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

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.