicine
2Research Institute for Lymphology of Federal
Research Center В«Institute of Cytology and Genetics SB RAS»
3Research Institute for Biochemistry of Federal Research Center for Fundamental and Translational Medicine
Keywords: G-CSF человека, apoA-I, клонирование, химерный ген, Pichia pastoris X-33, ионообменная хроматография, клетки костного мозга, проточная цитометрия, human G-CSF, apoA-I, cloning, chimeric gene, Pichia pastoris X-33, ion exchange chromatography, bone marrow cells, flow cytometry
Abstract
The aim of this work was to
design and study biological properties of the recombinant human
granulocyte colony stimulating factor (G-CSF), «linked» to
apolipoprotein A-I (apoA-I) by a peptide linker, for obtaining in
perspective a prolong form of the drug based on this cytokine. Material
and methods. The nucleotide sequences of the genes encoding G-CSF and
apoA-I were designed and optimized for expression in Pichia pastoris
yeast using several computer programs. The assembly of the gene coding
for the G-CSF-аpoA-I chimeric cytokine, its cloning in the pPICZα-A
vector, and expression in P. pastoris cells were performed using
standard genetic engineering methods. Purification of the chimeric
cytokine was carried out by two-stage ion-exchange chromatography. The
biological activity of the chimera was determined in vitro on rat and
human bone marrow cells (BMC) using flow cytometry, cell cycle analysis
and myelograms. Results. A recombinant P. pastoris X-33 yeast strain
producing a chimeric cytokine containing the amino acid sequence G-CSF
from the N-terminus, and mature human apoA-I from the C-terminus was
constructed. In experiments on BMC of rat, it was shown that
G-CSF-аpoA-I increases the number of granulocytes in 1.8-2 times less
compared with G-CSF. At the same time, the chimeric cytokine maintained
the viability of monocytic and lymphocytic cells. Unlike G-CSF, the
chimera increased the number of blast cells and normalized neutrophil
segmentation, reducing the number of anomalies 1.5 times more
efficiently. Conclusion. A new chimeric cytokine G-CSF-аpoA-I was
constructed, exhibiting the properties of not only a colony-stimulating
factor, but also a growth factor, supporting the viability of other
types of BMC.
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