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目的对人 IFN- α2 b基因进行突变和修饰 ,从翻译水平上调控 IFN- α2 b基因在大肠杆菌中的表达。方法采用PCR技术 ,人工合成寡核苷酸引物 ,对人 IFN- α2 b基因进行了改造 :在不改变氨基酸的前提下 ,将 5’端编码区的四个 G、C位点突变为 A、T;去除 3’端非编码区。将突变和修饰后的基因片段分别克隆入原核表达载体 p BV32 1,在大肠杆菌中进行表达。对表达产物进行活性效价测定。结果 3’端删除非编码区 ,表达水平比删除前提高 4倍 ;5’端四个位点突变 ,表达水平比突变前降低 2倍。结论 IFN-α2 b基因的 3’端非编码区抑制其在原核系统的表达 ,删除该区可提高表达水平
Objective To mutate and modify the human IFN-α2b gene and to regulate the expression of IFN-α2b gene in Escherichia coli at the translational level. Methods The human IFN-α2b gene was modified by PCR and synthetic oligonucleotide primers. The four G and C sites of the 5 ’end coding region were mutated to A without changing the amino acids. T; remove 3 ’non-coding region. The mutated and modified gene fragments were respectively cloned into the prokaryotic expression vector p BV32 1 and expressed in E. coli. The expressed product was assayed for activity titer. Results The non-coding region was deleted at the 3 ’end, and the expression level was increased by 4 times compared with that before deletion. The four positions at the 5’ end were mutated and the expression level was reduced by 2 times than that before the mutation. Conclusion The 3 ’untranslated region of IFN-α2b gene inhibits the expression of IFN-α2b gene in prokaryotic system. Deletion of this region can increase the expression level