人肝素酶蛋白的表达及其单克隆抗体的制备

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。

Expression of human heparanase protein and production of monoclonal antibodies against human heparanase

Objective To construct Prokaryotic expression vectors of human heparanase protein, to purify recombinant human heparanase protein and to prepare monoclonal antibodies against human heparanase. Methods cDNA of human heparanase was coloned into prokaryotic expression vector pET30a (+) and was further transfected into DE3. The recombinant protein of human heparanase was used immuned Balb/c female mouse. The spleen lymphocyte were fused with SP2/0 hybridoma to produce monoclonal antibody against human heparanaes. Western blotting and ELISA were carried out to preliminarily identify the monoclonal antibody produced by these hybridomas. Results Protein of human heparnase was purified from DE3 transfected with human heparanase cDNA prokaryotic expressing vector. After fusion with SP2/0 hybridoma, we selected 3 from 10 hybridoma cell lines which were able to produce monoclonal antibodies against human heparanase stablely. Conclusions We successfully prepare hybridomas which can stablely produce monoclonal antibodies against human heparanase.