Study On The Cloning/ Expression And Biological Functions Of Vascular Endothelial Growth Factor 165

Objective: To construct a procaryotic expression vector of vascularendothelial growth factor 165 (VEGF165) and to express VEGF165 inE.coli. To construct an eukaryotic expression vector of VEGF165 and tostudy the effect of VEGF165 on proliferation and apoptosis of K562. Methods: The VEGF165 fragment was amplified with RT-PCR fromhuman leukemic cell line TF1 , the amplified fragment was inserted intopUCmT and sequenced, then procaryotic expression vectorPET20b(+)-VEGF165 was constructed. After induced by IPTG,therecombinant VEGF165 was purified using Ni-NTA Resin column.SDS-PAGE analysed the expression and purification of VEGF165, andWestern Blot identified recombinant VEGF165. The VEGF165 fragmentwas amplified with RT-PCR from human leukemic cell line HL60 , theamplified fragment was inserted into pUCmT and sequenced, theneukaryotic expression vector pcDNA3.1(-)-VEGF165 was constructed andtransfected into K562 with lipofect technique. Its expression was identifiedby RT-PCR and Western Blot. The effect of VEGF165 on K562proliferation was observed by MTT trial and tumor colony formation.Apoptosis of K562 was induced by As2O3(3μM),then the effect ofVEGF165 on K562 apoptosis was observed by MTT trial and flowcytometric analysis. Results: 1.The sequence of VEGF165 fragment cloned was thesame as the reported VEGF165. 2. SDS-PAGE showed that VEGF165 wasexpressed in E.coli and purified successfully. 3.Western Blot showed thatrecombinant VEGF165 could combine with rabbit anti-human VEGFMoAb specially. 4. RT-PCR and Western Blot showed VEGF165 wasexpressed in K562. 5.MTT trial showed the proliferation of K562/V wasstronger than K562/M and the proliferation inhibition of K562/M withAs2O3 ( 3μM ) was more striking than K562/V(P<0.05).The colonyformation rate of K562/V and K562/M was 32.2±2.5% and 23.5±1.7%;Flow cytometric analysis showed that the apoptotic rates of K562/V withAs2O3(3μM)48h,72h were 5.1%,14.9%, K562/M were 12.3%,35.8%. Conclusion:VEGF165 was cloned, expressed in E.coli and purifiedsuccessfully, which laid the foundation for further studying of function ofVEGF165. Transfection of VEGF165 into K562 could increase itsproliferation and inhibit its apoptosis.