突变人干扰素-α2b基因5′端提高其在大肠杆菌中的表达  

High-level expression of recombinant human interferon-α2b in Escherichia coli by mutating its 5′ end

作　　者：王参军[1] 邹文艺[2] 张玲[3] 范清林[2] 宋礼华[1,2] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,合肥230032 [2]安徽安科生物工程(集团)股份有限公司,合肥230088 [3]安徽大学生命科学学院,合肥230039

出　　处：《安徽医科大学学报》2010年第2期149-153,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家"重大新药创制"科技重大专项(编号:2008ZX09203-003)

摘　　要：目的对人干扰素-α2b(hIFN-α2b)基因的5′端进行突变,构建重组人干扰素-α2b(rhIFN-α2b)原核高表达工程菌,以提高rhIFN-α2b的表达水平。方法依据大肠杆菌密码子偏好性,在不改变hIFN-α2b的氨基酸序列的情况下,定点突变hIFN-α2b基因5′端序列并PCR扩增出rhIFN-α2b′,克隆至温控型表达载体pJW2,转化大肠杆菌DH5α,筛选阳性重组子,温度诱导表达,表达产物进行SDS-PAGE、Western blot鉴定分析并以WISH-VSV系统检测其抗病毒活性。结果SDS-PAGE分析表明rhIFN-α2b表达量约占菌体总蛋白20.6%,Western blot表明rhIFN-α2b具有与hIFN-α2b相同的免疫原性,WISH细胞-VSV病毒系统证实其比活性达1.2×108IU/mg,与未突变工程菌一致。结论突变hIFN-α2b基因5′端可以提高其表达水平,并成功构建出rhIFN-α2b原核高表达工程菌。Objective To construct the prokaryotic expression system of recombinant human interferon-α2b （rhIFN-α2b） and to improve the expression level of rhIFN-α2b in Escherichia coli （E.coli） by mutating its 5′ end.Methods The 5′ end sequences of hIFN-α2b genes were mutated by site-directed mutagenesis and the sequences of hIFN-α2b genes were amplified with PCR according to the frequency table of gene codon in E.coli without changing their amion acid sequences.Then cloned into the temperature-controlled expression vector pJW2,transformed into competent cell of E.coli DH5α.The positive recombinant pJW2/rhIFN-α2b clones were screened and expressed under heat induction,the expression productions were investigated by SDS-PAGE,Western blot analysis and WISH-VSV system indentified anti-virus vitalism.Results The analysis of SDS-PAGE indicated that the rhIFN-α2b was expressed in inclusion bodies in E.coli with the yield accounting for 20.6% of total bacteria proteins.Western blot analyses showed that the expression productions had the immunogenicity of hIFN-α2b and WISH-VSV verified its specific activitity was able to obtain 1.2×10^8 IU/mg similar to that of no-mutated engineered strain.Conclusion The expression level of rhIFN-α2b may be increased by mutating its 5′ end,and the efficient expression system of rhIFN-α2b in E.coli construct successfully.

关 键 词：突变 干扰素-Α2B 基因表达 大肠杆菌 

分 类 号：R392.2[医药卫生—免疫学] R343.9[医药卫生—基础医学]