重组人内皮抑素的表达、纯化、复性方法研究

A Study of Expression,Purification,and Refolding in Recombinant Human Endostatin

[目的]摸索重组人内皮抑素(recombinanthumanendostatin,rhES)的最佳生产工艺路线,解决rhES在大肠杆菌中表达提取的蛋白变性问题。[方法](1)按所优化的条件进行rhES的表达,包涵体的提取、洗涤、变性、复性。(2)活性检测:观察rhES对裸鼠皮下移植瘤生长情况及病理组织学的影响。[结果](1)每升培养基可得可溶性rhES蛋白10.55mg,rhES在大肠杆菌中的表达量超过总菌体蛋白的60%,纯化后的蛋白纯度超过95%。(2)动物实验:rhES抑瘤效果显著,抑瘤率为45.86%,实验组与对照组在肿瘤体积、重量、裸鼠除瘤净重、瘤重/鼠净重4个指标上均有显著性差异(P<0.01)。光镜见实验组微血管密度降低,坏死面积增加,电镜见实验组凋亡细胞增加。[结论]本实验所优化的生产工艺路线可获得可溶性的、高纯度、高活性的rhES蛋白,解决了rhES在大肠杆菌中提取时的蛋白变性问题。

To explore the best technology of production in recombinant human endostatin(rhES),at the same time to slove the problem of degeneration of rhES expression and extraction in colibacillus.(1)The expression of rhES,extraction,wash,degeneration and refolding of inclusion bodies were carried out by the optimized route.(2)Activities examination:The influence of rhES on growth and pathology in tumor subcuta-neously implanted nude mice was observed.(1)10.55mg soluble rhES protein was obtained from cul-ture medium per litre.The expression of rhES protein is over60%of all the colibacillus thalli protein.The pu-rity of the protein after purification was over95%.(2)Nude mice experiment:in compared with the untreated controls,growth of the implanted tumor was significantly inhibited.Inhibition rate was45.86%.There was re-markable differences between the two groups in the tumor weight,tumor volume,net weight of the nude mice,and the ratio between tumor weight and the net weight(P<0.01).The density of microvessels decreased and the necrosis area increased with light microscopy and apoptosis of tumor cells increased with electron microscopy in the group of rhES treated.[Conclusion]Soluble,high purity and activity rhES protein could be obtained by the optimized technique route,meanwhile,the problem of protein degeneration of rhES extraction in colibacillus could be solved.