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目的探讨重组质粒pET-28a(+)/CTX-M-3分别在大肠埃希菌ER2566和BL21(DE3)中进行原核表达的最佳条件。方法分别在不同的宿主菌、诱导温度、诱导时间和诱导剂浓度中诱导pET-28a(+)/CTX-M-3融合表达载体,目的产物经SDS-PAGE和BandScan凝胶分析软件分析,以获得最佳表达条件。结果表达载体的最佳诱导条件是重组质粒在BL21(DE3)中18℃诱导24h,IPTG终浓度为0.8mmol/L;表达载体在最佳条件下表达时,目的蛋白的最高表达量占菌体总蛋白的33%。结论获得pET-28a(+)/CTX-M-3在大肠埃希菌中表达CTX-M-3型超广谱β-内酰胺酶(extended-spectrum β-lactamase,ESBLs)蛋白的最佳条件,为此酶的大量纯化奠定了基础。
Objective To investigate the optimal conditions for the prokaryotic expression of recombinant plasmid pET-28a (+) / CTX-M-3 in Escherichia coli ER2566 and BL21 (DE3). Methods The fusion expression vector pET-28a (+) / CTX-M-3 was induced in different host bacteria, induction temperature, induction time and inducer concentration, respectively. The target product was analyzed by SDS-PAGE and BandScan gel analysis software. Get the best expression condition. Results The optimal conditions for the expression of the recombinant plasmid were as follows: the recombinant plasmid was induced in BL21 (DE3) at 18 ℃ for 24 h and the final concentration of IPTG was 0.8 mmol / L. The highest expression of the target protein 33% of total protein. Conclusion The optimal conditions for the expression of CTX-M-3 extended-spectrum β-lactamase (ESBLs) protein in Escherichia coli pET-28a (+) / CTX- , Which laid the foundation for the massive purification of this enzyme.