酪氨酸磷酸酶PRL-3单抗的制备及特性鉴定

Preparation and Characterization of Monclonal Antibody against Protein Tyrosine Phosphatase PRL-3

目的制备并鉴定抗肝再生磷酸酶-3(Phosphatase of Regenerating Liver-3,PRL-3)单克隆抗体,为临床检测和以PRL-3为靶点的肿瘤治疗提供可能。方法运用杂交瘤融合技术制备PRL-3单克隆抗体,通过Western blot检测和免疫沉淀鉴定其与原核及真核PRL-3蛋白的反应性;重组并诱导表达PRL-3的6个截短体,Western blot分析单抗结合的大致抗原表位。结果共得到9株单抗,3株与PRL-1、PRL-2和PRL-3均反应,6株(9D8、9E2、9F4、11B2、4D3和4D10)只与PRL-3反应,其中4株特异性单抗可以与哺乳动物细胞表达的PRL-3反应;单抗9D8、9E2、9F4和11B2可以和PRL-3羧基末端(162-173位氨基酸)的短肽结合;4D3和4D10与中间部分(69～95位氨基酸)的短肽结合。结论抗体9D8、9E2、9F4、11B2、4D3和4D10特异性强、亲和力高,为临床检测提供了可靠工具,并为进一步的功能研究奠定了基础。

Objective To prepare and identify specific Phosphatase of Regenerating Liver-3 （PRL-3） monoclonal antibodies for clinical detection and for further therapeutic intervention. Methods Hybridoma technology was used to prepare PRL-3 monoclonal antibodies （MAbs）,and the specificities of MAbs against PRL-3 were evaluated by Western blot and mmunoprecipation. Six truncations of PRL-3 were cloned and expressed in prokaryotic cell for identifying the approximate epitope. The binding abilities of MAbs were analyzed by Western blot. Results Among 9 hybridoma clones obtained, 6 （9D8, 9E2, 9F4, 11B2, 4D3 and 4D10）could specifically bind to PRL-3,and 4-could react to PRL-3 protein in eukaryotic cells.Clone 9D8,9E2,9F4 and 11B2 cloud bind to the COOH terminal of PRL-3,and clone 4D3 and 4D10 cloud bind to 69-95 amino acids. Conclusion MAbs 9D8, 9E2, 9F4, 11B2, 4D3 and 4D10 can react only to PRL-3, which provides potential applications in and will be powerful and reliable tools for future study and clinical diagnosis and in future study.