【摘要】 为了鉴定重组UK114基因工程菌是否稳定表达,并进行初步的高细胞密度发酵试验。将重组基因工程菌BL21(DE3)/p GEX-4T-3-UK114进行菌种的传代培养并酶切检测;在保持菌种不变情况下,在5 L高密度发酵罐中,采用补料分批培养方法进行高密度发酵。结果表明,重组基因工程菌BL21(DE3)/p GEX-4T-3-UK114传代20代后质粒稳定存在,高密度发酵结束菌体浓度OD600大于50,融合蛋白量占菌体总蛋白量的31%,其含量达到2.1 g/L,工程菌获高效表达。更多还原

【Abstract】 To construct the engineering bacterial strain of the recombinant UK114 for the high effective expression, the recombinant vector p GEX-4T-3-UK114 was constructed,and transformed into E. coli BL21(DE3). The expression of fusion protein GST-UK114 was induced with IPTG. The recombinant prokaryotic expression vector was successfully constructed by the restriction enzymes digestion and gene sequencing. Fed-batch culture was used to obtain high density culture cell and produce GST-UK114 fusion protein by 5 L fermentor. Fusion protein GST-UK114 was expressed in solubility in E. coli BL21(DE3)in SDS-PAGE gel. The final density of grown bacterial was 51(OD600), the amount of fusion protein accounted for 31% of the total protein; the products of GST-UK114 was 2.1 g/L.The engineering bacterial strain of the recombinant UK114(BL21(DE3)/ p GEX-4T-3-UK114)was successfully constructed and expressed in prokaryotes by high cell density culture with high efficiency. The results provide a foundation for studing the biological activity of UK114 protein.更多还原