人血管生成素相关蛋白2单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibody against angiopoietin-related protein 2

目的：制备人血管生成素相关蛋白2（angiopoietin—related protein2，ARP2）的单克隆抗体。方法：用RT-PCR方法获取人脾脏组织ARP2基因序列，构建pET32a-ARP2，经电穿孔导入大肠杆菌诱导表达，获得可溶蛋白样品和包涵体蛋白样品，回收、纯化蛋白，免疫BALB／c小鼠，采用杂交瘤技术建立产生ARP2抗杂交瘤细胞株，Western印迹鉴定。结果：获得融合蛋白的相对分子质量为570000，与理论计算值相符，纯化后蛋白浓度〉90％，杂交瘤细胞培养上清抗体效价为1：10^4，腹水抗体效价为1：10^7～1：10^8，抗体亚型为IgG2a类，Western印迹示目的蛋白相对分子质量为570000，免疫组化显示该抗体能特异结合人ARP2。结论：制备的抗ARP2单克隆抗体可用于ARP2蛋白鉴定。

Objective：To prepare a monoclonal antibody against human angiopoietin-related protein 2（ARP2）. Methods： Human spleen ARP2 gene was obtained by RT-PCR. A pET32a-ARP2 plasmid was constructed and was incorporated into E. coli. The products were purified and were used to immunize 6-week-old BALB/c female mice. Hybridoma secreting antiARP2 monoclonal antibody was obtained by standard procedure. Mass production was carried out after specificity identification with Western blotting. Results： The fusion protein obtained by pET32a system had a relative molecular weight of about 570 000, which was in accordance with the theoretical value. The purity of the protein was more than 90% after purification. The antibody titer was 1 ： 104 in the hybridoma culture supernatant and 1 ： 107-10s in the ascites. The IgG2a type antibody had a relative molecular weight of about 570 000 by Western blot analysis. Immunohistochemistry method showed that the antibody bond with human ARP2. Conclusion： The prepared anti-human ARP2 monoclonal antibody in this study can be used for identification of ARP2 protein.