【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。更多还原

【Abstract】 To construct a recombinant plasmid p Cold II-HSP70 of chicken,expression,purify and detection of the quality of HSP70 protein was conducted. HSP70 gene which was amplified by PCR was inserted into p Cold II expression vector to generate p Cold II-HSP70. The p Cold II-HSP70 was then transformed into BL21( DE3) and Rosetta( DE3) p Lys S expressed on induction of IPTG at the different temperature and concentration. After purification,purity was detected by SEC-FPLC and HPLC,endotoxin level was detected by Limulus amebocyte lysate. The vector was successfully constructed as confirmed by enzyme digestion and DNA sequencing. The optimum condition was BL21( DE3) at 37 ℃ after induction of IPTG. The relative molecular weight of HSP70 protein was 71 988. 04,the purity was more than 90% and the endotoxin level less than 500 Eu / mg. Our results suggested that the purified HSP70 obtained with good quality providing a theoretical basis for researches on HSP70 at heat stress.更多还原