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目的 :利用大肠杆菌系统表达小鼠重组PeriostinC端肽。方法 :将编码小鼠PeriostinC端肽的基因片段克隆到大肠杆菌表达载体pRSET -B上 ,使其受控于T7启动子 ,构建成表达质粒pRS -B -Peri,以大肠杆菌E .coliBL2 1(DE3)为宿主菌。对工程菌用异丙基硫代 - β -D -半乳糖苷 (isopropylthio - β -D -galacto side ,IPTG)诱导表达 ,表达产物纯化后进行复性处理 ,再通过观察成骨细胞的伸展性检测其生物活性。结果 :经IPTG诱导 5h ,工程菌在SDS -PAGE上出现一条新蛋白带 ,相对分子质量 (Mr)与预期的一致 ,约为 36 0 0 0 ,主要以包涵体形式存在。所得包涵体经纯化和复性处理 ,得到纯度高于 95 %的有良好活性的小鼠重组PeriostinC端肽。结论 :小鼠重组PeriostinC端肽在大肠杆菌中得到表达并具有良好的生物学活性。
OBJECTIVE: To express mouse recombinant Periostin C telopeptide using E. coli system. Methods: The gene fragment encoding mouse periostin C telopeptide was cloned into E.coli expression vector pRSET-B, which was controlled by T7 promoter and constructed into expression plasmid pRS-B-Peri. The E.coli strain E. coli BL21 DE3) as host bacteria. The engineered bacteria were induced with isopropylthio - β - D - galactoide (IPTG), and the expressed product was purified and then renaturated. Then the osteoblasts were observed for their extensibility Test its biological activity. Results: After induced by IPTG for 5h, the engineered bacteria appeared a new protein band on SDS-PAGE. The relative molecular mass (Mr) was in agreement with the expectation, which was about 36,000, which was mainly in the form of inclusion body. The resulting inclusion bodies were purified and refolded to give recombinant mouse Periostin C telopeptide with good activity higher than 95%. Conclusion: The mouse recombinant Periostin C telopeptide is expressed in E. coli and has good biological activity.