庞丽君 刘道洁 林明华

    [摘要] 目的 构建血管内皮生长因子(VEGF)原核表达载体并诱导其表达蛋白，为后续进行VEGF相关研究奠定基础。 方法 通过逆转录聚合酶链式反应(RT-PCR)扩增得到目的片段，将DNA片段克隆至带组氨酸标签的pET-30a(+)载体上;构建的重组质粒经过菌液聚合酶链式反应(PCR)及测序方法鉴定;利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组质粒表达，通过蛋白质印迹法(Western blot)进行鉴定。 结果 以HepG2细胞为模板扩增出大小正确的片段;构建的pET-30a-VEGF重组质粒经菌液PCR及DNA测序证实序列正确;考马斯亮蓝染胶和Western blot结果显示IPTG诱导表达的融合蛋白分子量正确且条带单一。 结论 成功构建pET-30a-VEGF原核表达质粒，后续可进行大量诱导表达用于肝癌的诊疗相关研究。

    [关键词] 血管内皮生长因子;肝细胞癌;基因克隆;原核表达

    [中图分类号] R735.7 ? ? ? ? ?[文献标识码] A ? ? ? ? ?[文章编号] 1673-7210(2020)08(b)-0004-04

    [Abstract] Objective To construct a prokaryotic expression vector containing vascular endothelial growth factor (VEGF) and induce to express protein， it may lay a foundation for the follow-up study of VEGF. Methods Amplified The target fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) and the DNA fragment was cloned into pET-30a(+) vector with histidine tag. The recombinant plasmid was identified by polymerase chain reaction (PCR) and sequencing. The expression of recombinant plasmid was induced by isopropylthio-β-D-galactoside (IPTG) and identified by Western blot. Results The right size of the fragment was amplified from HepG2 cells. The recombinant plasmid pET-30a-VEGF was confirmed by PCR and DNA sequencing. Coomassie brilliant blue staining and Western blot results showed that the molecular weight of the fusion protein induced by IPTG was correct and the band was single. Conclusion The prokaryotic expression plasmid of pET-30a-VEGF is successfully constructed ......