人肝再生增强因子单克隆抗体的制备:以非纯化的大肠杆菌表达抗原筛选杂交瘤

Preparation of monoclonal antibody to human augmenter of liver regeneration:screening of hybridomas with unpurified antigen expressed by E.coli

目的 :利用杂交瘤技术、以非纯化的大肠杆菌表达重组蛋白作为筛选抗原 ,制备小鼠抗人肝再生增强因子(hALR)的单克隆抗体。方法 :用实验室纯化的重组hALR 硫氧环蛋白融合蛋白免疫BALB c小鼠 ;小鼠脾细胞与SP2 0骨髓瘤细胞融合后经HAT选择培养基筛选杂交瘤 ;以重组质粒pQE30 hALR与空质粒pQE30在大肠杆菌中诱导表达后的细菌裂解产物作为筛选抗原和对照抗原 ,用ELISA方法筛选能分泌抗hALR单克隆抗体的阳性杂交瘤细胞克隆 ;进一步以ELISA方法和免疫印迹方法检测该杂交瘤细胞产生的抗体对真核细胞表达的重组hALR及人体血清中天然hALR的反应性。结果 :成功筛选出一株能稳定分泌抗hALR单克隆抗体的杂交瘤细胞 ;其产生的抗体能对真核细胞表达的重组hALR及人体血清中天然的hALR发生特异的抗原抗体反应。结论 :大肠杆菌表达的重组蛋白在以空质粒表达产物作为对照下 ,不经过任何纯化步骤也能够用于单克隆抗体制备中的杂交瘤筛选 ;hALR单克隆抗体为深入研究hALR提供了研究手段。

Objective:To prepare monoclonal antibody(McAb) against human augmenter of liver regeneration(hALR) by hybridoma technique with unpurified recombinant protein expressed by E.coli as screening antigen.Methods:BALB/c mice were immunized with hALR thioredoxin(hALR Thi) fusion protein twice.The splenocytes were fused with myeloma cells by polyethylene glycol and the hybridomas were selected in HAT medium.The positive hybridomas secreting anti hALR McAb were screened by means of ELISA,with the lysates of E.coli containing the recombinant plasmid pQE30 hALR induced by IPTG as screening antigen and containing plasmid pQE30 as controlling antigen.The reactivity of McAb secreted by the positive hybridoma to hALR expressed in eukaryocyte and native hALR in human serum was confirmed by means of ELISA and Western blot methods.Results:A positive hybridoma was screened successfully;the specific antigen antibody reaction was confirmed between the McAb prepared and hALR expressed in eukaryocyte and native hALR in human serum.Conclusion:Without any purification procedure,recombinant protein expressed by E.coli can be used for screening bybridomas in preparing of McAb with the expressed product of empty plasmid as control;anti hALR McAb can be used to the further study of hALR.