Construction of eukaryotic expressing vector pCH503 of CH50 and its chemotaxis and anti-tumor function by expressionin vivo in mice

Abstract

An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function by in vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5'-terminal noncoding region and coding region of signal peptide of mouse IFN-gamma cDNA at 5' side and 3'-terminal noncoden region of human FN cDNA at 3' side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503 in vivo and cultured in vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%-60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can express in vivo in mice. The transfection of pCH503 in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor.