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目的:研究表达重组变形链球菌表面蛋白(rPAc)的工程菌pET20b(+)-AP/BL21(DE3)plysS的发酵条件。方法:通过摇瓶培养,进行发酵条件优化,在5L发酵罐中发酵pET20b(+)-AP/BL21(DE3)plysS工程菌,并对发酵产物进行SDS-PAGE、Western印迹分析鉴定。结果:确立了pET20b(+)-AP/BL21(DE3)plysS工程菌的高密度发酵工艺,即LB-2培养基,初始pH值为7.2,溶氧控制为30%以上,补料为10%甘油、5%酵母粉和5%蛋白胨。目的蛋白呈可溶性表达,表达量占菌体总蛋白的45%以上,菌体密度达到44。Western印迹分析显示,rPAc蛋白和抗PAc抗体有良好的结合活性。结论:通过发酵工艺优化,提高了rPAc蛋白的可溶性表达量,为进一步规模化研究和生产奠定了基础。
OBJECTIVE: To study the fermentation conditions of engineering strain pET20b (+) - AP / BL21 (DE3) plysS expressing the recombinant Streptococcus mutans surface protein (rPAc). Methods: Fermentation conditions of pET20b (+) - AP / BL21 (DE3) plysS in 5L fermenter were optimized by shake flask culture. The fermentation products were identified by SDS-PAGE and Western blot. Results: The high density fermentation process of pET20b (+) - AP / BL21 (DE3) plysS engineered bacteria was established, that is, LB-2 medium with initial pH of 7.2, dissolved oxygen control of 30% Glycerin, 5% yeast extract and 5% peptone. The target protein was expressed in soluble form, accounting for more than 45% of the total bacterial protein, cell density reached 44. Western blot analysis showed that rPAc protein and anti-PAc antibody had good binding activity. Conclusion: The optimization of fermentation process improves the soluble expression of rPAc protein, which lays the foundation for further scale research and production.