重组B7-H1IgV融合蛋白的表达、纯化及活性鉴定

Expression,Purification and Activity of Recombinant B7-H1IgV Fusion Protein

目的以B7-H1为靶点,研制肿瘤免疫治疗蛋白疫苗。方法将人B7-H1胞外片段IgV区基因插入pQE-30原核表达载体,转化大肠杆菌,经IPTG诱导表达。Ni2+-NTA亲和层析纯化蛋白,Westernblot鉴定。用纯化的rhB7-H1IgV蛋白免疫昆明小鼠,经ELISA、流式细胞术、免疫组化技术和CDC试验测定融合蛋白及其抗血清的生物学活性。结果所构建的pQE-30-TT-B7-H1IgV表达载体,能稳定表达rhB7-H1IgV蛋白,经纯化后免疫昆明小鼠,可获得高滴度抗B7-H1抗血清。经流式细胞术和免疫组化检测显示,其抗血清可与HT-29/B7-H1+及SP2/0肿瘤细胞结合,且在CDC试验中,可依赖补体杀伤HT-29/B7-H1+及SP2/0肿瘤细胞。结论rhB7-H1IgV融合蛋白不仅可引发小鼠体液免疫应答,而且其抗体还能与表达B7-H1的肿瘤细胞相结合,并介导补体依赖的体外杀伤作用。

Objective To prepare protein vaccine for immunotherapy of tumors using BT-H1 as a target. Methods Insert the gene encoding extracellular IgV domain of B7-H1 into prokaryotic expression vector pQE-30 ,then transform to E. coli for expression under induction of IPTG. Purify the expressed rhB7-H1IgV by Ni^2＋-NTA column chromatography and identify by Western blot. Immunize Kunming mice with purified rhB7-H1IgV protein and determine the activities of the fusion protein and its antiserum by ELISA,flow cytometry,immunohistochemical method and CDC test. Results rhB7-H1IgV protein was stably expressed by using the constructed recombinant plasmid pQE-30-TT-B7-H1IgV. High titer antisera against B7-H1 were obtained by immunizing Kunming mice with the purified rhB7-H1IgV protein. Both flow cytometry and immunohistochemical method showed specific binding activity of the antisera to HT-29/B7- H1^＋ cells as well as SP2/0 cells. CDC test proved complement-dependent killing activity of the antisera to both HT-29/B7-H1^＋ cells and SP2/0 cells. Conclusion The expressed rhB7-H1IgV fusion protein induced hmnoral immune response in mice. The induced antibody showed binding activity to the tumor cells in which B7-H1 was expressed,and mediated complement-dependent killing activity in vitro.