黄鳝醛酮还原酶基因的重组表达及多抗制备

发布时间：2018-06-18 03:47

本文选题：黄鳝 + 醛酮还原酶　；参考：《水产科学》2017年06期

【摘要】：为实现原核表达黄鳝醛酮还原酶基因并制备其多克隆抗体,笔者利用基因特异性引物自黄鳝肝脏cDNA中扩增黄鳝醛酮还原酶全长序列,将之克隆至原核表达载体pET-28a(+)中,构建了重组表达载体;经测序验证后的重组质粒转化大肠杆菌BL21(DE3)后进行IPTG诱导。利用Ni离子亲和柱纯化了重组蛋白,通过多次免疫新西兰兔制备了多克隆抗体;采用Western blot和免疫组化法鉴定兔抗醛酮还原酶多克隆抗体的特异性,用间接ELISA技术检测多抗的效价。结果发现成功构建pET/Eakr原核表达载体,并实现了重组蛋白的融合表达和纯化;Western blot检测表明制备的兔多克隆抗体不仅能特异性地识别来源于黄鳝4种组织的醛酮还原酶蛋白而且还可识别来源于黄颡鱼、团头鲂、乌鳢、鲫鱼和草鱼的同源蛋白。免疫组化结果提示该多抗可特异性识别黄鳝小肠、肝胰组织和脾脏的醛酮还原酶蛋白。制备的多抗效价达1∶6400。
[Abstract]:In order to express aldosterone reductase gene in prokaryotic expression and prepare polyclonal antibody, the full-length aldosterone reductase was amplified from the liver cDNA of Monopterus Albus by gene specific primers and cloned into the prokaryotic expression vector pET-28a (). The recombinant expression vector was constructed and transformed into E. coli BL21DE3 by sequencing. The recombinant protein was purified by Ni ion affinity column, polyclonal antibodies were prepared by immunizing New Zealand rabbits, and the specificity of polyclonal antibodies against aldehyde-ketone reductase was identified by Western blot and immunohistochemistry. The titers of polyclonal antibodies were detected by indirect Elisa. The results showed that pET-Eakr prokaryotic expression vector was successfully constructed. The fusion expression of the recombinant protein and the purification of the recombinant protein by Western blot showed that the rabbit polyclonal antibody could recognize not only aldehydes and ketone reductase proteins from four tissues of Monopterus Albus, but also from Pelteobagrus fulvidraco, bream, snakehead Argus. The homologous proteins of crucian carp and grass carp. The results of immunohistochemistry indicated that the polyclonal antibody could specifically identify aldehydes and ketone reductase proteins in small intestine, liver, pancreas and spleen of Monopterus Albus. The titer of the prepared polyclonal antibody is 1: 6400.
【作者单位】：长江大学生命科学学院;武汉大学生命科学学院;
【基金】：国家大学生创新创业训练计划项目(201510489007) 湿地生态与农业利用教育部工程研究中心开放课题项目(KF2015016)
【分类号】：S917.4

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