【摘要】 背景与目的:制备抗兔辅酶Ⅱ依赖性视黄醇脱氢/还原酶[NADP(H)-dependent retinol dehydrogenase/reductase,NRDR]的单克隆抗体(mAb),并鉴定其特性。材料与方法:以基因工程重组兔NRDR为抗原免疫BALB/c小鼠,用杂交瘤技术建立稳定分泌兔NRDRmAb的细胞株。以间接ELISA法筛选阳性克隆、鉴定Ig亚类、细胞培养上清及腹水效价,同时采用Westernblot方法检测mAb的特异性。结果:获得3株可分泌特异性mAb的杂交瘤细胞(NR1、NR2和NR5)。其抗体亚类均为IgG1,细胞培养上清效价依次为1∶20、1∶40和1∶20,腹水效价分别为1∶106、1∶107和1∶106。Westernblot结果显示NR1、NR2和NR5抗体均能有效识别兔肝组织中的NRDR及重组表达的兔NRDR。结论:成功地建立了稳定分泌兔NRDRmAb的杂交瘤细胞株,为NRDR生物学功能的进一步研究打下了基础。更多还原

【Abstract】 BACKGROUND AND AIM: To prepare specific monoclonal antibody (mAb) against rabbit NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) for further exploration of its structure and function. MATERIALS AND METHODS: The BALB/c mice were immunized with the recombinant rabbit NRDR expressed by genetic engineering. Splenocytes of immunized mice were collected and fused with the mouse myeloma cell line NS-1 cells. Hybridomas that secreted rabbit NRDR mAb were cloned with limited dilution method. Characteristics of mAb(Ig subclasses,titers and specificities) were identified and determined by indirect ELISA and Western blot. RESULTS: From over 80 positive hybridomas which secreted anti-rabbit-NRDR mAbs,three clones of hybridoma were obtained, and designated as NR1、NR2 and NR5. They were all of IgG1 subclass. The cell culture supernatant titers of NR1,NR2 and NR5 were1∶20,1∶40 and 1∶20,respectively. Ascite titers of NR1,NR2 and NR5 mAb were 1∶106, 1∶107 and 1∶106, respectively. Western blot analysis showed that mAbs had specific binding abilities with NRDR in rabbit liver and recombinant rabbit NRDR. CONCLUSION:The hybridomas secreting mAbs to rabbit NRDR were established successfully and primarily identified,which could lay the basis for further research on the biological structure and function of NRDR.更多还原