黄鳝醛酮还原酶基因的重组表达及多抗制备

Recombinant Expression and Polyclonal Antibody Preparation of an Aldo-keto Reductase from Swamp Eel

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。

A pair of gene-speeific primers were synthesized to isolate the sequence of AKR of swamp eel Monopterus albus from the liver cDNAs to recombinant expression of swamp eel aldo-keto reductase in Escherichia coli and to prepare polyclonal antibody against it. The purified products were subcloned into expression vector pET-28a（＋）. Subsequently, the confirmed plasmid pET/Eakr was transformed into BL21（DE3） competent cells. The positive clones were selected and were induced by IPTG. Then, the re- combinant protein was purified by one step affinity chromatography with a Ni-NTA agarose column. New Zealand white rabbits were immunized with the purified protein to generate polyclonal antibodies. West- ern-blotting and immunohistochemistry assay were performed to detect the specificity of the antibody. In- direct ELISA was used to examine the titer of the antibody. The results showed that the antiserum specif- ically recognized Eakr in four different tissues of swamp eel and reacted with homologous proteins in grass carp Ctenopharyngodon idella, yellow catfish Pelteobagrus fulvidraco, bluntnose black bream Megalo- brama arnblycephala , snakehead Channa argus and crucian carp Carassius auratus. Immunohistochemis- try assay revealed that the antiserum specifically reacted to Eakr in kidney, liver and intestine with anti- body titer of about 1-6400.