斑节对虾cyclinH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

cyclin H是细胞分裂周期调控中的重要因子,研究斑节对虾cyclin H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cyclin H的重组表达质粒p ET21a/cyclin H,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cyclin H重组蛋白获得了表达,纯化的蛋白经鉴定为cyclin H重组蛋白;在22℃和37℃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℃条件下,部分融合蛋白为可溶性表达;在37℃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。

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 Ni-chelating 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℃, 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.