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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 a cell line that can simultaneously target epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and human epidermal growth factor receptor 2 ) Of Fc fusion protein EVP1, and analysis of its multi-target binding function, lay the foundation for subsequent anti-tumor research. Methods: The coding sequence of Her-1 gene and Ig-like second domain of Flt-1 gene (Flt-1D2) was amplified by PCR. The Fc region of human IgG1 with point mutation was synthesized by whole sequence. Herin, The Flt-1D2 and Fc genes are in turn linked to form the Herin-Flt-1D2-Fc gene (named EVP1 gene). The EVP1 encoding gene was inserted into the plasmid pDC659 to construct the adenoviral shuttle plasmid pDC659-EVP1 carrying the EVP1 gene. The plasmid pDC659-EVP1 and adenoviral backbone vector pPE3-F35 were co-transfected into 293 cells and packaged to obtain the non-proliferating adenovirus Ad5 / 35-EVP1 expressing the EVP1 fusion protein. The virus was identified by PCR, amplified and purified using 50 % Tissue culture infectious dose (TCID50) method for the determination of virus titer. 293 cells were infected with adenovirus Ad5 / 35-EVP1 at MOI = 11 and cell culture supernatants were harvested after 4 days. The fusion protein EVP1 was purified by ammonium sulfate salting-out and Protein G affinity chromatography. The expression of fusion protein EVP1 in cell culture supernatant was detected by Western blotting. The content of fusion protein EVP1 in cell culture supernatant was detected by ELISA. Indirect immunofluorescence assay and Octet Red 96 biomolecular interaction analyzer were used to characterize and quantify the targeted binding characteristics of the fusion protein EVP1. Results: The adenovirus Ad5 / 35-EVP1 was successfully packaged with a titer of 5.4 × 109 PFU / ml. The adenovirus Ad5 / 35-EVP1-293 cell system could effectively express the fusion protein EVP1. At MOI = 11, the expression level of fusion protein EVP1 was (1 613.94 ± 24.65) ng / ml. The protein EVP1 has good binding ability with A431 cells highly expressing EGFR and SK-OV-3 cells highly expressing HER2, and the affinity constants Kd with EGFR, HER2 and VEGF are 4.55, 18.70 and 0.63 nmol / L. Conclusion: The multi-targeting fusion protein EVP1 was successfully prepared, which can efficiently bind VEGF and EGFR receptor family members and has good anti-tumor clinical value.