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1.0mmol/L,最佳诱导时机和诱导时间分别为0.58h、7h。
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 trans- formed into E. coli Rosetta and was induced to express under different temperatures, dura- tions, 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.
出处
《渔业科学进展》
CSCD
北大核心
2011年第5期44-50,共7页
Progress in Fishery Sciences
基金
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)
科技部农业科技成果转化资金项目(2010GB23260589)共同资助
关键词
脊尾白虾
热休克蛋白90
原核表达
Exopalaemon carinicaucla Heat shock protein 90 （HSP90） Prokaryotic expression
