重组人L型丙酮酸激酶N末端单克隆抗体的制备与鉴定

Preparation and Identification of a Monoclonal Antibody Against Recombinant Human L-type Pyruvate Kinase N Terminal

目的制备抗人L型丙酮酸激酶N末端(PK-N)的单克隆抗体并对其特异性进行鉴定。方法以PK-N-GST-tag表达蛋白作为免疫原免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,经多次筛选及克隆化,建立能稳定分泌抗PK-N单克隆抗体的杂交瘤细胞株。用ELISA、Western blot、免疫组化对此单克隆抗体特性进行鉴定。结果筛选到两株能稳定分泌抗PK-N单克隆抗体的细胞株,亚类鉴定重链为IgG2b,轻链为kappa链。ELISA法测定腹水效价分别为1∶409600和1∶102400,抗体相对亲和力分别为3.54×108L/mol和2.72×108L/mol。Western blot显示抗体能特异识别免疫原和人肝细胞株HL-7702中的天然丙酮酸激酶蛋白。免疫组化进一步显示了其在细胞及组织中的定位,均匀的分布于细胞的胞浆中。结论成功制备了抗PK-N单克隆抗体。

Objective To prepare and identify the monoclonal antibody(mAb) against pyruvate kinase N terminal(PK-N).Methods BALB/C mice were immunized with immunogen PK-N-GST-tag.Then the spleen cells were isolated and fused with SP2/0 cells.After several rounds of detecting and cloning,the hybridoma cell strains secreting anti-PK-N mAb were obtained.Its specificity was evaluated with ELISA and Western blot,and the titer,immunoglobulin subtype and affinity of the mAb were measured.Results Two cell strains of hybridoma,2B2E4G and 2C6F5,were obtained.The hybridoma cell strains secreting anti-PK-N mAb belonged to IgG2b subtype,with a mAb titer in ascetic fluid of 1∶409600 and 1∶102400,respectively.Their affinity reached 3.54×108 L/mol and 2.72×108 L/mol,respectively,as determined by ELISA.Western blot demonstrated that the mAb could specifically recognize the immunogen and the natural cell lysis protein.The cell immunohistochemistry proved that the antibody could recognize human L type pyruvate kinase expressed in the plasma of HL-7702 cell strain and paraffin slice of hepatoma.Conclusion The success in anti-PK-N mAb preparation provides a foundation for further studies into glycolysis in normal condition and metabolic diseases.