人中性粒细胞明胶酶相关脂质运载蛋白的原核表达和活性鉴定

发布时间：2018-09-14 19:07

【摘要】：目的对人源中性粒细胞明胶酶相关脂质运载蛋白(NGAL)进行原核表达、纯化,并验证其免疫学活性。方法通过化学合成法合成人源NGAL成熟肽c DNA序列,构建原核表达载体p Cold-NGAL,转化至E.coli BL21(DE3)plys S中。摸索p Cold-NGAL最佳诱导条件,表达并纯化重组蛋白;使用SDS-PAGE、Western blot法和免疫比浊法鉴定纯化后的重组NGAL蛋白的相对分子质量(Mr)和以及与NGAL单克隆抗体(m Ab)的结合活性。结果经双酶切和测序验证,原核表达载体p Cold-NGAL成功构建。优化诱导条件后,重组NGAL蛋白在E.coli BL21(DE3)plys S中以可溶性蛋白形式表达,Mr约为25 000。通过Ni2+亲和层析纯化获得高纯度重组NGAL蛋白,SDS-PAGE分析其纯度高于98.0%。经Western blot法和免疫比浊法鉴定,重组NGAL蛋白可与NGAL m Ab特异性结合。结论成功表达和纯化了重组NGAL蛋白,并具有免疫学活性。
[Abstract]:Objective to express and purify the human neutrophil gelatinase-associated lipid carrier protein (NGAL) in prokaryotic expression, and to verify its immunological activity. Methods the human NGAL mature peptide c DNA sequence was synthesized by chemical synthesis, and the prokaryotic expression vector p Cold-NGAL, was transformed into E.coli BL21 (DE3) plys S). The recombinant protein was expressed and purified under the optimal induction conditions of p Cold-NGAL, and the relative molecular weight (Mr) and the binding activity of the purified recombinant NGAL protein to NGAL monoclonal antibody (m Ab) were identified by SDS-PAGE,Western blot and immunoturbidimetry. Results the prokaryotic expression vector p Cold-NGAL was successfully constructed by double enzyme digestion and sequencing. After optimizing the induction conditions, the recombinant NGAL protein expressed in E.coli BL21 (DE3) plys S) as a soluble protein was about 25 000. High purity recombinant NGAL protein was purified by Ni2 affinity chromatography and its purity was higher than 98.0 by SDS-PAGE. The recombinant NGAL protein was identified by Western blot and immunoturbidimetry. The recombinant NGAL protein could specifically bind to NGAL m Ab. Conclusion the recombinant NGAL protein was successfully expressed and purified and has immunological activity.
【作者单位】：广州万孚生物技术股份有限公司自检型快速诊断国家地方联合工程实验室;
【基金】：国家高技术研究发展计划(863)(2011AA02A103) 广东省战略新兴产业核心技术攻关项目(2012A080800007)
【分类号】：R392

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