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目的:利用大肠埃希菌表达基于抗表皮生长因子受体(epidermal growth factor receptor,EGFR)单链抗体和靶向CD13环状NGR肽的双靶点重组融合蛋白ER(Fv)-NGR,研究其对肿瘤细胞的亲和能力和体内外抑制作用。方法:用基因工程的方法构建重组质粒pET-scfv-ngr,转入大肠埃希菌BL21,IPTG诱导表达带有His-tag标签肽的重组蛋白ER(Fv)-NGR,用Ni离子亲和柱进行纯化。运用细胞免疫荧光检测ER(Fv)-NGR与肿瘤细胞的结合;流式细胞术分析重组蛋白与肿瘤细胞的亲和力;克隆形成实验测定重组蛋白对肿瘤细胞体外增殖的抑制作用;利用人乳腺癌MCF-7裸鼠移植瘤模型对ER(Fv)-NGR的体内抗肿瘤活性进行研究。结果:重组蛋白ER(Fv)-NGR由抗EGFR单链抗体和3个连续的环状NGR肽构成,相对分子质量约为27×104,以包涵体形式在大肠埃希菌内表达,表达量约为5mg/L。细胞免疫荧光结果表明,重组蛋白与EGFR和CD13皆高表达MCF-7细胞有较强的结合能力,而与正常HEK293细胞结合较弱。ER(Fv)-NGR与MCF-7细胞结合的解离常数为8.4×10-7 nmol/L。ER(Fv)-NGR在体外对肿瘤细胞的增殖有抑制作用,作用于MCF-7和A549细胞的IC50值分别为1.2×10-6和4.7×10-6 mol/L。在体内对人乳腺癌MCF-7裸鼠移植瘤有生长抑制作用,在10mg/kg药物剂量下28d对照组与实验组瘤体积分别为(1.06±0.17)和(0.63±0.11)cm3,F=18.7,P=0.003;瘤质量分别为(0.95±0.12)和(0.52±0.07)g,F=42.7,P<0.001;抑瘤率为45.4%。结论:成功制备了基于单链抗体和靶向肽的双靶点融合蛋白ER(Fv)-NGR,其对肿瘤细胞具有良好的亲和力和体外增殖抑制作用,能够有效抑制裸鼠移植瘤的生长,为研制新型双靶点抗肿瘤药物提供依据。
OBJECTIVE: To use the double-target recombinant fusion protein ER (Fv) -NGR based on single chain antibody against epidermal growth factor receptor (EGFR) and CD13 cyclic NGR peptide in Escherichia coli. Affinity to tumor cells and in vitro and in vivo inhibition. Methods: The recombinant plasmid pET-scfv-ngr was constructed by genetic engineering and transformed into Escherichia coli BL21. The recombinant protein ER (Fv) -NGR with His-tag tag peptide was induced by IPTG. Using Ni ion affinity column Purification. The binding of ER (Fv) -NGR to tumor cells was detected by immunofluorescence assay. The affinity of the recombinant protein to tumor cells was analyzed by flow cytometry. The inhibitory effect of the recombinant protein on the proliferation of tumor cells was determined by clonogenic assay. -7 nude mouse xenograft model in vivo anti-tumor activity of ER (Fv) -NGR. Results: The recombinant protein ER (Fv) -NGR was composed of anti-EGFR single chain antibody and three consecutive NGR peptides. The relative molecular mass was about 27 × 104 and expressed as inclusion bodies in Escherichia coli. The expression level About 5mg / L. The results of immunofluorescence showed that the recombinant protein had strong binding ability with both EGFR and CD13 high expression MCF-7 cells, but weakly with normal HEK293 cells. The dissociation constant of ER (Fv) -NGR binding to MCF-7 cells was 8.4 × 10-7 nmol / L. ER (Fv) -NGR inhibited the proliferation of tumor cells in vitro, and the IC50 values of MCF-7 and A549 cells were 1.2 × 10-6 and 4.7 × 10-6 mol / L, respectively. The growth of MCF-7 xenografts in nude mice was inhibited in vivo by (1.06 ± 0.17) and (0.63 ± 0.11) cm3 at 28 days after treatment with 10 mg / kg of drug, respectively, with F = 18.7, P = 0.003 respectively. The tumor mass was (0.95 ± 0.12) and (0.52 ± 0.07) g respectively, F = 42.7, P <0.001. The tumor inhibition rate was 45.4%. CONCLUSION: The double-target fusion protein ER (Fv) -NGR based on single chain antibody and targeting peptide has been successfully prepared. It has good affinity to tumor cells and in vitro proliferation inhibition, which can effectively inhibit the growth of xenografts in nude mice. To provide a basis for the development of new dual-target anti-tumor drugs.