Lee N, Zhang S Q, Cozzitorto J, Yang J S, Testa D
Interferon Sciences, Inc., New Brunswick, NJ 08901.
Gene. 1987;58(1):77-86. doi: 10.1016/0378-1119(87)90031-x.
A plasmid (pNL015) was constructed to contain a human interferon alpha 1 (IFN-alpha 1) gene under the transcriptional control of the Escherichia coli lipoprotein promoter. The E. coli cells harboring this plasmid produce 2.8 x 10(4) units/ml of IFN. Secondary structure analysis of the transcripts produced by pNL015 showed that the coding region could base pair with the Shine-Dalgarno (SD) region with a delta G = -3.9kcal/mol. A new plasmid pNL008 was constructed by modifying pNL015 with an 11-bp deletion and a 2-bp insertion in the coding region, so that the SD region is not involved in the secondary structure. E. coli cells harboring pNL008 produce ten times more IFN activity than cells harboring pNL015. A series of experiments were carried out to show that the specific activities of IFN, differential rates of IFN transcription, protein degradation or mRNA degradation could not account for the difference observed in expression. A rigorous test on this model of translational inhibition was conducted by the construction of pNL017 with a single bp substitution which did not change the amino acid sequence of the IFN (synonymous codon substitution) but which increased the calculated energy of interaction with the SD sequence to delta G = -10.8 kcal/mol. The E. coli cells harboring pNL017 produced no detectable IFN activity.
构建了一种质粒（pNL015），使其在大肠杆菌脂蛋白启动子的转录控制下包含人干扰素α1（IFN-α1）基因。携带该质粒的大肠杆菌细胞可产生2.8×10⁴单位/毫升的干扰素。对pNL015产生的转录本进行二级结构分析表明，编码区可与Shine-Dalgarno（SD）区形成碱基对，ΔG = -3.9千卡/摩尔。通过在编码区进行11个碱基的缺失和2个碱基的插入对pNL015进行修饰，构建了一种新的质粒pNL008，从而使SD区不参与二级结构。携带pNL008的大肠杆菌细胞产生的干扰素活性比携带pNL015的细胞高10倍。进行了一系列实验以表明，干扰素的比活性、干扰素转录的差异速率、蛋白质降解或mRNA降解均不能解释所观察到的表达差异。通过构建pNL017进行了对这种翻译抑制模型的严格测试，pNL017有一个单碱基替换，该替换不改变干扰素的氨基酸序列（同义密码子替换），但增加了与SD序列相互作用的计算能量至ΔG = -10.8千卡/摩尔。携带pNL017的大肠杆菌细胞未产生可检测到的干扰素活性。
