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旨在构建鸡热休克蛋白70(heat shock protein 70,HSP70)表达质粒p ColdⅡ-HSP70,诱导表达、纯化后检测HSP70蛋白的质量。将目的基因扩增后克隆至表达载体p Cold II,构建重组载体p ColdⅡ-HSP70,在2种大肠杆菌菌株(BL21(DE3)和Rosetta(DE3)p Lys S中经IPTG在不同温度与不同浓度条件下诱导表达并纯化。经SEC-FPLC和HPLC进行纯度鉴定,采用鲎试剂检测蛋白内毒素含量(将HSP70蛋白按1 200、600、120倍稀释);Western blot及质谱方法检测蛋白分子量。结果显示:酶切和测序结果均证实HSP70表达载体构建正确,可诱导表达。经IPTG诱导表达后确定BL21(DE3)在37℃条件下表达蛋白效果最佳,经检测HSP70蛋白的相对分子质量为71 988.04,蛋白纯度大于90%,内毒素小于500 Eu/mg。结果表明:成功构建了大骨鸡HSP70基因的表达载体,获得了纯化的HSP70蛋白,为进一步研究HSP70在热应激中的作用机制提供了基础。
The aim of this study was to construct the expression plasmid HSP70 of cold shock protein 70 (HSP70), and induce the expression of HSP70 protein. The target gene was amplified and cloned into the expression vector pColdII to construct the recombinant vector pColdII-HSP70. The two kinds of E. coli strains (BL21 (DE3) and Rosetta (DE3) pLysS) were treated with IPTG at different temperatures and concentrations The expression of protein was detected by SEC-FPLC and HPLC, and the protein endotoxin content was detected by limulus reagent (HSP70 protein was diluted by 1 200, 600 and 120 times), and the protein molecular weight was detected by Western blot and mass spectrometry. The result of enzyme digestion and sequencing confirmed that HSP70 expression vector was constructed correctly and induced to express.The result of IPTG induced expression of BL21 (DE3) was the best at 37 ℃, and the relative molecular mass of HSP70 protein was 71 988.04, the purity of the protein was over 90% and the endotoxin was less than 500 Eu / mg.The results showed that HSP70 gene was successfully constructed and the purified HSP70 protein was obtained.To further investigate the mechanism of HSP70 in heat stress Provide the foundation.