人β神经生长因子基因稀有密码子及mRNA二级结构对其在大肠杆菌中表达的影响  

Changes of Rare Codon and mRNA Secondary Structure Affect Expression of Human β-ngf in Escherichia coli

作　　者：欧晓敏[1] 李少华[1] 刘农乐[1] 孙卫国[2] 赵强[1] 丁红梅[1] 王芳[1] 邵宁生[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]解放军309医院结核病研究室,北京100091

出　　处：《生物技术通讯》2011年第6期789-792,共4页Letters in Biotechnology

摘　　要：目的:研究人β神经生长因子(β-NGF)基因中稀有密码子及其mRNA二级结构对其在大肠杆菌中表达量的影响。方法:根据对人β-ngf中稀有密码子及其mRNA二级结构的研究,同义突变人β-ngf基因,通过PCR得到人β-ngf的5'端同义突变基因rh-β-ngfp32和全同义突变基因rh-β-ngfmu,将这2个序列克隆入载体pET3a中,得到重组质粒pET3a-ngfp32和pET3a-ngfmu,分别转化大肠杆菌BL21(DE3)感受态,IPTG诱导表达,收集菌体,SDS-PAGE检测其表达量的改变。结果:构建的pET3a-ngfp32和pET3a-ngfmu表达载体酶切和测序结果正确,SDS-PAGE结果显示,与在重组菌pET3a-NGF总蛋白中的表达量相比,目的蛋白rh-β-NGF在重组菌pET3a-NGFP32和pET3a-NGFmu中的表达量均明显增高,并且在重组菌pET3a-NGFmu中的表达量高于重组菌pET3a-NGFP32。结论:目的蛋白rh-β-NGF在重组菌pET3a-NGFP32和pET3a-NGFmu中表达量的增高,说明人β-ngf基因中稀有密码子和mRNA的二级结构对其在大肠杆菌中的表达有较为明显的影响,结果为构建rh-β-NGF的大肠杆菌工程菌株奠定了基础。Objective： To construct the recombinant human β-nerve growth factor（β-NGF） prokaryotic expression vector and study the effect of rare codon and the mRNA secondary structure on its expression in Escherichia coli.Methods： Based on the rare codon and the secondary structure of hβ-ngf mRNA,5＇ end of synonymous mutation of hβ-ngf gene,rh-β-ngfp32,and nonsense mutation gene rh-β-ngfmu were amplified by PCR.The two genes were cloned into the prokaryotic expression vector pET3a to construct the recombinant plasmid pET3a-ngfp32 and pET3a-ngfmu,respectively.The two recombinant plasmids were then transformed into E.coli BL21（DE3） and the expression of the genes were induced by IPTG followed by SDS-PAGE detection.Results： The two genes were successfully cloned into pET3a confirmed by restriction endonuleases digestion and sequencing identification.The expression level of the target protein rh-β-NGF were significantly increased both in pET3a-NGFP32 and pET3a-NGFmu transformed cells than the recombinant pET3a-NGF transformed cells.Conclusion： The expression of recombinant hβ-ngf was increased after the gene is designed according to the secondary structure of mRNA and high-frequency codon usage in the host cell.Our work indicated that the codon bias and the mRNA secondary structure have a significant effect on rh-β-NGF expression in E.coli.All the work above will benefit the further research on the construction of rh-β-NGF engineering bacterium.

关 键 词：人β神经生长因子 稀有密码子 MRNA二级结构 大肠杆菌 

分 类 号：Q78[生物学—分子生物学] Q75