EGF-Ang融合蛋白的构建、表达及活性研究

Construction and Expression of the EGF-Ang Fusion Protein and Its Biological Activity

目的:构建由人表皮生长因子(epidermal growth factor,EGF)和人血管生成素(angiogenin,Ang)组成的人源化的融合蛋白,并检测其对肿瘤细胞的靶向杀伤能力。方法:利用基因工程技术将EGF和Ang基因连接起来,克隆到高效表达载体pET28a(+)中,构建重组表达质粒pEGF-Ang,并在大肠杆菌中表达该融合蛋白(EGF-Ang)。经DEAE-Sepharose FF阴离子交换柱层析纯化后,用MTT法检测复性蛋白的细胞毒性。结果:SDS-PAGE和薄层扫描分析表明外源蛋白的表达量占菌体裂解蛋白总量的18.6％。细胞活性检测表明EGF-Ang重组蛋白能明显地抑制Hep2细胞的生长,而不影响MA104细胞的正常生长。结论:融合蛋白EGF-Ang在体外对过度表达EGFR的Hep2细胞具有明显的杀伤作用。

Objective: To construct a humanized fusion protein consisting of a EGF linked to a human angiogenin ( Ang) and evaluate the potential of the construct( EGF-Ang) to target and kill tumor cells. Methods: By using of genetic engineering techniques, we constructed a recombinant expressing plasmid pEGF-Ang, and expressed fusion protein accumulated in intracellular inclusion bodies. The recombinant EGF-Ang proteins were purified through DEAE-Sepharose FF chromatography in the denatured condition. After renaturing process, the cytotoxicity was measured with MTT colorimetric assay in vitro. Results: A novel fusion protein named EGF-Ang was constructed and expressed in E coli. Analysis of SDS-PAGE and thin layer scanning results showed that amount of expressed fusion protein was 18.6% in total lysis protein of bacteria. Cytotoxicity analysis showed that the fusion proteins could inhibit the growth of Hep2 cells but had little influence on the growth of MA104 cells. Conclusion:EGF-Ang fusion protein had definitely cytotoxicity to Hep2 cells which over expressed EGFR in vitro.