论文部分内容阅读
为了获得足够多纯度高且有活性的肝靶向肽-人内皮抑制素融合蛋白(HTP-r ES),首先研究了BL21/p ET21b-HTP-r ES重组菌株的生长曲线和最佳诱导时机;单因素分析不同p H值、不同诱导时间、不同诱导剂浓度、不同诱导温度时融合蛋白表达量;通过包涵体洗涤、复性、纯化,以获得高纯度的肝靶向肽-人内皮抑制素融合蛋白;最后采用流式细胞仪和MTT对融合蛋白进行活性鉴定。结果表明,BL21/p ET21b-HTP-r ES重组菌株在1.5–3.5 h处于对数生长期,培养基p H 8.0、IPTG终浓度0.06 mmol/L、42℃、诱导表达5 h为最佳表达条件。包涵体洗涤后纯度达60%,经复性、纯化后获得的目的蛋白纯度达到95%以上,对人肝癌细胞具有靶向性,能抑制人脐静脉内皮细胞的增殖。研究确立了融合蛋白最佳表达条件以及复性、纯化条件,为进一步研究其生物学活性及药物开发奠定基础。
In order to obtain enough pure and active liver-targeting peptide, human endostatin fusion protein (HTP-r ES), the growth curve of BL21 / p ET21b-HTP-r ES recombinant strain and the optimal induction time ; Single factor analysis of different p H value, different induction time, different concentrations of inducer, different induction temperature fusion protein expression; by inclusion body washing, renaturation, purification, in order to obtain high purity liver-targeting peptide - human endothelium inhibition Finally, the fusion protein was identified by flow cytometry and MTT. The results showed that the recombinant strain BL21 / p ET21b-HTP-r ES was in the logarithmic growth phase at 1.5-3.5 h, the medium p H 8.0, the final IPTG concentration 0.06 mmol / L, and the optimal expression was induced at 42 ℃ for 5 h condition. After washing, the purity of the inclusion body reaches 60%. After purification and purification, the purity of the target protein reaches more than 95%, which is targeted to human hepatoma cells and inhibits the proliferation of human umbilical vein endothelial cells. The study established the optimal expression conditions of fusion protein as well as renaturation and purification conditions, which laid the foundation for further study of its biological activity and drug development.