肝靶向肽-人内皮抑制素融合蛋白表达条件的优化及活性鉴定

Optimization of expression conditions and activity identification of hepatocyte-targeting peptide-human endostatin

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。

To obtain sufficient purified and active fusion protein-hepatocyte-targeting peptide-human endostatin（HTP-r ES）,we studied the growth curve and the optimal induction timing of BL21/p ET21b-HTP-r ES.Different conditions of p H value,induction time,induction concentration and induction temperature were optimized by univariate analysis.After washing,refolding and purifying,the activity of fusion protein was identified by flow cytometry and 3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide（MTT）.Results show that the logarithmic growth phase of BL21/p ET21b-HTP-r ES was from 1.5 h to 3.5 h,the optimum expression conditions were p H 8.0,0.06 mmol/L IPTG,at 42 for 5 h.The purity of ℃inclusion bodies was up to 60% after washing.The purity of target protein was more than 95% after refolding and purification.Our findings provide the foundation for further biological activity and drug development.