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目的获取大肠杆菌甲硫氨酸氨基肽酶(re-MAP)基因,构建重组大肠杆菌甲硫氨酸氨基肽酶的原核高表达载体pACYCDuet-1-MAP,利用大肠杆菌BL21作为宿主菌进行原核表达,并利用表达的reMAP切除重组人干扰素-α2b(rhIFN-α2b)N末端的初始甲硫氨酸。方法提取大肠杆菌BL21基因组作为模板,用PCR方法扩增得到大肠杆菌MAP基因,并克隆到载体质粒pGEM-7zf(-)中,将重组质粒pGEM-7zf(-)-MAP转化感受态大肠杆菌DH5α,蓝-白斑筛选及测序验证正确后,将reMAP基因克隆至表达载体质粒pACYCDuet-1,Nco I/BamH I双酶切及PCR验证正确后,将重组表达质粒pACYC-Duet-1-MAP转化至感受态大肠杆菌BL21中,筛选得到阳性克隆通过IPTG诱导进行可溶性表达,经SDS-PAGE和Western blot分析及活性检测后,通过两种方法作用rhIFN-α2b:①将纯化后reMAP和rhIFN-α2b在体外适当的缓冲液中作用;②构建re-MAP和rhIFN-α2b的共表达体系,实现在细胞内对rhIFN-α2b进行作用,并对两种作用方式的结果进行比较。结果 Nco I/BamH I双酶切、PCR验证及测序结果表明重组表达质粒pACYCDuet-1-MAP构建成功;SDS-PAGE和Western blot分析结果表明re-MAP和rhIFN-α2b均得到正确表达;酶活性检测结果表明reMAP具有水解N端甲硫氨酸的活性,切除MET的活性>30 pmol/(μg.min);两种方法作用后的rhIFN-α2b经氨基酸序列测定后显示N端起始甲硫氨酸均被切除,其中体内作用切除率>94%、体外作用切除率>96%。结论 reMAP可以用于rhIFN-α2b N末端的初始甲硫氨酸的切除。
OBJECTIVE: To obtain the recombinant plasmid pACYCDuet-1-MAP of Escherichia coli methionine aminopeptidase (re-MAP) and construct the prokaryotic expression vector pACYCDuet-1-MAP of Escherichia coli methionine aminopeptidase using E. coli BL21 as host strain for prokaryotic expression , And the initial methionine at the N-terminus of recombinant human interferon-α2b (rhIFN-α2b) was excised using the expressed reMAP. Methods The Escherichia coli BL21 genome was extracted and used as a template. The MAP gene of Escherichia coli was amplified by PCR and cloned into vector pGEM-7zf (-). The recombinant plasmid pGEM-7zf (-) - MAP was transformed into competent E. coli DH5α After blue-white blotting and sequencing validation, the recombinant plasmid pACYC-Duet-1-MAP was transformed into pACYCDuet-1 and Nco I / BamH I double digestion. Competent E. coli BL21, the positive clones were screened by IPTG induced soluble expression, by SDS-PAGE and Western blot analysis and activity detection, by two ways the role of rhIFN-α2b: ① the purified reMAP and rhIFN-α2b in In vitro appropriate buffer role; ② build re-MAP and rhIFN-α2b co-expression system to achieve intracellular rhIFN-α2b role, and the results of the two modes of action were compared. Results The Nco I / BamH I double digestion, PCR validation and sequencing showed that the recombinant plasmid pACYCDuet-1-MAP was constructed successfully. The results of SDS-PAGE and Western blot showed that both re-MAP and rhIFN-α2b were correctly expressed. The results showed that reMAP had the activity of hydrolyzing N-terminal methionine and the activity of excising MET was> 30 pmol / (μg.min). The amino acid sequences of rhIFN-α2b showed that N-terminal methionine Adenine were excised, in which the role of excision rate of> 94%, in vitro excision> 96%. Conclusion reMAP can be used for the excision of the initial methionine at the N terminus of rhIFN-α2b.