分子佐剂TBhsp和MT在肝细胞再生磷酸酶-3(PRL-3)单克隆抗体制备中的作用

Role of molecular adjuvants TBhsp and MT in the preparation of monoclonal antibody against phosphatase of regenerating liver-3

目的探讨结核分枝杆菌热休克蛋白(TBhsp)和结核分枝杆菌T细胞刺激表位(MT)在肝细胞再生磷酸酶-3(PRL-3)单克隆抗体(mAb)制备过程中的佐剂作用。方法构建对照质粒pET28a-PRL-3和佐剂-PRL-3融合蛋白表达质粒pET28a-PRL-3-MT、pET28a-TBhsp-PRL-3和pET28a-TBhsp-PRL-3-MT,并纯化其表达蛋白,分别免疫BALB/c小鼠,ELISA检测并比较各组的抗血清效价,选取效价最高的小鼠,采用杂交瘤技术制备PRL-3 mAb,并进行类和亚类鉴定。结果成功构建了上述4种PRL-3相关的重组表达质粒,并表达纯化出相应的融合蛋白,其中PRL-3-MT融合蛋白免疫组抗体效价高于其他组;用该组小鼠进行细胞融合并筛选获得均为IgG类的10株分泌PRL-3 mAb的细胞株。结论 MT在PRL-3 mAb制备中可以发挥较为明显的免疫佐剂作用。

Objective To explore the adjuvant effects of Mycobacterium tuberculosis heat shock protein （TBhsp） and Mycobactedum tuberculosis T cell stimulatory epitope （MT） in the preparation of the monoclonal antibody （mAb） against phosphatase of regenerating liver-3 （PRL-3）. Methods The prokaryotic expression vectors pET28a were used for the construction of pET28a-PRL-3 （control plasmids）, pET28a-PRL-3-MT, pET28a-TBhsp-PRL-3 and pET28a- TBhsp-PRL-3-MT （recombinant plasmids）. The various fusion proteins expressed in bacteria were purified and utilized to immunize the BALB/c mice, respectively. The serum level of specific anti-PRL-3 antibody was measured by ELISA. The mouse with the highest serum level of anti-PRL-3 antibody was selected to prepare mAb with hybridoma technique. The subclasses of mAb were identified. Results The PRL-3 fusion proteins expressed from the four expression plasmids were purified successfully. The serum level of anti-PRL-3 antibody from the mice immunized with PRL-3-MT was the highest compared with the other immune groups. Ten hybridoma cell lines secreting anti-PRL-3 mAb were obtained after cell fusion and ELISA primary screening, and all of the mAb subclasses were IgG. Conclusion MT is a potentially effective molecular adjuvant in preparation of mAb specific for PRL-3.