HCA518的蛋白表达、纯化和单克隆抗体的制备

Expression,purification and preparation of monoclonal antibody of HCA518 protein

目的:对HCA518蛋白进行表达和纯化,并制备HCA518蛋白的单克隆抗体。方法:采用基因重组技术在原核系统表达HCA518蛋白,金属镍螯合的Ni-NAT亲和层析柱进行蛋白纯化;杂交瘤技术建立分泌HCA518单克隆抗体的杂交瘤细胞株;ELISA方法进行单克隆抗体细胞株的筛选;Westem blot方法鉴定单克隆抗体的特异性;免疫荧光法对HCA518蛋白进行定位。结果:成功表达和纯化了HCA518重组蛋白,其纯度达98％。共筛选出2株分泌HCA518单克隆抗体的杂交瘤细胞株,制备腹水并纯化了HCA518的单克隆抗体,腹水中单克隆抗体的效价分别为1×10-4、5×10-4,属于IgG2b亚型和IgM,该抗体能与重组蛋白和肿瘤细胞核蛋白(P100)发生特异性反应,免疫荧光显示HCA518蛋白主要定位于细胞核中。结论:建立了稳定分泌HCA518单克隆抗体的杂交瘤细胞株,制备的单克隆抗体特异性好,对进一步应用于检测肿瘤组织中HCA518蛋白和判断肿瘤细胞的恶性增生程度预测肿瘤预后具有重要的实用价值。

Objective：To express and purify HCAS18 protein and prepare its monoclonal antibody （MeAb）. Methods： The HCA518 protein was expressed with gene recombinant technique in prokaryotic system and purified with nickel chelate nitrilotriaeetie acid（Ni-NTA） affinity chromatography column. Hybridoma cell lines that secreted anti-HCA518 McAb were established by cells fusion and screened by enzyme linked immunosorbent assay（ELISA）. The specificity of anti-HCA518 McAb was identified by Western blot assay. The HCA518 protein in tumor cells was stained by immunoflourescence assay. Results： Rcombinant HCA518 protein was expressed with a purity of 98%. Two hybridoma cell lines was selected and anti-HCA518 McAb was purified from mice ascites. The titers of antiHCAS18 MeAb in ascites were 1 ×10^-4 and 5×10^-4 respectively. The antibody belonged to IgG2b subtype and igM. Anti-HCAS18 MeAb specifically reacted with recombinant HCASI8 protein and tumor cells＇nuclear protein（P100）. The HCA518 protein was mainly located in cell nucleus. Conclusion： Stable hybridoma cell lines that secreted anti-HCA518 MeAb have been established and antiHCAS18 McAb was prepared with high specificity. It has important significance for detecting HCA518 protein in tumor tissues and determining malignant proliferation status of tumor cells and predicting its prognosis.