RESUMO
<p><b>OBJECTIVE</b>To observe the effect of human vascular endothelial growth factor (VEGF) fragment (3 approximately 4 exon) in E. coli on anti-angiogenesis.</p><p><b>METHODS</b>Through RT-PCR amplification, endonuclease cut and DNA sequence analysis identification, hVEGF fragment cDNA was inserted into E. coli expression vector pTrcHis2A. The prokaryotic expression plasmid pTrcHis2A/VEGF(3 approximately 4) was constructed and transformed into TOP10F.</p><p><b>RESULTS</b>After 8hr isopropy-beta-D-thiogalactoside (IPTG) induction, VEGF fragment was expressed in 15% of total proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expressed protein was highly antigenic and specific. The VEGF fragment was further purified by affinity, which could inhibit HUVEC proliferation and neovascularization of the chick chorioallantoic membrane.</p><p><b>CONCLUSION</b>VEGF fragment is anti-angiogenetic, which may potentially be used in oncologico-biological targeting therapy.</p>
Assuntos
Humanos , Inibidores da Angiogênese , Farmacologia , Clonagem Molecular , Fatores de Crescimento Endotelial , Genética , Farmacologia , Escherichia coli , Genética , Expressão Gênica , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular , Genética , Farmacologia , Isopropiltiogalactosídeo , Farmacologia , Linfocinas , Genética , Farmacologia , Fragmentos de Peptídeos , Genética , Farmacologia , Plasmídeos , Genética , Reação em Cadeia da Polimerase , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Objective:To develop an inducible system for expression of GraB in E.coli for the purpose of investion of its function on genome DNA fragment.Methods:Amplified granzyme B cDNA by RT-PCR via extracting lymphocyte total RNA.Identified with endonuclease cut and DNA sequence analysis,the gene was inserted into E.coli expression vector pTrcHis2A,the pokaryotic expression plasmid pTrcHis2A/GraB was constructed and transformed into TOP10F.Results:After 8 h of IPTG in duction, the GraB was expressed 15% of total proteins by SDS-PAGE.Western blot assay proved the expressed hGraB to be of good antigenicity and high specificity.The recombinant protein purified by affinity chromatography has effect on GraB substrate.GraB was found to work directly on the genome DNA fragments,promoting its split.Conclusion:It is found that there may exist certain novel pathway for GraB-induced apoptosis.