目的 克隆、表达和纯化融合蛋白IVT, 并检测其生物学活性。 方法 采用基因工程手段将具有免疫功能、抗血管生成功能和诱导肿瘤细胞凋亡功能的3个基因片段进行融合构建了融合基因IVT。将其插入pPIC9 表达质粒中, 转化至毕赤酵母GS115(his4), 筛选出的表达株经甲醇诱导, 表达了重组融合蛋白IVT, 并得以纯化。采用MTT法观察IVT对CTLL-2细胞、HUVEC和NCI-H446细胞增殖的影响;通过Transwell实验观察IVT对HUVEC迁移的影响;采用Hoechst染色检测IVT对NCI-H446细胞凋亡的影响。结果 IVT能促进CTLL-2细胞增殖、抑制HUVEC和NCI-H446细胞生长、降低HUVEC细胞迁移能力以及诱导NCI-H446细胞凋亡。结论 IVT具有多种生物学活性,为开发多功能的新型融合蛋白打下了基础。
Abstract
To clone, express and purify a fusion protein IVT, and detect its biological activity. METHODS A fusion gene IVT with immunal function, anti-angiogenesis and apoptosis-inducing activities was constructed by genetic engineering means. pPIC9-IVT was constructed and transformed into Pichia pastoris GS115(his4). The recombinant fusion protein IVT was expressed in yeast engineering strain and purified from the culture supernatant. MTT assay was used to observe the effect of the IVT on the proliferation of CTLL-2 cells, human umbilical vein endothelial cells(HUVECs)and NCI-H446 cells.Transwell migration assay was used to observe the impact of the IVT on cell migration ability of HUVECs.The apoptosis of NCI-H446 cells was examined by Hoechst staining. RESULTS The IVT promoted CTLL-2 cells proliferation, inhibited HUVECs and NCI-H446 cells proliferation, decreased HUVECs migration and induced apoptosis of NCI-H446 cells.CONCLUSION The IVT has a variety of biological activities. This study laid a foundation for the development of novel multi-functional fusion proteins.
关键词
免疫 /
抗血管生成 /
凋亡 /
融合蛋白IVT /
增殖
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Key words
immune /
anti-angiogenesis /
apoptosis /
fusion protein IVT /
proliferation
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中图分类号：
Q816
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参考文献
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