肿瘤相关抗原MART-1蛋白原核表达载体的构建、表达纯化和活性鉴定

Construction,expression,purification and identification of prokaryotic expression vector of MART-1 fusion protein

目的构建编码肿瘤相关抗原MART-1融合基因的原核表达载体,在大肠埃希菌中表达His-MART-1融合蛋白,并对蛋白进行纯化和免疫原性鉴定。方法采用PCR法扩增编码MART-1的基因片段,将该基因片段克隆到含有His标签的pET-28b原核表达载体上,重组质粒经双酶切、PCR及测序鉴定正确后转化大肠埃希菌,IPTG诱导融合蛋白表达,用镍离子亲和层析法纯化融合蛋白并进行脱盐,SDS-PAGE电泳和Western blotting法鉴定。通过ELISA法检测经树突状细胞提呈后的His-MART-1融合蛋白对特异性CD4+T细胞的刺激作用。结果重组质粒经双酶切、PCR和测序鉴定证明载体构建成功。His-MART-1融合蛋白经表达纯化后,分子量约13kD,与预期值相符。Western blotting证实该融合蛋白可与抗His单克隆抗体发生特异性结合。His-MART-1融合蛋白经树突状细胞提呈后能刺激MART-1特异性CD4+T细胞分泌IFN-γ。结论成功构建了编码MART-1基因原核表达载体pET-28b-MART-1,表达及纯化获得该融合蛋白,并证实其具有良好的免疫原性。

Objective To construct a prokaryotic expression plasmid containing a fusion gene of MART-1 expressing the His-MART-1 fusion protein in E.coli,and to purify the protein and identify the immunogenicity of His-MART-1.Methods The MART-1 coding sequence was amplified by polymerase chain reaction（PCR）,and then cloned into the prokaryotic expression vector（pET-28b） containing His tag.The constructed vector,verified by restriction endonuclease digestion,PCR and DNA sequencing,was then transformed into E.coli for expression.The expression of MART-1 recombinant protein was induced by IPTG in E.coli,purified with Ni2＋-NTA affinity chromatography method,and identified by SDS-PAGE and Western blotting.ELISA was used to detect the IFN-γ expression secreted by the His-MART-1 specific CD4＋ T cells which recognized the His-MART-1 fusion protein presented by dendritic cells（DCs）.Results The successful construction of recombinant plasmid was confirmed by restriction digestion,PCR and sequencing.The molecular weight of the purified fusion protein was identified as 13kD by SDS-PAGE,which was identical to the expected value.It was confirmed by western blotting that His-MART-1 fusion protein could be recognized by His monoclonal antibody.ELISA analysis showed that His-MART-1 fusion protein presented by DCs could induce IFN-γ secretion of MART-1 specific CD4＋ T cells.Conclusion The recombinant plasmid of pET-28b-MART-1 has been successfully constructed.The expressed His-MART-1 fusion protein has been purified and the immunogenicity of inducing responses between DCs and CD4＋ T cells has been determined.