人血管内皮钙黏蛋白5单克隆抗体的制备及应用

Preparation and application of monoclonal antibody against human vascular endothelial cadherin 5

目的制备并鉴定人血管内皮钙黏蛋白5(VECDH5)的单克隆抗体(m Ab)。方法用重组原核人VECDH5蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合;间接ELISA筛选阳性克隆,得到稳定分泌抗VECDH5抗体的杂交瘤细胞株,ELISA测定其效价;Western blot法、流式细胞术、免疫荧光细胞化学染色、免疫组织化学染色鉴定其特异性及抗原表位。结果制备并获得1株可分泌特异性抗VECDH5的杂交瘤细胞株(2C11)。ELISA测定腹水效价高达1∶10 000。多种方法均证实该抗体能特异性识别人VECDH5蛋白,并可在上述不同方法中应用。经抗原表位鉴定,研究制备的2C11识别的氨基酸序列为LDREVVPWYNLTVEA。结论成功制备了亲和力高、特异型好的抗人VECDH5的m Ab。

Objective To prepare and identify monoclonal antibody（ m Ab） against human vascular endothelial cadherin 5（ VECDH5）. Methods BALB/c mice were immunized with recombinant VECDH5 protein for preparing m Ab using hybridoma technique. The positive clones were confirmed and selected by indirect ELISA for titer determination. Western blotting,flow cytometry,immunofluorescence staining, immunohistochemical staining were performed to identify the specificity and epitope. Results One hybridoma cell strain secreting specific m Ab against VECDH5 was obtained（ 2C11）. ELISA showed that the titer of the ascites was 1 ∶ 10 000. Western blotting,flow cytometry,immunofluorescence,immunohistochemistry demonstrated that the m Ab could specifically recognize and bind VECDH5. Epitope identification showed that the amino acid sequence recognized by 2C11 was LDREVVPWYNLTVEA. Conclusion We have prepared m Ab against human VECDH5,which has good binding ability and specificity.