人甲型流感病毒H1N1亚型NS1蛋白的原核表达及纯化

发布时间：2018-08-06 15:38

【摘要】：目的表达并纯化人甲型流感病毒H1N1亚型NS1蛋白。方法用RT-PCR法从人甲型流感病毒株A/PR/8/34(H1N1)中扩增NS1基因,克隆入原核表达载体pTXB1,构建重组原核表达质粒pTXB1/NS1,经酶切鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达形式和表达量。经几丁质柱亲和层析纯化表达蛋白,串联飞行时间质谱仪检测其相对分子质量。结果所构建的重组表达质粒pTXB1/NS1序列完整,插入的基因片段全长690bp。以1.0mmol/LIPTG37℃诱导4h,重组蛋白表达量最高,占菌体总蛋白的50%以上。破菌上清及沉淀中均有目的蛋白表达。纯化的NS1蛋白纯度达95%以上,相对分子质量约为26000。结论已成功表达并纯化了人甲型流感病毒H1N1亚型NS1蛋白,为其进一步的研究奠定了基础。
[Abstract]:Objective to express and purify human influenza A virus H1N1 subtype NS1 protein. Methods the NS1 gene was amplified by RT-PCR from human influenza A virus strain A/PR/8/34 (H1N1) and cloned into the prokaryotic expression vector pTXB1. The recombinant prokaryotic expression plasmid pTXB1 / NS1 was constructed. The recombinant plasmid pTXB1 / NS1 was identified by restriction endonuclease digestion, and then transformed into E. coli BL21 (DE3) BL21 (DE3) to induce expression by SDS-PAGE. The expressed protein was purified by chitosan column affinity chromatography and its molecular weight was determined by tandem time of flight mass spectrometer. Results the pTXB1/NS1 sequence of the recombinant expression plasmid was complete and the inserted gene fragment was 690 BP. When induced at 1.0mmol/LIPTG37 鈩，

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