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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 human epidermal growth factor (EGF) and human angiogenin (Ang) and to test its targeting activity to tumor cells. METHODS: EGF and Ang genes were ligated by using gene engineering technology, cloned into pET28a (+) vector and constructed recombinant plasmid pEGF-Ang. The fusion protein (EGF-Ang) was expressed in E. coli. After purification by DEAE-Sepharose FF anion exchange chromatography, the cytotoxicity of the renaturation protein was detected by MTT assay. Results: The results of SDS-PAGE and TLC showed that the expression of exogenous protein accounted for 18.6% of the total bacterial lysate protein. Cell viability assay showed that EGF-Ang recombinant protein could significantly inhibit the growth of Hep2 cells without affecting the normal growth of MA104 cells. CONCLUSION: The fusion protein EGF-Ang has obvious killing effect on Hep2 cells overexpressing EGFR in vitro.