| 郭 松， 傅明骏， 赵 超，等.斑节对虾cyclin H基因的原核表达和蛋白纯化[J].广东农业科学,2015,42(3):125-130 |
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斑节对虾cyclin H基因的原核表达和蛋白纯化 |
| Prokaryotic expression and protein purification of cyclin H in giant tiger shrimp Penaeus monodon |
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| DOI： |
| 中文关键词: 斑节对虾 细胞周期蛋白 H 原核表达 |
| 英文关键词: Penaeus monodon cyclin H prokaryotic expression |
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| 摘要点击次数: 1465 |
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| 中文摘要: |
| cyclinH 是细胞分裂周期调控中的重要因子，研究斑节对虾 cyclinH 对阐明斑节对虾卵巢发育机
制有重要意义。构建了斑节对虾 cyclinH 的重组表达质粒 pET21a/cyclinH，利用原核表达技术进行诱导表达获
得目的蛋白，并用亲和层析技术进行纯化，用 SDS-PAGE、Western blot 和质谱联用分析诱导和纯化结果。 结果
表明，斑节对虾 cyclinH 重组蛋白获得了表达，纯化的蛋白经鉴定为 cyclinH 重组蛋白；在 22益和 37益培养条
件下， 重组蛋白在 LA 和 YTGA 两种培养基上均能被诱导表达；22益条件下， 部分融合蛋白为可溶性表达；在
37益培养条件下 LA 培养基获得较好的效果，且均以包涵体形式存在。 |
| 英文摘要: |
| The research on cyclin H (Pmcyclin H) is necessary for understanding the mechanisms involving ovarian developmental processes of giant tiger shrimp (Penaeus monodon). A high level prokaryotic expression of Pmcyclin H in E.coli BL21 (DE3) was set up, Pmcyclin H gene was cloned into expression vector pET-21a, which then was determined by double -endonuclease digestion and DNA sequencing. The recombinant vector was transformed into E.coli BL21 (DE3), and was induced to express fusion protein by 0.6 mmoL/L IPTG. The quality of expression product was identified by SDS-PAGE and Western blot; the recombinant protein was purified through Nichelating affinity chromatography, and the purified protein was identified by SDS-PAGE gel scan analysis and mass spectrometry. At 22%, both LA and YTGA medium could be used, Pmcyclin H protein was more abundantly expressed as an insoluble than soluble protein; at 37益, LA medium was better, and all the product was an insoluble protein. This study provides a fundamental condition supporting researches on structure, function and biological activity of cyclin H of P. monodon. |
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