大骨鸡HSP70蛋白的表达、纯化及质量检测

发布时间：2018-07-16 09:30

【摘要】：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。
[Abstract]:The aim of this study was to construct the expression plasmid P Cold II -HSP70 of the chicken heat shock protein 70 (heat shock protein 70, HSP70) expression plasmid, and to detect the quality of HSP70 protein after purification. The expression was induced and purified under different temperatures and different concentrations. The purity of the protein was identified by SEC-FPLC and HPLC. The content of protein endotoxin was detected by limulus reagent (diluted HSP70 protein at 1200600120 times). The molecular weight of the protein was detected by Western blot and mass spectrometry. The results showed that the results of enzyme digestion and sequencing confirmed that the HSP70 expression vector was constructed positive. The expression of BL21 (DE3) at 37 centigrade was the best, the relative molecular weight of the HSP70 protein was 71988.04, the protein purity was more than 90% and the endotoxin was less than 500 Eu/mg.. The results showed that the expression vector of the HSP70 gene was successfully constructed and the purified HSP70 protein was obtained, and the purified HSP70 protein was obtained. Further studies on the mechanism of HSP70 in heat stress provide the basis.
【作者单位】：锦州医科大学生命科学研究院;南京农业大学动物医学院;锦州医科大学畜牧兽医学院;辽宁省畜产品质量与安全工程重点实验室;
【基金】：辽宁省自然科学基金项目(2015020765) 辽宁省科学技术计划项目(2015211003) 辽宁医学院第二批领军人物团队项目
【分类号】：S831

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