起始密码下游序列与16srRNA3′端序列的匹配性对基因表达效率的影响

目的 :研究起始密码下游序列与 16srRNA 3′端 +146 9——— +1482局部序列间的匹配关系对基因表达效率的影响。方法 :我们一方面对人IL_2、人IL_8、人IL_6 ,人GM_CSF、人G_CSF、人IL_1ra、人IL_9等全长cDNA序列及 5′末端局部序列与 16srRNA 3′端 +146 9——— +1482序列进行匹配性进行分析 ,寻找序列匹配性与表达效率间的关系 ;另一方面 ,对人IL_6、人GM_CSFcDNA 5′端序列进行沉默突变 ,增强其与 16srRNA 3′端 +146 9——— +1482序列间的匹配性 ,并克隆入pKpL4表达型质粒载体 ,通过大肠杆菌进行表达 ,实验验证序列匹配性与基因表达效率的关系。结果 :分析发现起始密码下游局部序列与 16srRNA 3′端 +146 9——— +1482核酸匹配性越好 ,则目的蛋白表达效率越高 ;目的基因编码区内与 16srRNA 3′ +146 9——— +1482间的最大匹配区域越靠近 5′末端 ,目的蛋白表达效率越高。通过对人IL_6、人GM_CSFcDNA 5′末端序列进行沉默突变 ,改善其与 16srRNA 3′端 +146 9——— +1482序列的匹配性后 ,目的蛋白的表达量均呈不同程度地提高。结论 :提高起始密码下游序列与 16srRNA 3′端 +146 9——— +1482局部序列间的匹配性 ,可增强目的基因的表达效率。

Enhanced translation of 16s rRNA pairing region of mRNA

In order to study the correlation between the downstream of initation codon and the efficiency of the initation signals in E.coli.We analysed the complementary between the nucleotides+1469——+1482 of the 16s rRNA to the cDNA sequences of human IL_1ra,IL_2,IL_6,IL_8,IL_9,GMCSF,GCSF,etc that we had been cloned and expressed in E.coli,and found that the nearer the apparent base pairing region to initation codon,the higher the target gene expressed.Subsequently ,the 5′_coding region of the human IL_6 cDNA and human GM_CSF cDNA were silent mutated to rised the base complementary to nucleotides+1469——+1482 of the 16s rRNA and expressed in E.coli,result that the expressing level of human IL_6 and human GM_CSF from 12% rised to 13.8%,and 0 to 3% respectively.Shown that the apparent base pairing region specifies a stimulatory interaction during the translation step.