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

作　　者：袁辉[1] 刘笑迪[2] 谭毅[1] 林绍强[1] 杨树林[3] 李校[1] 

机构地区：[1]温州医学院生物与天然药物研究院,浙江温州325035 [2]吉林农业大学生物工程技术研究院,吉林长春130118 [3]南京理工大学生物工程研究所,江苏南京210094

出　　处：《温州医学院学报》2007年第4期303-308,共6页Journal of Wenzhou Medical College

基　　金：国家十五重大生物医药专题滚动立项项目"重组人成纤维细胞生长因子改构体新药研究"(2004AA2Z3C60);2005年度温州市科技局"重组生长因子的中试研究"立项资助项目(Y2005B025);温州医学院引进人才启动基金资助项目(2005-170)。

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。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 of β -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.

关 键 词：重组人酸性成纤维细胞生长因子改构体 质粒稳定性 大肠杆菌 诱导 酶切图谱 

分 类 号：Q78[生物学—分子生物学]