摘要
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。
AIM: To express HLA-A2 heavy chain (HC) and light chain β2m in bacteria and to prepare soluble HLA-A2-peptide complex. METHODS: HC-and β2m-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 chro-matography. Then HC and β2m 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 β2m 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℃ 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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2003年第6期538-540,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划(863)资助(No.2001AA215121)
