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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 induce the expression of two subunits of HLA-A2, heavy chain (HC) and light chain (β2m), and to bind their purified products and specific peptides to HLA-A2-peptide complex. Methods: HC engineering bacteria and β2m engineering bacteria were induced by 0.5 mmol / L IPTG for 5 h. Purification by ultrasonic sonication and patented technology to obtain purified inclusion bodies, renaturation, ultrafiltration concentration, purification using DEAE Sepharose Fast Flow anion exchange chromatography. HC and β2m were then separately bound to two specific peptides, purified by gel filtration on Superdex 75 and initially identified using their native monoclonal antibody (mAb) W6 / 32. Results: The induced levels of HC engineered bacteria and β2m engineered bacteria were 43% and 47%, respectively. The purity of HC and β2m purified reached 95%. The two specific peptides and HC and β2m can be assembled into HLA-A2-peptide complex. The purified HLA-A2-peptide complex can be stored at 4 ° C for about 2 months and can specifically bind to mAb W6 / 32 . CONCLUSION: Both HLA-A2 HC-engineered bacteria and light-chain β2-m engineering bacteria have higher expression levels and can form stable soluble HLA-A2-peptide complexes with the corresponding specific peptides. To further study cytotoxic T lymphocytes Cell (CTL) recognition and response laid the foundation.