利用CEA_(694-702)-β2m融合蛋白制备HLA-CEA_(694-702)复合体  

Preparation of HLA-CEA_(694-702) complex with CEA_(694-702)-β2m fusion protein

作　　者：孟凡岩[1] 沈传来[1] 郭薇[1] 缪凤琴[1] 谢维[1] 张建琼[1] 

机构地区：[1]东南大学发育与疾病相关基因教育部重点实验室,江苏南京210009

出　　处：《东南大学学报（医学版）》2009年第5期366-370,共5页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(30571703);国家杰出青年科学基金资助项目(30325017);东南大学优秀博士学位论文基金资助

摘　　要：目的:获得大量CEA694-702-β2-微球蛋白(β2m)融合蛋白,并制备HLA-CEA694-702复合体。方法:通过引物设计在β2m基因的5′端引入CEA694-702序列和L inker序列,并将目的基因克隆到质粒pET28 a中,在BL21(DE3)中表达。CEA694-702-β2m和HLA重链蛋白用金属螯合亲和层析法进行纯化,并经稀释复性法复性。制备的HLA-CEA694-702复合体用PEG20000浓缩,应用ELISA鉴定其构象,并用尺寸排阻色谱纯化和鉴定。结果:HLA重链蛋白和CEA694-702-β2m蛋白经金属螯合亲和层析法纯化后纯度提高。ELISA鉴定结果表明稀释复性后HLA-CEA694-702复合体形成了正确的构象。利用尺寸排阻色谱对HLA-CEA694-702复合体成功地进行了纯化。结论:利用CEA694-702-β2m融合蛋白成功制备出HLA-CEA694-702复合体,为HLA-CEA694-702四聚体的制备奠定了基础。Objective To gain CEA694-702-β2m fusion protein and to generate HLA-CEA694.-702 complex. Methods The sequences of CEA694-702 and Linker were fused at the 5＇-terminus of the β2m gene by the primer design, and the resulted CEA694-702-β2m gene was inserted into plasmid vector pET28a. CEA694-702-β2m protein was overexpressed in BL21 （DE3） and purified by IMAC. HLA-CEA694-702 complex generated by dilution refolding was concentrated with PEG20000 and purified by size exclusion chromatography. The conformation of HLA-CEA694-702 complex was identified by ELISA. Results The purities of HC and CEA694-702-β2m proteins were enhanced after purified by IMAC. ELISA confirmed the correct conformation of HLA-CEA694-702 complex. The purity of the HLA-CEA694-702 complex was successfully purified by SEC. Conclusion HLA-CEA694-702 complex is successfully prepared with CEA694-702-β2m fusion protein,and will facilitate the generation of HLA-CEA694-702 tetramer.

关 键 词：Β2-微球蛋白 癌胚抗原 稀释复性法 HLA-肽复合体 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]