| 摘要: |
| 将脊尾白虾热休克蛋白90基因克隆到原核表达载体pET-30a中，经酶切验证和DNA测序鉴定后，将重组质粒转化表达宿主大肠杆菌Rosetta，优化温度、时间、IPTG和OD600表达条件进行诱导表达，收集菌液，进行SDS-PAGE和质谱检测，并用Quantity one软件分析蛋白表达水平。结果表明，成功构建了含脊尾白虾HSP90基因的重组表达载体pET-30a-HSP90，表达目的蛋白相对分子量为82.7kD，为HSP90蛋白。通过条件优化认为重组菌株Rosetta/pET-30a-HSP90的最佳诱导温度为37 ℃，最佳IPTG浓度为1.0 mmol/L，最佳诱导时机和诱导时间分别为0.58 h、7 h。 |
| 关键词: 脊尾白虾 热休克蛋白90 原核表达 |
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| 基金项目:公益性行业（农业）科研专项（nyhyzx07-042，200803012）、国家虾产业技术体系（CARS-47）和科技部农业科技成果转化资金项目（2010GB23260589）共同资助 |
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| High level prokaryotic expression and identification of heat shock protein 90 in Exopalaemon carinicauda |
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| Abstract: |
| The aim of this study was to set up a high level prokaryotic expression of heat shock protein 90(HSP90) of Exopalaemon carinicauda in E. coli Rosetta. The HSP90 gene of E. carinicauda was cloned into prokaryotic expression vector pET-30a, which was confirmed by double-endonuclease digestion and DNA sequencing. The recombinant vector was transformed into E. coli Rosetta and was induced to express under different temperatures, durations, IPTG concentrations and OD600. The expressed product was identified by SDS-PAGE and an 82.7 kD protein (determined by mass spectrometry) was found. The expression level varied under different conditions. The optimal expression was achieved under induction conditions of 1 mmol/L IPTG,37℃, OD600=0.58, and 7 h. |
| Key words: Exopalaemon carinicauda Heat shock protein 90 (HSP90) Prokaryotic expression |
