【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。更多还原

【Abstract】 Objective To express and purify the NS1 protein of human influenza A virus subtype H1N1. Methods NS1 gene was amplified from human influenza A virus strain A / PR / 8 / 34 (H1N1)by RT-PCR and cloned into prokaryotic expression vector pTXB1. The constructed recombinant plasmid pTXB1 / NS1 was identified by restriction analysis and transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed protein was analyzed by SDS-PAGE, then purified by chitin affin-ity chromatography and determined for relative molecular mass by MALDI-TOF, SA. Results Restriction map showed that the gene fragment at a full-length of 690 bp was inserted into the constructed recombinant plasmid pTXB1 / NS1. After induction of recombi-nant E. coli with 1.0 mmol / L IPTG at 37℃ for 4 h, the target protein reached the maximum expression level, which contained more than 50% of total somatic protein. The target protein was expressed in both ultrasonic supernatant and precipitate of recombinant E. coli, and reached a purity of more than 95% after purification. The relative molecular mass of expressed product was about 26 000. Conclusion The NS1 protein of human influenza A virus subtype H1N1 was successfully expressed and purified, which laid a foun-dation of further study on function of NS1 protein and NS1-based antiviral drugs.更多还原