人热休克蛋白70基因的原核表达

目的 表达人热休克蛋白70（HSP70）并进行鉴定。方法 用PCR方法扩增人HSP70基因片段，经T-A克隆法克隆到载体pUCm-T中，并进行DNA测序，将人HSP70基因片段从载体pUCm-T中酶切后，构建重组表达载体pGEX-4T-1-HSP70，并转化大肠杆菌JM109，用IPTG诱导，收集细菌，菌体裂解后进行SDS-PAGE及Western blot检测。结果 人HSP70基因的PCR产物约为1.9kb。序列测定结果证实，所获目的序列与文献3]报道的相一致，经EcoRⅠ和XhoⅠ酶切鉴定证实。人HSP70基因已成功地克隆到表达载体pGEX-4T-1中，构建的表达载体pGEX-4T-1-HSP70，能很好地在大肠杆菌中表达相对分子质量（Mr)为96000并具有抗原特性的融合蛋白，结论 成功地克隆并表达了HSP70基因，为研究HSP70的结构。功能与临床应用提供了必要条件。

Procaryotic,expression of human heat shock protein 70 gene

Aim To express human heat shock protein 70(HSP70) in E.coli . Methods The cDNA fragment encoding hHSP70 was amplified by PCR. The PCR product was cloned into vector pUCm T and was sequenced. The hHSP70 gene was subcloned into expression vector pGEX 4T 1. The recombinant vector pGEX 4T 1 HSP70 was transformed into E. coli JM109, then hHSP70 was expressed via the induction of IPTG. The expressed product was analyzed by SDS PAGE and Western blot. Results The size of PCR product of hHSP70 gene was about 1.9 kb . Sequencing result revealed the sequence of amplified hHSP70 gene was identical with that in GenBank. Restrictive enzyme ( Eco R I and Xho I) digestion analysis showed that hHSP70 gene recombinant expression vector pGEX 4T 1 hHSP70 was constructed successfully. Expressed product was a fusion protein with Mr 96 000, and it could be recognized by anti HSP70 antibody. Conclusion The hHSP70 gene has been cloned and expressed. The study provides a necessary condition for studying structure, function and clinical application of hHSP70.