利用移码突变和终止密码子提高下游目的基因的表达水平

Improving Gene Expression by Introducing Upstream Frameshift and Stop Codon

目的:在基因结构复杂的慢病毒载体插入报告基因并提高目的基因表达量。方法:慢病毒载体上有复杂的基因排列,为了不影响慢病毒载体的活性,必须尽量保留原有的基因,替换不必要的基因。首先将报告基因萤光素酶插入慢病毒载体替换基因Env后,结果检测不到报告基因的表达。为了提高报告基因的表达水平,将报告基因的读码框向后移动一个碱基,同时在其上游增加一个终止密码子,然后检测报告基因的表达水平。结果:通过移动报告基因的读码框同时在上游增加终止密码子,使报告基因的表达水平大大提高。结论:在构建基因表达载体时,通过改变目的基因与上游起始密码子ATG之间的相对位置以及增加终止密码子,可以大幅提高目的基因的表达水平。

Objectives：To improve the expression of gene of interest in a lentiviral vector with complicated gene distribution.Methods：Lentiviruses have complicated gene distributions such as overlapping Env,Rre,Rev and Tat on their genomes.To avoid damaging the reproduction efficiency of these vectors,it is better to retain as much as possible the original gene structure when replacing the unwanted Env gene with a gene of interest.A reporter gene luc was first inserted into the lentiviral vector in place of the Env gene.The resultant plasmid did not express detectable luciferase activity.In order to improve the target gene expression,a frameshift mutation and a stop codon upstream the luc gene were introduced.Results：After the introduction of the upstream frameshift mutation and a stop codon,the luciferase expression was greatly enhanced.Conclusion：By introducing frameshift mutation and stop codon in the upstream reading frame,the expression of the downstream open reading frame could be largely increased.