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目的:制备抗醛糖还原酶(AR)的单克隆抗体(mAb),并与本室制备的抗醛糖还原酶相似蛋白(ARL-1)mAb进行比较。方法:经RT-PCR获得AR基因,将基因插入pGEX-4T-1(His)6C中,构建重组质粒pGEX-4T-1(His)6C-AR,以重组质粒转化E.coliRosetta诱导表达GST-AR蛋白。以纯化的GST-AR蛋白免疫BALB/c小鼠,采用杂交瘤技术制备mAb。应用间接ELISA和Western blot方法对mAb进行筛选和鉴定。使用Clustalx和Antheprot软件,比较AR与ARL-1的同源性,表达GST-dAR[80~142氨基酸(aa)],与ARL-1差异较大;并分析AR的抗原性,表达GST-dA1(1~79aa)、GST-dA2(80~99aa)、GST-dA3(111~142aa)、GST-dA4(143~316aa)。利用AR全长及截短蛋白,采用Western blot分析制备的抗AR mAb识别AR抗原的部位。结果:获得3株稳定分泌抗AR mAb的杂交瘤细胞系ARB3、AR7B3G4和ARF10。3株抗GST-AR的mAb均为IgG1(κ型),腹水mAb效价为1∶4×105,细胞培养上清mAb效价为1∶1×104,3株mAb均可与胎盘组织中的AR蛋白起反应,而与GST-ARL-1和GST蛋白无交叉反应。它们分别为抗GST-dA1、GST-dA3和GST-dA4蛋白的mAb。结论:成功地制备了3株特异性抗AR mAb,可分别识别AR的1~79、111~142、143~316位氨基酸。将它们与抗ARL-1mAb联合应用,将有助于进一步研究AR与ARL-1蛋白的功能,并为深入探讨AR、ARL-1与相关疾病的关系及进行大规模的流行病学调查提供了有力的工具。
OBJECTIVE: To prepare monoclonal antibodies (mAbs) against aldose reductase (AR) and to compare with anti-aldose reductase similar protein (ARL-1) mAb prepared in our laboratory. METHODS: The AR gene was obtained by RT-PCR and inserted into pGEX-4T-1 (His) 6C to construct recombinant plasmid pGEX-4T-1 (His) 6C-AR. The recombinant plasmid was transformed into E.coli Rosetta to induce the expression of GST- AR protein. BALB / c mice were immunized with purified GST-AR protein and mAbs were prepared using hybridoma technology. The mAbs were screened and identified by indirect ELISA and Western blot. The Clustalx and Antheprot softwares were used to compare the homology of AR with ARL-1 and express GST-dAR [80-142 amino acids (aa)], which was significantly different from that of ARL-1. The antigenicity of AR was analyzed and the expression of GST-dA1 (1 ~ 79aa), GST-dA2 (80 ~ 99aa), GST-dA3 (111 ~ 142aa) and GST-dA4 (143 ~ 316aa). Using AR full-length and truncated proteins, the anti-AR mAbs identified by Western blot were used to identify AR antigen sites. Results: The mAbs of ARB3, AR7B3G4 and ARF10.3 which were obtained from 3 hybridoma cell lines secreting anti-AR mAb were all IgG1 (κ-type), the titer of ascites mAb was 1 × 4 × 105, Supernatant mAb titers of 1: 1 × 104, 3 strains of mAbs can react with AR protein in placenta tissue without cross-reaction with GST-ARL-1 and GST protein. They are mAbs against GST-dA1, GST-dA3 and GST-dA4 proteins, respectively. CONCLUSION: Three anti-AR mAbs were successfully prepared, which can recognize amino acids 1 to 79, 111 to 142 and 143 to 316 of AR, respectively. Their combination with anti-ARL-1 mAb will be helpful to further study the function of AR and ARL-1 protein, and provides a further study on the relationship between AR, ARL-1 and related diseases and large-scale epidemiological investigation Powerful tool.