兔抗醛-酮还原酶家族1成员B10(AKR1B10)多克隆抗体的制备及鉴定

Preparation and identification of rabbit anti-AKR1B10 polyclonal antibody

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 rabbit anti-aldo-keto reductase family 1 member B10（AKR1B10） polyclonal antibody and identify its specificity.Methods AKR1B10 c DNA was amplified by reverse transcription PCR（RT-PCR） and inserted into prokaryotic expression vector p ET-15 b to form recombinant plasmid p ET-15b-AKR1B10.The recombinant plasmid p ET-15b-AKR1B10 was transformed into E.coli DH5α.Isopropylthio-β-D-galactoside（IPTG） was used to induce the expression of the recombinant protein His-tagged AKR1B10 in E.coli DH5α.The expression products from different clones of E.coli DH5αwere identified by SDS-PAGE.The positive bacteria were picked out and amplified.His-Tag-AKR1B10 protein was purified from the expression product of the positive clones by His-tagged purification column.The purified recombinant protein His-Tag-AKR1B10 was used to immunize New Zealand white rabbits.Antisera were acquired after two months.Anti-AKR1B10 polyclonal antibodies were purified by antigen purification column with AKR1B10 recombinant protein.Lastly,the purified polyclonal antibodies were identified by SDS-PAGE,ELISA,Western blotting.Results The recombinant plasmid p ET-15b-AKR1B10 was constructed successfully, and the recombinant protein His-Tag-AKR1B10 with high purity was acquired.The purified polyclonal antibodies were able to specifically recognize AKR1B10 protein.Conclusion The rabbit anti-AKR1B10 polyclonal antibodies is prepared successfully with high specificity.