多靶向VEGF-EGFR的Fc融合蛋白EVP1的构建及其结合特性

Construction of Fc fusion protein EVP1 targeting members of VEGF-EGFR and its binding affinity

目的:构建能同时靶向表皮生长因子受体(epidermal growth factor receptor,EGFR)、血管内皮生长因子(vascular endothelial growth factor,VEGF)和人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)的Fc融合蛋白EVP1,并分析其多靶向结合功能,为后续的抗肿瘤研究奠定基础。方法:利用PCR方法扩增Herin基因和Flt-1基因Ig样第二结构域(Flt-1D2)编码序列,全序列合成带有点突变的人IgG1的Fc段编码序列,利用重组PCR方法将Herin、Flt-1D2和Fc基因依次连接,构成Herin-Flt-1D2-Fc基因(命名为EVP1基因)。再将EVP1编码基因插入质粒pDC659,构建携带EVP1基因的腺病毒穿梭质粒pDC659-EVP1。将质粒pDC659-EVP1与腺病毒骨架载体pPE3-F35共转染至293细胞,包装获得表达EVP1融合蛋白的非增殖型腺病毒Ad5/35-EVP1,经PCR鉴定,扩增、纯化病毒,采用50%组织培养感染剂量(TCID50)法测定病毒滴度。用MOI=11的腺病毒Ad5/35-EVP1感染293细胞,4 d后收集细胞培养上清液。采用硫酸铵盐析法和Protein G亲和层析对融合蛋白EVP1进行纯化。Western blotting检测细胞培养上清液中融合蛋白EVP1的表达,ELISA法检测细胞培养上清液中融合蛋白EVP1的含量。采用间接免疫荧光实验和Octet Red 96生物分子相互作用分析仪对融合蛋白EVP1的靶向结合特性进行定性和定量检测。结果:成功包装出腺病毒Ad5/35-EVP1,滴度为5.4×109PFU/ml。腺病毒Ad5/35-EVP1-293细胞系统能有效表达融合蛋白EVP1;MOI=11时,融合蛋白EVP1的表达量为(1 613.94±24.65)ng/ml。蛋白EVP1与高表达EGFR的A431细胞和高表达HER2的人卵巢癌SK-OV-3细胞有良好的结合能力,与抗原EGFR、HER2、VEGF结合的亲和力常数Kd分别为4.55、18.70和0.63 nmol/L。结论:成功制备多靶向融合蛋白EVP1,它能高效结合VEGF、EGFR受体家族成员,具有良好的抗肿瘤临床应用价值。

Objective： To construct the Fc fusion protein EVP1 with high affinity to bind epidermal growth factor receptor（EGFR）,vascular endothelial growth factor（VEGF）,and human epidermal growth factor receptor 2（HER2）,and to analyze its multi-target binding affinity for further study of antitumor therapy.Methods： The coding sequences of Herin and FLT-1D2（the second Ig-like domain of FLT-1 gene） were amplified by PCR individually,and joined to synthesize human IgG1 Fc fragment with point mutations via overlap PCR.Then,the fused open reading frame of Herin-Flt-1D2Fc（named as EVP1） was inserted into pDC659 plasmid,an adenoviral shuttle vector.The plasmid of pDC659-EVP1 and pPE3-F35 were co-transfected into 293 cells to package Ad5 /35-EVP1,a replication-incompetent adenovirus carrying the EVP1 coding gene.After identification,amplification,purification and titration determination,the recombinant virus Ad5 /35-EVP1 was used to transfect 293 cells at a MOI = 11.Four days post transfection,culture supernatant was collected to enrich and purify the fusion protein EVP1 through ammonium sulfate salting out method and protein G affinity chromatography.Subsequently,Western blotting and ELISA were performed to detect the expression of EVP1.Finally,the binding affinities of EVP1 to EGFR,HER2 and VEGF were both qualitatively identified via indirect immunofluorescent assay and quantitatively determined on the Octet Red 96 Biomolecular Interaction Analysis System.Results： The recombinant adenovirus Ad5 /35-EVP1 was constructed successfully,and its titer was 5.4 × 10 9 PFU/ml.With the expression system of Ad5 /35-EVP1 and 293 cells,the fusion protein EVP1 could be effectively produced with the expression level being（1 613.94 ±24.65） ng/ml when MOI =11.The obtained EVP1 could bind EGFR on A431 cells and HER2 on SK-OV3 cells with high affinity,and the affinity constants K d of EGFR,HER2 and VEGF were 4.55,18.70,and 0.63 nmol/L,respectively.Conclusion： The fusion protein EVP1 was constructed successfully,and EVP1 has high binding-affinity to VEGF and EGFR family members,showing a good clinical potent in targeted cancer therapy.