人干扰素ε的基因合成、原核表达及质谱表征

Gene synthesis,prokaryotic expression of human interferon ε and its characterization by mass spectrometry

首先人工合成了人源干扰素ε的基因,经密码子优化后分别构建表达载体Ifn-ε/pET-21a(+)和Ifn-ε/pET-22b(+)。将表达载体转化入大肠埃希菌中表达,优化表达条件筛选出两株优势表达菌Ifn-ε/pET-21a/BL21和Ifn-ε/pET-22b/BL21。通过发酵、提取、纯化,成功地从Ifn-ε/pET-22b/BL21表达菌中得到纯度较高的IFN-ε,通过质谱对其结构进行确证。研究建立了人干扰素ε高效表达体系,为获取大量蛋白进行结构和作用机制研究奠定基础。此外通过基因合成建立的表达体系为后期以表达的方式对蛋白的氨基酸序列进行突变进而研究构效关系奠定基础。

First of all,the gene of human Ifn-εwas synthesized,the expression vectors of Ifn-ε/pET-21a(+)and Ifn-ε/pET-22b(+)were constructed respectively after codon optimization.The expression vector was transformed into Escherichia coli and the expression conditions were further optimized.Two dominant strains Ifn-ε/pET-21a/BL21A and Ifn-ε/pET-22b/BL21A were obtained.At last,IFN-εwith high purity was isolated from Ifn-ε/pET-22b/BL21A throgh fermentation,extraction,purification,and its structure was confirmed by mass spectrometry.An effective expression system of human IFN-εhas been established to provide large quantity of protein for the study of its structure and mechanism.Moreover,the establishment of expression system through gene synthesis laid the foundation for the study of structure-activity relationship by amino acid mutation.