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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NCIH446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。
Objective To clone, express and purify the fusion protein IVT and test its biological activity. Methods Three gene fragments with immunological function, anti-angiogenic function and apoptosis-inducing function were fused by genetic engineering to construct the fusion gene IVT. It was inserted into pPIC9 expression plasmid and transformed into Pichia pastoris GS115 (his4). The selected strains were induced by methanol and expressed recombinant fusion protein IVT and purified. The effect of IVT on the proliferation of CTLL-2 cells, HUVECs and NCI-H446 cells was observed by MTT assay. The effect of IVT on the migration of HUVECs was observed by Transwell assay. The effect of IVT on the apoptosis of NCIH446 cells was detected by Hoechst staining. Results IVT could promote the proliferation of CTLL-2 cells, inhibit the growth of HUVECs and NCI-H446 cells, decrease the migration ability of HUVECs and induce the apoptosis of NCI-H446 cells. Conclusion IVT has a variety of biological activity, laying a foundation for the development of a new multi-functional fusion protein.