抗膜联蛋白Ⅰ单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against annexinⅠ

目的：制备抗膜联蛋白Ⅰ单克隆抗体（mAb），对其进行特性鉴定，为在恶性肿瘤诊断和治疗中的应用打下基础。方法：构建膜联蛋白Ⅰ基因480bp的原核表达载体pQE30ANXI，异丙基硫代-β-D-半乳糖苷（IPTG）诱导蛋白表达，并用Ni—NTA树脂亲和层析柱纯化蛋白。用纯化的膜联蛋白I作为抗原免疫BALB／c小鼠，取免疫小鼠的脾细胞与SP2／0骨髓瘤细胞融合，经过HAT、HT选择培养及有限稀释法进行克隆化，制备抗膜联蛋白Ⅰ蛋白的单克隆抗体，用ELISA、Western blot及免疫组织化学染色法进行特性鉴定。结果：获得5株分泌抗膜联蛋白ImAb的杂交瘤细胞，分别命名为1A8、1D2、1E2、4E7和5G3，所产抗体，均为IgGl亚型，轻链均为K链，亲和力为（2．3～3．03）×10^8L／mol。经Western blot证实获得的mAbs可以特异性识别膜联蛋白Ⅰ抗原；免疫组织化学染色结果也显示这些mAbs可以特异识别肿瘤组织中的膜联蛋白Ⅰ。结论：成功地获得了5株抗膜联蛋白Ⅰ的mAbs，为进一步在肿瘤诊断和治疗中应用膜联蛋白Ⅰ抗体打下了基础。

Objective：To prepare monoclonal antibody （mAb） against annexin Ⅰ and identify its properties to establish the basis for future application in malignant tumor diagnosis and therapy. Methods： Prokaryotic expression vector pQE-30 of Annexin Ⅰ gene （1-480 bp） was successfully constructed. The transformed bacteria bearing pQE30-annexin Ⅰ plasmids were induced by IPTG to express fusion proteins. The expression products was purified by affinity chromatography using Ni-NTA resin. Purified annexin Ⅰ protein was used as the antigen to immunize BALB/c mice. Splenocytes from the immunized mice were fused with Sp2/0 cells. Monoclonal hybridoma cell lines were obtained by selective culture with the HAT/HT culture systems and isolated by limiting dilution. The mAbs were characterized by ELISA, Western blot and immunohistochemical staining. Results： Five hybridoma cell lines named 1A8, 1D2, 1E2, 4E7 and 5G3 were obtained. All of the mAbs belonged to IgG1 where light chains. The affinity constants of mAbs are in the range of 2.3 to 3.03 × 10^8 L/tool. Western blot analysis confirmed that the five mAbs could recognize Annexin Ⅰ antigen, but had no cross-reaction with other antigens. Immunohistochemical results showed that the mAbs could recognize Annexin Ⅰ antigen in tumor tissues. Conclusion： Five hybridoma cell lines secreting mAbs against Annexin Ⅰ protein were obtained successfully, which laid the groundwork for its future application in tumor diagnosis and therapy.