摘要
Objective To express human vascular endothelial growth factor (hVEGF-{165}) cDNA in %Pichia pastroris, %purify the expressed product and detect the biological activity of it. Methods By inserting hVEGF-{165} cDNA coding 165 amino acid residues into %Pichia pastoris% expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF-{165} was constructed and transformed to yeast host strain KM71, then multiple_copy insert transformants were screened out and cultured in flasks, and hVEGF-{165} was expressed under the induction of 1% methanol. Results SDS_PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by Heparin_Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC. Conclusion hVEGF-{165} was successfully expressed. The study opened up a wide prospect for the application of VEGF-{165} in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.
Objective To express human vascular endothelial growth factor (hVEGF-{165}) cDNA in %Pichia pastroris, %purify the expressed product and detect the biological activity of it. Methods By inserting hVEGF-{165} cDNA coding 165 amino acid residues into %Pichia pastoris% expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF-{165} was constructed and transformed to yeast host strain KM71, then multiple_copy insert transformants were screened out and cultured in flasks, and hVEGF-{165} was expressed under the induction of 1% methanol. Results SDS_PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by Heparin_Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC. Conclusion hVEGF-{165} was successfully expressed. The study opened up a wide prospect for the application of VEGF-{165} in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.
基金
theNationalHighTechnology"86 3"ProgramofChina
No.1 0 2_0 8_0 1_0 3andthe GuangdongProvinceNaturalScienceFund
No .0 0 1 0 98
