CAJ | 학술논문

目的:利用基工程方法构建无标签人性脂联素球状结构(gAd)基的核表达载体,并对重组蛋白进行诱导表达、纯化及鉴定.方法:从正常人脂肪组织里提取总 RNA,反转录合成 cDNA,经 PCR 扩增、酶切后连入pET-22b(+)载体构建重组质粒 pET-22b(+)-gAd,转化大肠杆菌 BL21(DE3)感受态细胞,经低温、低浓度 IPTG 诱导使其可溶性表达,采用硫酸铵沉淀、凝胶过滤层析和离子交换层析三步分离纯化,得到不带任何标签的人性gAd;运用 SDS-PAGE、Western 印迹、HPLC 对重组蛋白进行鉴定,通过对 AMP 激活的蛋白激酶(AMPK)的磷酸化水平检测纯化蛋白的生物学活性.结果:构建了核表达载体 pET-22b(+)-gAd,实现了人性 gAd 在核细胞中的可溶性表达,纯化的蛋白经 SDS-PAGE 和 Western 印迹分析证实为 gAd,HPLC 分析蛋白纯度达到95%以上;通过对 AMPK磷酸化水平的检测,证明纯化的 gAd 具有高生物学活性.结论:重组表达和纯化了无标签、高生物学活性的人性脂联素球状结构,为其进一步的理论研究、生产开发奠定了基础.
목적:이용기공정방법구건무표첨인성지련소구상결구(gAd)기적핵표체재체,병대중조단백진행유도표체、순화급감정.방법:종정상인지방조직리제취총 RNA,반전록합성 cDNA,경 PCR 확증、매절후련입pET-22b(+)재체구건중조질립 pET-22b(+)-gAd,전화대장간균 BL21(DE3)감수태세포,경저온、저농도 IPTG 유도사기가용성표체,채용류산안침정、응효과려층석화리자교환층석삼보분리순화,득도불대임하표첨적인성gAd;운용 SDS-PAGE、Western 인적、HPLC 대중조단백진행감정,통과대 AMP 격활적단백격매(AMPK)적린산화수평검측순화단백적생물학활성.결과:구건료핵표체재체 pET-22b(+)-gAd,실현료인성 gAd 재핵세포중적가용성표체,순화적단백경 SDS-PAGE 화 Western 인적분석증실위 gAd,HPLC 분석단백순도체도95%이상;통과대 AMPK린산화수평적검측,증명순화적 gAd 구유고생물학활성.결론:중조표체화순화료무표첨、고생물학활성적인성지련소구상결구,위기진일보적이론연구、생산개발전정료기출.
Objective: To construct prokaryotic expression vector pET-22b(+)-gAd of human globular domain of adiponectin(gAd) gene, express and purify no-tagged recombinant human gAd protein. Methods: Total RNA was extracted from human adipose tissue. The gAd coding sequence was subcloned into the pET-22b(+) after ampli?fied by PCR. The recombinant plasmids were transformed into E.coli BL21(DE3), and the gAd was soluble ex?pressed in suitable temperature, by IPTG induction. The expressed gAd was purified by three-step procedure: am?monium sulfate precipitation, gel filtration chromatography and anion exchange chromatography. SDS-PAGE, West?ern blot, HPLC and the ability to induce the phosphorylation of AMPK in HUVEC were used for identification, pu?rity and biological activity assay. Results: The human gAd coding sequence was cloned into pET-22b(+) vector. After expression and purification, the purity of gAd was more than 95%. The recombinant human gAd significantly induced the phosphorylation of AMPK in HUVEC. Conclusion: The no-tagged recombinant human gAd was suc?cessfully expressed and purified by prokaryotic expression system and three-step purification procedure with high purity and biological activity..