CAJ | 학술논문

目的：为了获得真核表达的重组蛋白OCILRP2-Fc和OCILRP2特异性抗体，用于OCILRP2生物学功能的进一步研究。方法：构建了融合人IgG Fc段的小鼠OCILRP2真核表达载体pIg-CD5-OCILRP2，将pIg-CD5-OCILRP2转染CHO细胞，G418筛选稳定表达细胞株；Protein A 亲和层析从 CHO 细胞培养上清中纯化重组蛋白OCILRP2-Fc 并免疫家兔制备OCILRP2多克隆抗体。 ELISA检测抗血清和多克隆抗体的效价，Western blot 验证多克隆抗体的特异性，并应用该抗体检测OCILRP2在树突状细胞（ Dendritic cells ，DC）中的表达。结果：ELISA结果表明OCILRP2-Fc稳定表达细胞株经过多次传代培养后仍然具有较高的蛋白表达水平；SDS-聚丙烯酰胺凝胶电泳结果显示纯化后的蛋白只有单一条带出现，且该条带可以被抗人IgG检测到；重组蛋白OCILRP2-Fc免疫家兔获得的抗血清和纯化后得到的OCILRP2多克隆抗体效价分别为1∶1280000和1∶320000，Western blot证明该抗体可以和免疫原OCILRP2-Fc及NIH/3T3细胞中表达的OCILRP2特异性结合。应用该抗体检测OCILRP2在 DC 的表达发现 OCILRP2在成熟 DC 的表达明显高于不成熟 DC。结论：获得了真核表达的重组蛋白OCILRP2-Fc和高效价的OCILRP2特异性抗体，为进一步研究OCILRP2在免疫应答中的功能奠定了基础。
목적：위료획득진핵표체적중조단백OCILRP2-Fc화OCILRP2특이성항체，용우OCILRP2생물학공능적진일보연구。방법：구건료융합인IgG Fc단적소서OCILRP2진핵표체재체pIg-CD5-OCILRP2，장pIg-CD5-OCILRP2전염CHO세포，G418사선은정표체세포주；Protein A 친화층석종 CHO 세포배양상청중순화중조단백OCILRP2-Fc 병면역가토제비OCILRP2다극륭항체。 ELISA검측항혈청화다극륭항체적효개，Western blot 험증다극륭항체적특이성，병응용해항체검측OCILRP2재수돌상세포（ Dendritic cells ，DC）중적표체。결과：ELISA결과표명OCILRP2-Fc은정표체세포주경과다차전대배양후잉연구유교고적단백표체수평；SDS-취병희선알응효전영결과현시순화후적단백지유단일조대출현，차해조대가이피항인IgG검측도；중조단백OCILRP2-Fc면역가토획득적항혈청화순화후득도적OCILRP2다극륭항체효개분별위1∶1280000화1∶320000，Western blot증명해항체가이화면역원OCILRP2-Fc급NIH/3T3세포중표체적OCILRP2특이성결합。응용해항체검측OCILRP2재 DC 적표체발현 OCILRP2재성숙 DC 적표체명현고우불성숙 DC。결론：획득료진핵표체적중조단백OCILRP2-Fc화고효개적OCILRP2특이성항체，위진일보연구OCILRP2재면역응답중적공능전정료기출。
Objective:In order to get recombinant protein OCILRP 2-Fc and anti-OCILRP2 antibody for further study of OCILRP2.Methods:Eukaryotic expression vector pIg-CD5-OCILRP2 which fused with extracellular domain of OCILRP 2 and human IgG1 Fc fragment was constructed.G418 was used for stable expression cell strain after pIg-CD5-OCILRP2 transfected into CHO cells.Recombinant protein OCILRP 2-Fc purified from CHO cell supernatant was used to immunize rabbit and anti-OCILRP2 polyclonal antibody was purified from rabbit serum by using protein G column.Results: ELISA data showed that we got a high-titer anti-serum and anti-OCILRP2 antibody purified from the rabbit serum.Western blot indicated this antibody could specifically bind to OCILRP 2-Fc and OCILRP2 in NIH/3T3 lysate.OCILRP2 expression in murine bone marrow derived dendritic cells ( DC) was detected by this polyclonal antibody ,too.Compared to immature DC ,OCILRP2 expression was elevated in LPS induced-mature DC.Conclusion: This study has offered an available tool and provided a clue for further study of the roles of OCILRP 2 in immune response.