抗兔辅酶Ⅱ依赖性视黄醇脱氢／还原酶单克隆抗体的制备和鉴定

doi:10.3969/j.issn.1004-616x.2008.03.017

癌变·畸变·突变 ›› 2008, Vol. 20 ›› Issue (3): 235-238.doi: 10.3969/j.issn.1004-616x.2008.03.017

抗兔辅酶Ⅱ依赖性视黄醇脱氢／还原酶单克隆抗体的制备和鉴定

杜牡丹宋旭红刘戈飞梁　斌张巧霞李　蕊谢健平甘雪琼黄东阳

汕头大学医学院分子生物学中心，广东　汕头, 515041

收稿日期:2008-01-17 修回日期:2008-02-27 出版日期:2008-05-30 发布日期:2008-05-30
通讯作者: 黄东阳

Preparation and Characterizati-on of Monoclonal Antibody against Rabbit NADP(H)-dependent Retinol Dehydrogenase／Reductase

DU Mu-dan,SONG Xu-hong, LIU Ge-fei, LIANG Bin, ZHANG Qiao-xia, LI Rui, XIE Jian-ping,GAN Xue-qiong, HUANG Dong-yang

Center for Molecular Biology，Shantou University Medical College， Shantou 515041，Guangdong，China

Received:2008-01-17 Revised:2008-02-27 Online:2008-05-30 Published:2008-05-30
Contact: HUANG Dong-yang

摘要/Abstract

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NRDR mAb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Western blot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。Western blot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NRDR mAb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。

关键词: 兔辅酶Ⅱ依赖性视黄醇脱氢／还原酶, 单克隆抗体, 杂交瘤

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.

Key words: rabbit NRDR, monoclonal antibody(mAb), characterization, ratinoic acid

杜牡丹宋旭红刘戈飞梁　斌张巧霞李　蕊谢健平甘雪琼黄东阳. 抗兔辅酶Ⅱ依赖性视黄醇脱氢／还原酶单克隆抗体的制备和鉴定[J]. 癌变·畸变·突变, 2008, 20(3): 235-238.

DU Mu-dan, SONG Xu-hong, LIU Ge-fei, LIANG Bin, ZHANG Qiao-xia, LI Rui, XIE Jian-ping, GAN Xue-qiong, HUANG Dong-yang. Preparation and Characterizati-on of Monoclonal Antibody against Rabbit NADP(H)-dependent Retinol Dehydrogenase／Reductase[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2008, 20(3): 235-238.

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抗兔辅酶Ⅱ依赖性视黄醇脱氢／还原酶单克隆抗体的制备和鉴定

Preparation and Characterizati-on of Monoclonal Antibody against Rabbit NADP(H)-dependent Retinol Dehydrogenase／Reductase

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