论文部分内容阅读
目的制备一种以人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)为靶点的由抗体重链超变区多肽与力达霉素组成的强化融合蛋白LDP-Hr-AE,并初步探讨其抗肿瘤活性。方法通过聚合酶链式反应扩增出抗人表皮生长因子受体2抗体C6.5重链CDR3区的20个氨基酸残基基因,将其与力达霉素辅基蛋白基因连接构建出融合蛋白基因ldp-Hr,转化至大肠杆菌中进行诱导表达。融合蛋白LDP-Hr采用HisTrap Ni2+亲和色谱柱进行分离纯化,高效液相色谱法检测其纯度。细胞免疫荧光法和基于流式细胞术的亲和实验分析融合蛋白与肿瘤细胞的结合活性。纯化的LDP-Hr蛋白与力达霉素发色团在体外进行组装,构建出强化融合蛋白LDP-Hr-AE。采用MTT法检测强化融合蛋白对肿瘤细胞的杀伤活性,Annex-in V-FITC/PI双染结合流式细胞术的方法检测LDP-Hr-AE蛋白对细胞凋亡的影响。结果成功构建并表达了融合蛋白LDP-Hr,目的蛋白以可溶形式分泌至大肠杆菌培养液和周质腔中,产量可达每升发酵液40 mg活性蛋白,纯化后的蛋白经高效液相色谱法检测纯度为97.4%。细胞免疫荧光实验和基于流式细胞术的亲和实验检测结果均显示LDP-Hr蛋白与HER2高表达的肿瘤细胞系(如SK-BR-3和SK-OV-3细胞)都有很强的结合活性。LDP-Hr蛋白与力达霉素发色团在体外组装后经高效液相色谱法检测350 nm处出现特定吸收峰,表明强化融合蛋白LDP-Hr-AE构建成功。采用MTT法检测了强化融合蛋白的体外杀伤活性,结果显示,LDP-Hr-AE对肿瘤细胞有强烈的杀伤作用,且其杀伤活性要高于力达霉素。细胞凋亡检测结果也表明LDP-Hr-AE在极低的浓度下(如0.1 nmol.L-1)即可强烈的诱导细胞发生凋亡,且凋亡的比率随着蛋白浓度的升高而增加。结论本实验制备的强化融合蛋白LDP-Hr-AE可以特异的与人表皮生长因子受体2结合,对肿瘤细胞具有强烈的杀伤活性和诱导凋亡能力,具有发展为抗肿瘤靶向药物的潜能。
Objective To prepare an enhanced fusion protein LDP-Hr-AE composed of antibody heavy chain hypervariable region polypeptide and lidamycin targeting human epidermal growth factor receptor 2 (HER2) And preliminary study of its anti-tumor activity. Methods The 20 amino acid residues of the anti-human epidermal growth factor receptor 2 antibody C6.5 heavy chain CDR3 region were amplified by polymerase chain reaction and ligated with the lectin repair protein to construct the fusion protein Gene ldp-Hr, transformed into E. coli for induction of expression. The fusion protein LDP-Hr was purified by HisTrap Ni2 + affinity chromatography column and its purity was determined by high performance liquid chromatography. Cell immunofluorescence and flow cytometry-based affinity assays analyzed the binding activity of the fusion protein to tumor cells. The purified LDP-Hr protein was assembled with rindamycin chromophore in vitro to construct the enhanced fusion protein LDP-Hr-AE. The effect of LDP-Hr-AE protein on the apoptosis of tumor cells was detected by MTT assay and Annexin V-FITC / PI double staining combined with flow cytometry. Results The fusion protein LDP-Hr was successfully constructed and expressed. The target protein was secreted into Escherichia coli culture medium and periplasmic space in a soluble form. The yield reached 40 mg per liter of fermentation broth. The purified protein was purified by high performance liquid chromatography Purity was 97.4% by chromatography. Cell immunofluorescence assay and flow cytometry-based affinity assay showed that both LDP-Hr protein and HER2-overexpressing tumor cell lines (such as SK-BR-3 and SK-OV-3 cells) Binding activity. LDP-Hr protein and lidamycin chromophore assembled in vitro after the high-performance liquid chromatography detected specific absorption peak at 350 nm, indicating that the enhanced fusion protein LDP-Hr-AE constructed successfully. MTT assay was used to detect the in vitro cytotoxicity of the fusion protein. The results showed that LDP-Hr-AE had a strong killing effect on tumor cells, and its killing activity was higher than that of doxycycline. The results of apoptosis assay also showed that LDP-Hr-AE strongly induced cell apoptosis at extremely low concentration (eg 0.1 nmol.L-1), and the rate of apoptosis increased with the increase of protein concentration increase. CONCLUSION: The enhanced fusion protein LDP-Hr-AE prepared in this study can specifically bind to human epidermal growth factor receptor 2 and has strong killing activity and apoptosis-inducing ability on tumor cells and has the potential of developing anti-tumor targeting drugs .