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目的人α防御素主要由中性粒细胞(HNP1、HNP2和HNP3)和小肠潘氏细胞(HD5和HD6)分泌。HNP1-3可以被作为肿瘤标志物,小剂量的α防御素可以促进肿瘤的增长,大剂量的α防御素可以裂解杀死肿瘤细胞。制备人防御素(human defensin-5,HD5或D5)、力达霉素辅基蛋白(lidamycin apoprotein,LDP)与EGFR配体寡肽(EGFR-directing ligand peptide,Ec)组合的融合蛋白Ec-LDP-D5,并初步探讨其抗胰腺癌活性。方法采用基因工程的方法制备融合蛋白基因,并将其表达纯化得到融合蛋白Ec-LDP-D5。融合蛋白与细胞表面EGFR的亲和活性采用ELISA和细胞免疫荧光化学法;采用CCK-8法测定融合蛋白对人胰腺癌细胞PNAC-1和Aspc-1的体外杀伤活性;流式细胞术检测Ec-LDP-D5蛋白对细胞凋亡的影响。结果成功构建并表达融合蛋白Ec-LDP-D5,产物主要以包涵体的形式存在,目的蛋白经纯化、复性后,每升发酵液可以获得1mg纯度达到85%。Ec-LDP-D5可与高表达EGFR的PNAC-1和Aspc-1细胞表面结合。Ec-LDP-D5对PNAC-1和Aspc-1肿瘤细胞有强烈的杀伤作用,Ec-LDP-D5蛋白对Aspc-1肿瘤细胞抑制率为76.8%,与Ec-LDP的抑制率49.4%相比,差异有统计学意义,P=0.038;对PANC-1细胞抑制率为64.6%,与Ec-LDP的抑制率28.3%相比,差异有统计学意义,P=0.042。Ec-LDP-D5在8μmol/L的浓度下即可强烈的诱导细胞发生凋亡,细胞凋亡率(24.6±0.56)%,与Ec-LDP的凋亡率相比P=0.011。结论本实验制备的防御素融合蛋白Ec-LDP-D5可以与高表达EGFR的PNAC-1和Aspc胰腺癌细胞结合,对胰腺癌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。
The human alpha defensins are mainly secreted by neutrophils (HNP1, HNP2 and HNP3) and small intestine Paneth cells (HD5 and HD6). HNP1-3 can be used as a tumor marker, a small dose of α-defensins can promote tumor growth, high doses of α-defensins can kill and kill tumor cells. Preparation of fusion protein Ec-LDP combining human defensin-5 (HD5 or D5), lidamycin apoprotein (LDP) and EGFR-directing ligand peptide (Ec) -D5, and preliminary study of its anti-pancreatic cancer activity. Methods The fusion protein gene was prepared by genetic engineering, and its fusion protein Ec-LDP-D5 was expressed and purified. The affinity of the fusion protein to the cell surface EGFR was determined by ELISA and immunofluorescence staining. The cytotoxicity of the fusion protein to PNAC-1 and Aspc-1 was detected by CCK-8. The cytotoxicity of Ec Effect of LDP-D5 Protein on Apoptosis. Results The fusion protein Ec-LDP-D5 was successfully constructed and expressed in the form of inclusion bodies. After purification and renaturation of the target protein, the purity of 1 mg reached to 85% per liter of fermentation broth. Ec-LDP-D5 binds to the surface of PNAC-1 and Aspc-1 cells that express EGFR overexpressing EGFR. Ec-LDP-D5 had a strong killing effect on PNAC-1 and Aspc-1 tumor cells. The inhibition rate of Ec-LDP-D5 protein to Aspc-1 tumor cells was 76.8%, which was significantly lower than that of Ec-LDP , The difference was statistically significant, P = 0.038; the inhibition rate of PANC-1 cells was 64.6%, which was significantly different from the inhibition rate of Ec-LDP of 28.3%, P = 0.042. Ec-LDP-D5 strongly induced cell apoptosis at a concentration of 8 μmol / L, with a apoptotic rate of (24.6 ± 0.56)%, which was significantly lower than that of Ec-LDP (P = 0.011). Conclusions The defensin fusion protein Ec-LDP-D5 prepared in this study can bind to PNAC-1 and Aspc pancreatic cancer cells with high expression of EGFR and has strong anti-tumor activity and apoptosis-inducing ability against pancreatic cancer cells. The potential of targeting drugs.