人血管内皮生长因子的克隆表达及活性鉴定

Cloning, Expression and Activity Detection of Vascular Endothelial Growth Factor

【目的】克隆人血管内皮生长因子 16 5基因 ,并测定蛋白质的表达及鉴定其活性。【方法】用PCR法从人心脏cDNA文库中钓取目的基因VEGF16 5 ,插入质粒PUC19中并测序鉴定。构建真核表达重组质粒AdtrackCMV VEGF16 5转染2 93细胞 ,用RNA斑点杂交和Westernblot方法检测VEGF16 5基因表达 ,并通过Miles试验检测VEGF16 5蛋白活性。【结果】PCR产物为 5 82bp ,测序结果表明其序列正确 ,在RNA和蛋白水平检测到VEGF16 5基因表达 ,VEGF16 5蛋白相对分子质量为 2 2ku ,具有生物学活性。【结论】成功地克隆了有表达生物学活性的VEGF16

To clone, express and detect activity of VEGF165. Using human heart cDNA library as template, amplified the VEGF gene by PCR. The PCR product was ligated to PUC19 plasmid and sequenced. A eukaryotic expression plasmid AdtrackCMV harbouring VEGF165 was constructed., then transformed into 293 cells. RNA dot blot and Western blotting were performed to demonstrated whether the tranfermants expressed VEGF165 at mRNA and protein level respectively. Furthermore the bio activity of VEGF165 was preliminarily detected with Miles test. Sequence of 582bp VEGF165 cDNA was proved correct by sequencer analysis. The expression of VEGF165 mRNA was identified by RNA dot blot, Western blotting indicated that the molecular weight of VEGF165 protein was 22ku. VEGF165 also is of vascular permeability. [Conclusion] VEGF165 gene has been successfully cloned and expressed,which makes a basis for the further study in vivo.