小鼠抗西方马脑炎病毒E2蛋白胞外区(E2ecto)单克隆抗体的制备

Preparation of mouse monoclonal antibodies against the ectodomain of Western equine encephalitis virus E2(E2ecto)protein

目的制备针对西方马脑炎病毒(WEEV)E2包膜糖蛋白胞外区(E2ecto)的小鼠特异性单克隆抗体(mAb)。方法构建表达WEEV E2ecto的原核表达质粒pET-28a-WEEV E2ecto,转化BL21(DE3)感受态细胞后,IPTG诱导表达,主要为包涵体形式。包涵体纯化后,通过变性、复性、超滤等制备得到E2ecto蛋白;以QuickAntibody-Mouse5W为佐剂,将E2ecto蛋白肌肉免疫BALB/c小鼠,间隔21 d加强1次。免疫后35 d,取效价超过1×104的小鼠腹腔接种1次E2ecto蛋白,3 d后取小鼠脾细胞与SP2/0细胞融合。通过有限稀释法筛选稳定分泌特异性mAb的杂交瘤细胞,接种小鼠腹腔后制备腹水;利用ELISA检测抗体亚型及腹水中抗体效价的水平;将真核表达质粒pCAGGS-WEEV-CE3E2E1转染BHK-21细胞,通过免疫荧光法(IFA)鉴定上述mAb的生物学活性;利用东方马脑炎病毒(EEEV)、委内瑞拉马脑炎病毒(VEEV)的E2ecto蛋白进行ELISA鉴定上述抗体的特异性。结果成功表达及复性获得了纯化的WEEV E2ecto蛋白;制备了3G6G10、3D7G2、3B9E8、3D5B7四株mAb,其亚型分别为IgG2c(κ)、IgM(κ)、IgM(κ)、IgG1(κ);3G6G10、3B9E8、3D7G2腹水抗体效价均为105,3D5B7达到107;该4株抗体与VEEV和EEEV等其他脑炎甲病毒无交叉反应。结论成功制备了4株特异性结合WEEVE2ecto的小鼠mAb。

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2(E2ecto)glycoprotein of Western equine encephalitis virus(WEEV).Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21(DE3)competent cells.E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies.Then the purified E2ecto protein was prepared by denaturation,renaturation and ultrafiltration.BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route,boosted once at an interval of 21 days.At 35 days post-immunization,mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally,and spleen cells were fused with SP2/0 cells three days later.Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method,and ascites were prepared after intraperitoneal inoculation of hybridoma cells.The subtypes and titers of the antibodies in ascites were assayed by ELISA.The biological activity of the mAb was identified by immunofluorescence assay(IFA)on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1.The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV.Results Purified WEEV E2ecto protein was successfully expressed and obtained.Four monoclonal antibodies,3G6G10,3D7G2,3B9E8 and 3D5B7,were prepared,and their subtypes were IgG2c(κ),IgM(κ),IgM(κ)and IgG1(κ),respectively.The titers of ascites antibodies 3G6G10,3B9E8 and 3D7G2 were 105,and 3D5B7 reached 107.None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV.Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.