原核融合表达载体的设计、构建及应用

Design,construction,and application of a novel prokaryotic fusion expression vector

目的利用EspA(肠出血性大肠杆菌O157:H7Ⅲ型分泌蛋白A)作为融合伴体分子,构建一种高效原核融合表达载体。方法在EspA的羧基端设计一Linker,包括柔韧区,肠激酶酶切位点和多克隆位点。PCR分别扩增EspA、Linker基因,利用重叠延伸PCR技术获得二者融合基因,T-A克隆后插入表达载体pET-28a(+),构建高效原核表达载体-pEspA。分别将IL-24、GFP等六种基因克隆入pEspA,转化E.coli BL21(DE3),IPTG诱导融合蛋白表达,SDS-PAGE检测融合蛋白。以EspA单克隆抗体亲和层析柱纯化融合蛋白,融合蛋白经肠激酶酶切后再通过Ni2+亲和层析柱获得外源蛋白。分别利用MTT法和紫外光激发实验鉴定IL-24、GFP生物学活性。结果构建的表达载体pEspA可高效表达外源目的蛋白,使本身不表达或表达量低的目的蛋白获得高效表达。先以EspA单克隆抗体亲和层析一步纯化获得高纯度的融合蛋白,融合蛋白经肠激酶酶切后再以Ni2+亲和层析柱获得了外源蛋白,同时生物学活性实验显示纯化的IL-24、GFP具有较好的生物学活性。结论成功构建了基于EspA的新型高效原核融合表达载体,为蛋白的融合表达及纯化又提供了一种可供选择的工具。

Objective To construct a novel prokaryotic fusion expression vector using EHEC O157：H7 Ⅲ secreting protein A （EspA） as a thsion partner. Methods A linker containing YAPODP sequence, MCS, and sites for entemkinase was designed at the C-terminus of EspA. The coding genes of EspA and linker were amplified by PCR. The cDNA fragment encoding espA-linker was obtained by overlap PCR, and then cloned into pMD18-T which was an A-T cloning vector. Thereafter target genes were inserted into multi-cloning site of pET-28a（ ＋ ） to construct effieient fusion expression vector pEspA. Six genes, including IL-24 and GFP, were cloned into pEspA, respectively. The expression of fusion protein were conducted after transforming into E. coli BL21. The expression quantity and style of fusion protein were analyzed by SDS-PAGE; the fusion protein was purified by affinity chromatography column fixed with EspA-speeific monoclonal antibody. Two kinds of fusion proteins were digested by enterokinase （EK）, to obtain fragments which then were separated by nicke-laffinity chromatography. MTT assay and ultraviolet light provocation experiments were carried out to test the biological aetivity of the IL-24 and GFP, respectively. Results pEspA fusion expression vector exhibited capability of tacilitating the expression of foreign protein. The expressed fusion protein was purified by affinity chromatography colunm fixed with EspA monoclonal antibody easily. EspA and foreign proteins were separated by nickelaffinity chrumatography column. Cell proliferation experiments and ultraviolet light provocation experiments peoved that IL-24 and GFP retains biological activity. Conclusion The newly designed prokaryotic expression vector pEspA has been successfully constructed, which provide a alternation for fusion expression and purification of protein.