可溶性HLA-A2-肽组装复合物的研制及初步鉴定

目的:诱导表达HLA-A2的两个亚基-重链(HC)和轻链β2m,并将其纯化产物和特异性肽段结合为HLA-A2-肽复合体。方法:将HC工程菌和β2m工程菌经0.5 mmol/L IPTG诱导5 h。经超声破菌及专利技术处理获得精制包涵体,再复性、 超滤浓缩后,用DEAE Sepharose Fast Flow阴离子交换层析进行纯化。然后,将HC、β2m与两条特异性肽段分别结合,用Superdex 75凝胶过滤纯化,并用其天然结构的单克隆抗体(mAb)W6/32进行初步鉴定。结果:HC工程菌和β2m工程菌诱导表达的水平分别为43％和47％,HC和β2m纯化后的纯度均达到95％。两条特异性肽段与HC和β2m可组装成HLA-A2-肽复合体,纯化的HLA-A2-肽复合物在4℃可保存2个月左右,并可与mAb W6/32特异性结合。结论:HLA-A2的HC工程菌和轻链β2m工程菌均具有较高的表达水平,与相应的特异性肽段可形成稳定的可溶性HLA-A2-肽复合物,为进一步研究细胞毒性T淋巴细胞(CTL)的识别和应答奠定了基础。

Preparation and preliminary characterization of soluble HLA-A2-peptide complex

AIM: To express HLA-A2 heavy chain (HC) and light chain beta(2m) in bacteria and to prepare soluble HLA-A2-peptide complex. METHODS: HC and beta(2m) expressing engineering bacteria was induced for 5 h with 0.5 mmol/L IPTG.After sonification of the bacteria,crude inclusion body was obtained which was then refined, renaturated, ultrafiltrated, and purified by DEAE Sepherose Fast Flow anion exchange chromatography. Then HC and beta(2m) were connected with two specific peptides, purified through Superdex 75 gel filtration, and identified with mAb W6/32 which can recognize native HLA-A2. RESULTS: The expression rates of HC and beta(2m) in engineering bacteria was both about 50%.The purity of both expression products reached to 95%.Moreover, the two HLA-A2-peptide complexes could be recognized by mAb W6/32, even after being stored at 4 degrees Celsius for 2 months. CONCLUSION: The two soluble HLA-A2-peptide complexes prepared by us are stable and lay the foundation for further research of CTL recognition and response.