CAJ | 학술논문

目的获得纯度较高的WISP-l蛋白结构域,为进一步升发靶向WISP-1蛋白的药物奠定基础.方法将编码WISP-l蛋白4个结构域的基因克隆到pFN19A蛋白表达载体中,4个重组质粒分别在大肠埃希菌中诱导表达对应的WISP-1蛋白结构域.用Halo树脂共价层析法纯化目的蛋白.结果获得了4个重组质粒pFN19A Halo-IGFBP/VWFC/TSP/CTCK.并在KRX敏感菌株中诱导表达目的蛋白,通过优化诱导条件获得4个高表达结构域蛋白.SDS-PAGE和Western印迹特异性条带鉴定纯化蛋白的质量大小与预期一致.经BCA蛋白浓度监测,E2中重组蛋白浓度可达到500～ 1500 mg/L.结论通过Halo树脂共价层析法,获得了纯度较高的WISP-14个结构域蛋白,为研究WISP-1蛋白在肝癌等肿瘤发生发展过程中的关键功能结构域打下基础.
목적 획득순도교고적WISP-l단백결구역,위진일보승발파향WISP-1단백적약물전정기출.방법 장편마WISP-l단백4개결구역적기인극륭도pFN19A단백표체재체중,4개중조질립분별재대장애희균중유도표체대응적WISP-1단백결구역.용Halo수지공개층석법순화목적단백.결과 획득료4개중조질립pFN19A Halo-IGFBP/VWFC/TSP/CTCK.병재KRX민감균주중유도표체목적단백,통과우화유도조건획득4개고표체결구역단백.SDS-PAGE화Western인적특이성조대감정순화단백적질량대소여예기일치.경BCA단백농도감측,E2중중조단백농도가체도500～ 1500 mg/L.결론 통과Halo수지공개층석법,획득료순도교고적WISP-14개결구역단백,위연구WISP-1단백재간암등종류발생발전과정중적관건공능결구역타하기출.
Objective To achieve the pure recombinant proteins of WISP-1 domains and lay a foundation of further study on the targeting drug of protein WISP-1.Methods The genes which encoded four domains of WISP-1 protein with a halo tag at N-terminus (pFN19A vector)were cloned and the corresponding proteins were expressed in E.coli.The recombinant proteins were covalently immobilized onto HaloLink resins and purified by enzyme cleavage.Results The recombinant plasmids,namely pFN19A Halo-IGFBP,Halo-VWTC,Halo-TSP,Halo-CTCK,were transformed into the KRX competent cells to express proteins respectively.The induction conditions were optimized for high expression of the target proteins.Purified domain proteins were validated by SDS-PAGE and Western-blot,the results showed that purified domains had the expected molecular weights.The concentration of recombinant protein in E2 could reach 500-1500 mg/L by BCA protein concentration monitoring.Conclusions Four domains of WISP-1 protein are successfully purified by chromatography.These proteins are very important to better understand the key functional domains of WISP-1 in the development of the malignant tumors,such as hepatocellular carcinoma.