H5N1亚型禽流感病毒NS1基因的克隆及原核表达

Cloning and Procaryotic Expression of NSl Gene of Avian Influenza Virus H5N1 Subtype

克隆、表达和纯化禽流感病毒H5N1 NS1基因序列,为筛选与NS1相互作用的宿主蛋白以及深入研究NS1蛋白的功能打下基础.根据GenBank中收录的H5N1亚型禽流感病毒NS1基因序列,设计并合成一对特异性引物,利用RT-PCR方法扩增AIV NS1基因,将酶切处理后的基因片段定向克隆到原核表达载体pET-28a载体上,经酶切分析及序列测定正确后,鉴定出NS1基因的阳性重组子.阳性质粒转化大肠杆菌BL21(DE3)感受态细胞,用1 mmol/L IPTG诱导表达,表达产物进行SDS-PAGE检测,获得预期蛋白的表达,通过Ni-NTA树脂蛋白纯化系统对NS1蛋白进行纯化.结果成功克隆H5N1亚型AIV的NSl基因,其核苷酸序列长度为678 bp,重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致,表达产物在上清及包涵体中均有表达.经纯化,目的蛋白纯度高达90%,成功表达纯化出28KD的NS1融合蛋白并鉴定其免疫学活性.成功克隆和表达了禽流感病毒H5N1 NS1基因序列,为进一步研究NS1蛋白的生物学功能奠定了坚实基础.

To clone,express and purify the NS1 Protein of avian influenza virus H5N1,it could be useful for screening host proteins that interact with the NS1 protein and the further study of NSl gene function.According to the sequence of nonstructural protein NSl gene in GenBank,a pair of specific primers was designed.AIV NS1 gene was amplified by RT-PCR,was inserted into pET-28a,the constructed recombinant plasmid was analyzed.Then The positive plasmids were transformed into E.coli BL2 1(DE3) competent cell and induced by l mmol/L IPTG.Analysis of SDS-PAGE showed that recombinant fusion protein was successfully expressed.The fusion protein was purified by Ni-NTA His Bind Resins.NSl gene of H5N1 subtype AIV was successfully cloned and sequenced.Sequence length of this gene was 678bp.SDS-PAGE result showed that the gene could express in the supernatant and inclusion bodies as same as we expect.After purification,the purity of product was above 90 %,its molecular weight was 28 kD.The NS1 protein of avian influenza virus H5N1 had been successful cloned and expressed.The study provides a sound basis for further study on biological function of NS1.