Volume 14 Issue 4
Dec.  2001
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MA LI, WANG XIAO-NING, ZHANG Zhi-qing, CHEN Ai-jun, YAO LI-HONG. Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity[J]. Biomedical and Environmental Sciences, 2001, 14(4): 302-311.
Citation: MA LI, WANG XIAO-NING, ZHANG Zhi-qing, CHEN Ai-jun, YAO LI-HONG. Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity[J]. Biomedical and Environmental Sciences, 2001, 14(4): 302-311.

Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity

Funds:  国家高技术研究发展计划(863计划)(102-08-01-03)%the Guangdong Province Natural Science Fund(001098)
  • Objective To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it. Methods  By inserting hVEGF165 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/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 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  hVEGF165 was successfully expressed. The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.
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    沈阳化工大学材料科学与工程学院 沈阳 110142

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Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity

Funds:  国家高技术研究发展计划(863计划)(102-08-01-03)%the Guangdong Province Natural Science Fund(001098)

Abstract: Objective To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it. Methods  By inserting hVEGF165 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/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 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  hVEGF165 was successfully expressed. The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.

MA LI, WANG XIAO-NING, ZHANG Zhi-qing, CHEN Ai-jun, YAO LI-HONG. Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity[J]. Biomedical and Environmental Sciences, 2001, 14(4): 302-311.
Citation: MA LI, WANG XIAO-NING, ZHANG Zhi-qing, CHEN Ai-jun, YAO LI-HONG. Expression of Human Vascular Endothelial Growth Factor (VEGF165) in Pichia pastoris and Its Biological Activity[J]. Biomedical and Environmental Sciences, 2001, 14(4): 302-311.

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