重组人B7-H4IgV融合蛋白的表达、纯化及活性

目的表达、纯化重组人B7-H4IgV融合蛋白,并检测其活性。方法将人B7-H4胞外片段IgV区基因插入pQE-30原核表达载体,转化大肠杆菌,IPTG诱导表达。表达产物经Ni2+-NTA柱亲和层析纯化,并进行Westernblot鉴定。用纯化的rhB7-H4IgV蛋白免疫昆明小鼠,经ELISA、流式细胞术测定小鼠抗血清的活性;免疫SP2/0荷瘤小鼠,观察对肿瘤细胞生长的抑制作用。结果表达的rhB7-H4IgV蛋白约占菌体总蛋白的25%,纯化后蛋白纯度为93%,浓度约为0.5mg/ml,与抗His单抗可产生特异性反应条带。免疫昆明小鼠产生的抗血清效价可达1∶10000,可与SP2/0肿瘤细胞结合。rhB7-H4IgV蛋白对SP2/0移植瘤生长具有一定的抑制作用。结论已成功表达并纯化了重组人B7-H4IgV融合蛋白,纯化蛋白可诱发小鼠体内免疫应答,产生的抗体能与表达B7-H4的肿瘤细胞SP2/0结合,rhB7-H4IgV蛋白在一定程度上能抑制SP2/0移植瘤的生长。

Expression,Purification and Activity of Recombinant Human B7-H4IgV Fusion Protein

Objective To express recombinant human B7-H4IgV(rhB7-H4IgV) fusion protein and determine its activity. Methods Insert the gene encoding extracellular IgV domain of hB7-H4 into prokaryotic expression vector pQE-30. Transform the constructed recombinant plasmid to E. coli for expression under induction of IPTG. Purify the expressed product by Ni2+-NTA affinity chromatography and identify by Western blot. Immunize KM mice with the purified rhB7-H4IgV protein and determine the activity of antisera by ELISA and flow cytometry. Immunize tumor-bearing mice(inoculated with SP2/0 cells) with rhB7-H4IgV and observe the inhibiting effect on growth of SP2/0 cells. Results The expressed rhB7-H4IgV, at a concentration of about 0. 5 mg/ml, contained about 25% of total somatic protein and reached a purity of about 93% after purification. Western blot showed specific reaction band of expressed protein with McAb against His. The antisera of mice immunized with rhB7-H4IgV reached a titer of 1 ∶ 10 000 and bound to SP2/0 cells. The expressed rhB7-H4IgV protein showed a certain inhibiting effect on the growth of SP2/0 cells. Conclusion Recombinant hB7-H4IgV protein was successfully expressed. The purified expressed protein induced immune response in mice and inhibited the growth of SP2/0 cells at a certain degree.