Cecropin-X发酵过程中工程菌质粒稳定性的研究

Study on Plasmid Stability of Recombinant Escherichia coli Producing Cecropin-X During Fermentation

通过连续斜面转接实验 (5 0次 )检测重组Cecropin X工程菌质粒的结构稳定性 ,采用 30L自动控制发酵罐观察不同培养基 ,有无选择压力和溶氧高低等培养条件对质粒分裂稳定性的影响。结果表明 ,该工程菌质粒具有结构稳定性和分裂不稳定性。当外源蛋白表达时 ,便会出现分裂不稳定性 ,表达量越高 ,质粒丢失情况越严重。经1 2h的培养 ,当采用TB和 2×LB作为发酵培养基时 ,带质粒的菌为 6 8 5 %和 98 0 % ,包涵体得率有很大差异 ,干重分别为 1 2 4和 0 4 0g L。发酵培养基中 (TB)氨苄青霉素浓度为 0和 1 0 0 μg mL时 ,带质粒的菌为 6 8 5 %和 92 0 % ,包涵体得率基本一致 ,干重分别为 1 2 4和 1 2 0g L。通过改变搅拌速度来调节溶氧量 ,当转速为 1 5 0和 2 5 0r min时 ,带质粒的菌为 6 8 5 %和 1 0 0 % ,包涵体得率有较大差异 ,干重分别为 1 2 4和 0 71g L。

In the view of engineering application, the plasmid stability of pET11d-TNFb-Cecropin-X, harbored in BL21(DE3), was investigated. The results showed that the plasmid had structural stability and segregational instability. Plasmid segregational instability was aggravated by high-level expression. Segregational stability of the plasmid was investigated in a 30L-fermentor. In 2×LB or in a condition of high-level dissolved O 2, the rate of plasmid harboring cell was almost maintain 100% with a low yield of inclusion body, 0 40 and 0 71g/L respectively. In TB while the ampicillin was absence or not, the rate of plasmid harboring cell were 68 5% and 92 0% respectively, and the yield of inclusion body were 1 24 and 1 20g/L respectively.