抗GPNMB单克隆抗体的制备及双抗体夹心ELISA方法的建立

Preparation of Monoclonal Antibodies against GPNMB and Establishment of Double Antibody Sandwich ELISA

目的:制备抗GPNMB单克隆抗体,建立双抗体夹心ELISA检测GPNMB在胶质母细胞瘤中的表达情况。方法:以新鲜胶质母细胞瘤组织为材料,提取总RNA,通过RT-PCR扩增得到GPNMB cDNA序列,经NcoI和XhoI双酶切后,克隆入PET-28a(+)进行原核表达,经蛋白纯化得GPNMB-6×His融合蛋白;以该融合蛋白免疫BALB/c小鼠,淋巴细胞杂交瘤法制备单克隆抗体;采用Western-blot、双抗体夹心ELISA以制备所得的抗GPNMB抗体检测胶质母细胞瘤细胞系U251、U373、SHG44和星形胶质细胞系SVG12中GPNMB的表达。结果:成功构建了GPNMB原核表达载体,获得了GPNMB-6×His重组蛋白;制备得到了G203、F105和M306共3株抗GPNMB的单克隆抗体,腹水ELISA效价分别为1:8000、1:8000、1:5000,抗体亚类分别为IgG2、IgG1和IgG1,进一步实验证实单克隆抗体G203和M306可用于双抗体夹心ELISA检测不同胶质细胞瘤细胞系中GPNMB的表达。结论:成功制备出抗GPNMB单克隆抗体,效价及特异性良好,并证实GPNMB在胶质母细胞瘤细胞系中高表达,为进一步研究GPNMB在胶质母细胞瘤中的作用奠定了基础。

Objective：Anti-GPNMB monoclonal antibodies were prepared,and the expression of GPNMB in glioblastoma was detected by double antibody sandwich ELISA.Methods：Total RNA was extracted from glioblastoma tissue.Reverse transcription-polymerase chain reaction（RT-PCR） was performed to obtain the GPNMB gene.After double digested with NcoI and XhoI,the GPNMB gene segment was inserted into PET-28a（＋） vector and the recombinant protein GPNMB-6×His was expressed in E.coli.BL21.Monoclonal antibodies to GPNMB were got by immunizing the BALB/c mice with purified GPNMB-6×His emulsified in Freund＇s adjuvant.Then,the monoclonal antibodies were applied using double antibody sandwich ELISA and Western-blot for the detection of GPNMB in the U251,U373,SHG44 and SVG12 cell lines.Results：A recombinant plasmid with the target gene was successfully constructed and expressed recombinant GPNMB-6×His in E.coli.BL21.Three monoclonal antibodies were obtained,and the ELISA titers of ascetic fluids were 1：8000,1：8000,and 1：5000,respectively.We also revealed the monoclonal antibodies G203 and M306 could be applied on the double antibody sandwich ELISA for the detection of GPNMB in glioblastoma cell lines.Conclusion：Mice anti-GPNMB monoclonal antibodies with high specificity were obtained.The monoclonal antibodies provided good tools for further studying functional characterization of GPNMB.