新肿瘤睾丸抗原Ropporin重组蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of ropporin and generation of its rabbit polyclonal antisera

目的:构建新肿瘤睾丸抗原Ropporin原核表达载体,表达和纯化人Ropporin重组蛋白,并制备其兔抗血清.方法:人Ropporin全长cDNA克隆入pQE30载体,在大肠杆菌中利用IPTG诱导表达并纯化人Ropporin重组蛋白.利用人Ropporin重组蛋白免疫兔,制备人Ropporin多克隆抗体.结果:以构建的pQE30-Ropporin质粒转化大肠杆菌后,在IPTG诱导下表达和纯化得到相对分子质量与鼠Ropporin蛋白一致的蛋白质,利用其免疫动物后,Western blot检测显示该蛋白具有良好的免疫原性,制备的兔抗血清效价>1∶512000,对该蛋白具有高度特异性.结论:利用大肠杆菌原核表达系统表达和纯化了人Ropporin重组蛋白,并成功制备了人Ropporin多克隆抗体.

AIM：To prepare human ropporin（a novel cancer/testis antigen）recombinant protein using a prokaryotic expression system and generate its rabbit polyclonal antisera. METHODS：The full-length cDNA of the human ropporin gene was subcloned into the pQE30 vector and transformed into competent Escherichia coli（E.coli）JM109 cells.After transformed E.coli was induced using IPTG（isopropylβ-D1-thiogalactopyranoside）,human recombinantropporin protein was extracted,purified by Nickel sepharose affinity chromatography,and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.Rabbits were immunized with the purified protein to prepare antisera.The specificity of antisera was determined by Western blot. RESULTS：SDS-PAGE analysis showed that a protein with similar molecular weight to mouse popporin protein was purified from transformed E.coli.Western blot using anti-His tag monoclonal antibody showed that the prepared protein contained a His-tag.Polyclonal antisera were successfully generated by immunization of rabbits with human recombinant ropporin protein. The titers of antisera were more than 1∶512 000. Western blot analysis confirmed the specificity of the antisera obtained. CONCLUSION：Human recombinant ropporin protein was successfully prepared using a prokaryotic expression system,and its rabbit polyclonal antisera were also successfully generated. The antisera obtained can be used to detect the expression of ropporin protein in various types of tumors and investigate its role in malignant processes.