节点文献
重组人酸性成纤维细胞生长因子改构体在大肠杆菌中的表达
Research on the Expression of Recombinant Human aFGF Mutant in Escherichia coli
【摘要】 目的:提高重组人成纤维细胞生长因子改构体在大肠杆菌工程菌表达体系E.coliBL21(DE3)/pET3c-haFGF中的表达水平。方法:从菌种和培养基筛选做起,选择了全合成的YH培养基做大肠杆菌表达体系E.coliBL21(DE3)/pET3c-haFGF的发酵培养基,并从多角度研究了其质粒的稳定性,设计了生长期不添加抗生素、诱导前离心去除β-内酰胺酶同时添加氨苄青霉素的方法,通过密码子偏爱性和定点PCR突变技术改变β-内酰胺酶的表达强度。结果:经50代传代试验,质粒分配稳定性在90%以上,PstI酶切图谱显示结构稳定性达100%,表达水平维持在25%~28%。LBAmp平板揭示了诱导过程中工程菌丧失表达能力的部分原因,诱导前离心的实验使表达水平提高13.68%,在IPTG浓度0.3mmol/L、氨苄青霉素添加量50μg/ml时,表达效率提高了25%。结论:本研究建立的工艺操作较为简便易行,易于放大,具有较大的经济意义。
【Abstract】 Objective: To raise the level of the expression of the recombinant human acidic fibroblast growth factor in Escherichia coli BL21(DE3) rhaFGF mutant gene bearing system. Methods: The synthetic YH medium was selected as the fermentation medium through the screen of strain and medium; the plasmid structural stability and so on all measured, the rhaFGF then was produced by the addition of Amp upon induction before the centrifugation of the broth; at last the expression strength ofβ-lactamases was changed according to the code-bias and site-directed PCR technology. Results: The synthetic medium YH was selected as the fermentation broth for the E.coli expression system; it was found that the plasmid segrative stability was above 90% after 50 generation with no selective pressure, the expression level kept within 25%~28%, and the 100% stability of PstI digestion graph showed some extent of structural stability of the system. LB agar plates experiment revealed part of the reason why system lost expression ability during induction; the expression level was raised 13.68% through the centrifugation upon induction, and improved 25% under the condition of IPTG 0.3 mmol/L and Ampicilin 50 ug/ml. Conclusion: The process established in this paper is simple and easy to sacle up,and shows a distinct economics benefits.
【Key words】 recombinant human aFGF mutant; plasmid stability; Escherichia coli; induction, restriction digestion graph;
- 【文献出处】 温州医学院学报 ,Journal of Wenzhou Medical College , 编辑部邮箱 ,2007年04期
- 【分类号】Q78
- 【被引频次】1
- 【下载频次】229