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目的制备兔抗醛-酮还原酶家族1成员B10(AKR1B10)多克隆抗体,并鉴定其特异性。方法应用逆转录PCR法扩增AKR1B10全基因,将扩增产物连接到原核表达载体p ET-15b上,构建重组质粒p ET-15b-AKR1B10,将其转化至大肠杆菌E.coli DH5α中。用异丙基硫代β-D-半乳糖苷(IPTG)诱导其表达,表达产物经SDS-PAGE分析鉴定出阳性者进行克隆,扩大培养,提取重组蛋白,用His-tag纯化柱纯化重组蛋白。将纯化的重组蛋白皮下注射到新西兰白兔,加强免疫得到抗血清。用AKR1B10重组蛋白制备抗体纯化柱,从抗血清中纯化出抗AKR1B10多克隆抗体,并进行SDS-PAGE、ELISA、Western blot法鉴定。结果成功构建了重组质粒p ET-15b-AKR1B10,并获得高纯度的重组蛋白His-Tag-AKR1B10,制备的多克隆抗体相对特异性地识别AKR1B10。结论兔抗AKR1B10多克隆抗体制备成功,特异性较好。
Objective To prepare polyclonal antibody against rabbit aldehyde - ketoreductase family 1 B10 (AKR1B10) and identify its specificity. Methods AKR1B10 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The amplified product was ligated into prokaryotic expression vector p ET-15b. The recombinant plasmid p ET-15b-AKR1B10 was constructed and transformed into E.coli DH5α. The recombinant protein was induced by isopropyl thio-β-D-galactoside (IPTG). The positive clones were identified by SDS-PAGE and cloned. The recombinant protein was amplified and purified by His-tag purification column . The purified recombinant protein was injected subcutaneously into New Zealand White rabbits to enhance immunity to obtain antiserum. AKR1B10 recombinant protein was used to prepare antibody purification column. The anti-AKR1B10 polyclonal antibody was purified from antiserum and identified by SDS-PAGE, ELISA and Western blot. Results The recombinant plasmid p ET-15b-AKR1B10 was successfully constructed and the recombinant protein His-Tag-AKR1B10 was obtained. The prepared polyclonal antibody recognized AKR1B10 relatively specifically. Conclusion The rabbit anti-AKR1B10 polyclonal antibody was successfully prepared and its specificity was good.