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| 大肠杆菌表达重组人生长激素的纯化 |
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刘晓宏,粟永萍,王蒙,李淑蓉,艾国平,王军平,李广阔,程天民
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第三军医大学全军复合伤研究所,重庆,400038
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| 摘要: |
| 目的:获得具有生物学活性的重组人生长激素(rhGH).方法:PBV-GH/DH5α菌体经超声破菌、反复洗涤后获得包涵体.将包涵体变性、复性,用硫酸铵盐析,离子交换层析和凝胶层析进行纯化.产物经SDS-PAGE、HPLC、N末端15个氨基酸序列检测验证.结果:终产物rhGH纯度达98.2%,比活性大于3.0 IU/mg.分子量为22kDa,N末端氨基酸序列与DNA序列推导的氨基酸序列完全一致.结论:从自构建的PBV-GH/DH5α工程菌中获得高纯度、高活性重组人生长激素.其纯化工艺为中试生产提供可靠依据. |
| 关键词: 重组人生长激素 复性 纯化 |
| DOI： |
| 分类号:R378.21 |
| 基金项目: |
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| Purification of Recombinant Human Growth Hormone in E.coli |
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| Abstract: |
| Recombinant human growth hormone (rhGH) engineering bacterial strain PBV-rhGH/DH5α was disrupted and washed to achieve expressed inclusion bodies. Then expressed inclusion bodies were denatured and renatured. rhGH was purified by salting out, ion exchange and sephacryl S-100 gel filtration chromatography. The purified protein was identified by SDS-PAGE, HPLC and sequencing analysis of 15 amino acids in N-terminal. The purity and specific activity of the purified rhGH reached 98% and 3.0 IU/mg respectively. The molecular mass was 22KD. N-terminal amino acid analysis was consistent with amino acid sequence based on DNA sequence. This research has laid solid foundation for pilot-scale production of rhGH. |
| Key words: recombinant human growth hormone(rhGH) renaturation purification |
