鼠抗人B7-H6单克隆抗体的制备及其生物学特性鉴定

Preparation and identification of mouse anti-human B7-H6 monoclonal antibodies

目的：制备鼠抗人B7-H6单克隆抗体并对其生物学特性进行鉴定。方法构建B7-H6真核表达系统,转染小鼠成纤维细胞 NIH/3T3,潮霉素筛选获得稳转细胞株2H8,腹腔免疫BALB/c小鼠,采用杂交瘤技术进行细胞融合,用流式细胞术筛选阳性克隆,有限稀释法进行克隆化培养阳性杂交并鉴定其亚型。杂交瘤细胞株腹腔注射小鼠获得腹水型抗体,经G-sepharose柱采用亲和层析法对小鼠腹水中的抗体进行纯化,应用蛋白印迹和流式细胞术分析抗体的特异性。结果细胞融合后筛选培养出2株能够稳定分泌抗B7-H6单克隆抗体的杂交瘤细胞株2D12和9D1,单抗亚类鉴定均属于IgG2a,轻链为κ型。蛋白质印迹和流式细胞术证明其与 B7-H6均有特异性反应。结论成功制备出高特异性抗人B7-H6单克隆抗体。

Objective To prepare mouse anti-human monoclonal antibodies against B7-H6 and to identify their biological characteristics. Methods The B7-H6 gene was cloned by RT-PCR from a human lung adenocarcinoma cell line （ A549 ） and then subcloned into the eukaryote expression vector pCMV3 to construct the recombinant vector pCMV3-B7-H6. The recombinant vector pCMV3-B7-H6 that was verified with enzyme digestion and gene sequencing was transfected into NIH/3T3 cells by electroporation. BALB/c mice were immunized with the successfully transfected cells named 2H8 through intraperitoneal injection. The monoclonal antibodies against human B7-H6 with the advantages of high affinity and specificity were pre-pared by using hybridoma technology. Western blot assay and flow cytometry analysis were used to identify the specificity of prepared monoclonal antibodies. Results The recombinant eukaryotic expression vector encoding B7-H6 was successfully constructed. Two hybridoma clones that stably secreted monoclonal anti-bodies against B7-H6 were screened out by using flow cytometry analysis and the monoclonal antibodies se-creted by them were belonged to IgG2a isotype. Specific reactions between B7-H6 and the secreted mono-clonal antibodies were confirmed by Western blot assay and flow cytometry analysis. Conclusion The mon-oclonal antibodies which recognized B7-H6 specifically were prepared successfully.