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目的构建人肝素酶蛋白的原核表达载体,表达并纯化重组人肝素酶蛋白和制备其单克隆抗体。方法将人肝素酶cDNA克隆至原核表达载体pET30a(+),经大肠杆菌表达、纯化后,获得的肝素酶纯化蛋白免疫小鼠,取其脾淋巴细胞与SP2/0细胞融合,制备能产生肝素酶单克隆抗体的杂交瘤细胞株,进一步采用Western blotting、ELISA等技术对制备的单克隆抗体进行初步鉴定。结果原核表达重组质粒在大肠杆菌中能高效表达人肝素酶蛋白,并进一步从10株杂交瘤细胞中选取了3株能稳定分泌人肝素酶单抗的杂交瘤细胞株。结论成功制备了3株人肝素酶特异性单克隆抗体。
Objective To construct prokaryotic expression vector of human heparanase protein, express and purify recombinant human heparanase protein and prepare its monoclonal antibody. Methods Human heparinase cDNA was cloned into prokaryotic expression vector pET30a (+) and expressed in E.coli. After purification, the purified heparinase was used to immunize mice and spleen lymphocytes were fused with SP2 / 0 cells to prepare Hybridoma cell lines producing heparanase monoclonal antibodies were further identified by Western blotting and ELISA techniques. Results The prokaryotic recombinant plasmid could efficiently express human heparinase protein in E. coli and further selected three hybridoma cell lines which could stably secrete human heparanase mAb from 10 hybridoma cells. Conclusion Three human heparanase-specific monoclonal antibodies were successfully prepared.