重组人纤维连接蛋白羧基端细胞结合域单克隆抗体的制备与鉴定

Preparation and dentification of monoclonal antibodies against carboxy-terminal cell-binding domain of human fibronectin

目的:制备人纤维连接蛋白(fibronectin,FN)羧基端细胞结合域的单克隆抗体,并以该抗体检测心脏瓣膜基质蛋白中FN蛋白的表达情况。方法:用RT-PcR方法获取人FN羧基端细胞结合域基因序列,通过pET32质粒表达获得TRX融合蛋白。并以纯化的融合蛋白作为抗原免疫BALB／c小鼠后,按常规方法制备成单克隆抗体,获得分泌抗FN单克隆抗体的杂交瘤细胞株。结果:蛋白电泳结果显示,所获TRX融合蛋白条带与理论计算值相符,免疫动物后获得2株分泌抗FN羧基端细胞结合域不同位点单克隆抗体的杂交瘤细胞株,抗体亚型为IgC2a,免疫组化显示该抗体能特异性地结合人心脏瓣膜基质中的FN。结论:成功制备了分泌抗FN单克隆抗体,为FN结构、功能和机制的研究提供了有效工具。

Objective:To prepare the monoclonal antibodies against carboxy-terminal cell-binding domain of fibronectin (FN), and to use the product for detection of FN protein in heart valve matrix. Methods: The target DNA sequence of FN carboxy-terminal cell-binding domain was obtained by RT-PCR and the TRX fusion protein was expressed with pET32 system. The 6-week-old BALB/c female mice were immunized with recombinant fusion protein TRX-FN to prepare monoclonal antibodies and to obtain hybridoma secreting anti-FN monoclonal antibody. Results: Two lines of hybrids secreting monoclonal antibodies against different site of FN were established , both belonging to IgG2a subtypes. These monoclonal antibodies specifically demonstrated extracellular matrix of heart valves. Conclusion: The monoclonal antibodies against FN has been successfully established, which provide an useful tool in the studies of extracellular matrix of heart valve.