人5-脂氧合酶重组蛋白的纯化及其多克隆抗体的制备  

Purification of Recombinant Human 5-Lipoxygenase Protein and Preparation of Its Polyclonal Antibody

作　　者：王晓玲 郭明飞 史铁伟 高丽枫 王文涛 WANG Xiaoling;GUO Mingfei;SHI Tiewei(.Department of Pathogenic Biology and Immunology,College of Basic Medical Sciences,Chifeng University,Chifeng City,Inner Mongolia 024000)

机构地区：[1]赤峰学院基础医学院病原生物学与免疫学教研室,内蒙古自治区赤峰市024000

出　　处：《医学理论与实践》2024年第11期1805-1807,1817,共4页The Journal of Medical Theory and Practice

基　　金：内蒙古自治区自然科学基金(2020BS08005)。

摘　　要：目的:原核表达并纯化人5-脂氧合酶(5LO)△112蛋白,将其免疫新西兰大白兔制备5-LO多克隆抗体。方法:使用DNAMAN设计重组引物,PCR扩增N端缺失112个氨基酸的5-LO截短DNA片段,将其重组到KpnⅠ和EcoRⅠ酶切的pET30a(+)质粒中,重组质粒pET30a-5LO△112转化大肠杆菌Rosetta(DE3),培养至OD_(600)为0.4时加入IPTG以诱导5LO△112重组蛋白表达,目的蛋白经纯化后免疫新西兰大白兔制备其多克隆抗体。结果:使用BugBuster蛋白提取试剂裂解细菌并获得纯度较高的包涵体,包涵体用含6mol/L盐酸胍的溶液变性溶解,再经Ni-NTA agarose亲和纯化得到高纯度的5LO△112重组蛋白。用该蛋白免疫新西兰大白兔获得5-LO的特异性抗体,该抗体的效价为1∶51200。结论:使用6mol/L盐酸胍溶解包涵体蛋白结合Ni-NTA agarose亲和纯化的方法成功获得了纯度较高的5LO△112重组蛋白,并用此蛋白制备了高效价的多克隆抗体。Objective:We purified the 5LO△112 inclusion body protein expressed in Escherichia coli and prepared a polyclonal antibody against 5-LO by immunizing New Zealand white rabbit with it.Methods:We designed primers using DNAMAN software,amplified 5LO△112 DNA fragment by PCR,and cloned it into pET30a(+)vector digested by KpnⅠand EcoRⅠ.Transform the recombinant plasmid pET30a-5LO△112 into E.coli Rosetta(DE3),and add IPTG to induce the expression of 5LO△112 recombinant protein when E.coli is cultured to an OD _(600) of 0.4.The target protein was purified and used to immunize New Zealand white rabbits to generate polyclonal antibodies.Results:We used BugBuster protein extraction reagent to lyse bacteria and obtain high-purity inclusion bodies.The inclusion body was dissolved in a solution containing 6mol/L guanidine hydrochloride,and then purified by Ni-NTA agarose to obtain high-purity 5LO△112 recombinant protein.Immunize New Zealand white rabbit with this protein to obtain a specific antibody against 5-LO,with a titer of 1∶51200.Conclusion:We successfully purified high-purity 5LO△112 recombinant protein using 6mol/L guanidine hydrochloride and Ni NTA agarose,and prepared high titer rabbit derived polyclonal antibodies using this protein.

关 键 词：人5-脂氧合酶 包涵体蛋白 亲和纯化 多克隆抗体制备 

分 类 号：R-33[医药卫生]