肿瘤蛋白D52的原核表达及单克隆抗体的制备

Prokaryotic expression of tumor protein D52 and preparation of TPD52 monoclonal antibody

目的重组表达纯化人肿瘤蛋白D52(TPD52),制备其单克隆抗体。方法利用PCR将pFlag-CMV2-Tpd52质粒中的Tpd52全长基因序列扩增,通过酶切、连接插入表达载体pET-28a(+)中,构建重组质粒pET-28a-Tpd52,用IPTG诱导目的基因在E.coli中表达。表达的TPD52蛋白经纯化后作为抗原免疫小鼠,提取免疫小鼠脾细胞和骨髓瘤细胞(SP2/0)融合生成杂交瘤细胞。ELISA筛选分泌TPD52抗体的杂交瘤细胞,Western印迹(WB)筛选抗体特异性较好的杂交瘤细胞,再经有限稀释法分选出单克隆细胞株并用WB鉴定抗体特异性。以抗体特异性较好的单克隆杂交瘤细胞株进行腹水抗体制备,ELISA检测腹水抗体的效价及亚型。通过WB比较制备获得的抗体与商品化抗体并检测不同细胞系中TPD52蛋白的表达情况。结果重组质粒经测序确定插入Tpd52目的基因;SDS-PAGE分析表明,IPTG诱导6 h后E.coli中表达了大量TPD52融合蛋白;ELISA结果获得了阳性融合细胞,WB确定多克隆抗体和单克隆抗体均能与TPD52蛋白发生特异性反应;ELISA和WB结果表明,获得了高效价的TPD52腹水抗体。结论重组质粒pET-28a-Tpd52在E.coli中高效表达,成功制备了特异性、灵敏度高的TPD52蛋白的单克隆和多克隆抗体。

Objective To recombinantly express and purify human tumor protein D52(TPD52)protein,and prepare its monoclonal antibody.Methods The full-length Tpd52 gene sequence in the pFlag-CMV2-Tpd52 plasmid was ampli-fied by PCR and inserted into the expression vector pET-28 a(+)via digestion and ligation to construct a recombinant plas-mid pET-28 a-Tpd52.The target gene was induced by IPTG expressed in E.coli.The purified TPD52 protein was used as an antigen to immunize mice.Spleen cells and myeloma cells(SP2/0)of the immunized mice were extracted and fused to gen-erate hybridoma cells.Hybridoma cells capable of secreting TPD52 antibody were screened by ELISA,while hybridoma cells with better antibody specificity were screened by Western blot(WB).Monoclonal cell lines were selected with the limiting dilution method,and monoclonal antibodies were identified by WB.Monoclonal hybridoma cell lines with better antibody specificity were used to prepare ascites antibodies,the titers and subtypes of which were detected by ELISA meth-od.The prepared antibodies were compared with commercial antibodies by WB,and the expression of TPD52 protein in different cell lines was detected by WB.Results The recombinant plasmid was sequenced to confirm that the target gene of Tpd52 was inserted.SDS-PAGE analysis showed that a large number of TPD52 fusion proteins were expressed in E.coli after six hours of IPTG induction.ELISA results showed that positive fusion cells were obtained.WB experiments con-firmed that both polyclonal and monoclonal antibodies could specifically react with TPD52 protein.ELISA and WB results showed that a high titer TPD52 ascites antibody was obtained.Conclusion The recombinant plasmid pET-28 a-Tpd52 is highly expressed in E.coli,and monoclonal and polyclonal antibodies of TPD52 protein with high specificity and sensitivi-ty are successfully prepared.