按痘苗病毒优势密码子改造HIV-1 gag基因提高表达水平的研究

Optimizing the Codon Usage of HIV-1 gag Gene According to the Codon Bias of Vaccinia Virus Improves the gag Gene Expression

为确定痘苗病毒密码子偏向性与基因表达的关系及其在痘苗病毒与宿主细胞相互作用过程中的作用,按痘苗病毒的优势密码子对HIV-1gag基因进行改造,并对合成基因与野生型HIV-1gag基因在痘苗病毒载体系统的表达水平进行了研究。结果显示:①各目的基因分别正向插入了痘苗病毒TK区7.5k启动子下游;②免疫荧光检测显示,改造前后的gag基因均能够很好地在痘苗病毒中表达;③Westernblot检测显示,在相同感染量时,改造后的gag基因具有更高的表达水平;④流式细胞术检测显示,密码子改造后的gag基因较野生型gag基因表达水平提高约17%。上述结果表明:按照痘苗病毒优势密码子进行外源基因改造,可作为提高外源基因在痘苗病毒中表达的策略,同时提示,密码子偏向性是痘苗病毒与宿主细胞相互作用的重要调控因素。

To determine the relationship between codon bias of vaccinia virus (VV) and gene expression, and the role of codon bias in interaction between VV and host cells, we designed and synthesized a HIV-1 gag gene according to the codon bias of VV. The expression of wild-type and synthetic HIV-1 gag genes were analyzed in VV expression system. Data showed that: Each target gene was exactly inserted into the vaccinia virus TK fragment and controlled by 7.5k promoter; Immunofluorescence indicated well expression of wild-type and synthetic HIV-1 gag genes recombined in VV in HeLa cells;Western blot showed higher expression of the synthetic HIV-1 gag gene at the comparable infection level;Flow cytometry showed that the expression level of synthetic HIV-1 gag gene was 17% higher than that of wild-type HIV-1 gag gene in vaccinia virus expression system. The results above suggest that optimizing the codon usage of target gene according to the codon bias of VV might be a strategy to improve the expression of target gene in VV expression system. In addition, the codon bias might play an important role in the interaction between VV and host cells.