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目的 :在大肠杆菌系统中表达有活性的人 β 防御素 3(hBD3)。 方法 :从pGEM T hBD3重组质粒PCR获得hBD3成熟肽编码区基因 ,插入pGEX 4T 1构建pGEX 4T 1 hBD3融合表达载体 ,转化E .coliDH5α宿主菌 ,进行IPTG诱导 ,诱导菌经超声裂解后获得包涵体 ,包涵体经溶解、变性、复性和纯化处理后获得GST hBD3融合蛋白 ,再经凝血酶切割 ,获得纯化的重组hBD3(rhBD3)。对rhBD3采用最小抑菌浓度测定法测定其对金黄色葡萄球菌和大肠杆菌的抗菌活性。结果 :以与GST融合表达的方式 ,运用pGEX 4T 1系统在大肠杆菌中表达了hBD3,融合蛋白以包涵体形式存在。融合蛋白经凝血酶处理使rhBD3释放出来 ,其对金黄色葡萄球菌的MIC为 4 μg/ml;对大肠杆菌的MIC为 8μg/ml。 结论 :采用pGEX 4T 1融合表达系统在大肠杆菌中成功地表达了有活性的rhBD3。
OBJECTIVE: To express human β-defensin 3 (hBD3) in E. coli. Methods: The coding region of mature peptide of hBD3 was obtained by PCR from pGEM T hBD3 recombinant plasmid and inserted into pGEX 4T 1 to construct pGEX 4T 1 hBD3 fusion expression vector. The recombinant plasmid was transformed into host E. coli DH5α and induced by IPTG. Inclusion body The inclusion body was dissolved, denatured, refolded and purified to obtain GST hBD3 fusion protein, which was then cut by thrombin to obtain purified recombinant hBD3 (rhBD3). The antibacterial activity of rhBD3 against Staphylococcus aureus and Escherichia coli was determined by the minimum inhibitory concentration assay. Results: In the way of fusion expression with GST, hGD3 was expressed in E. coli using pGEX 4T 1 system, and the fusion protein existed as inclusion body. The rhBD3 was released by thrombin treatment of the fusion protein with a MIC of 4 μg / ml against Staphylococcus aureus and a MIC of 8 μg / ml against E. coli. Conclusion: pGEX 4T 1 fusion expression system was successfully expressed in E. coli rhBD3.