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为了解H5N1亚型流感病毒株的ns1基因特性及其规模制备NS1蛋白,首先将病毒在鸡胚中传代,从收获的尿囊液中提取RNA,采用RT-PCR技术扩增流感病毒全长ns基因。测序显示H5N1亚型流感病毒NS1cDNA全长678bp,编码225个氨基酸。BLAST分析表明,Qa/ST/852/01(H5N1)病毒株ns1基因与近年来从华南地区分离的禽H5N1毒株的ns1基因有很高的同源性。之后采用PCR方法扩增ns1基因的cDNA片段,将其克隆到pGEX-4T-3载体中,与谷胱甘肽巯基转移酶(GST)基因融合,构建重组质粒pGEX-4T-3/NS1cDNA,转化大肠杆菌BL21(DE3)并进行诱导表达。SDS-PAGE和凝胶扫描分析,GST-NS1融合蛋白在大肠杆菌中获得了高效表达,并且以可溶形式存在,重组融合蛋白的表达量占菌体总蛋白的28.5%,表达产物经亲和层析纯化后蛋白质纯度达96%以上。经免疫印记证实重组融合蛋白可以被GST特异性抗体所识别。该表达载体的构建为获得大量NS1蛋白进行功能研究及抗体制备提供了基础。
In order to understand the characteristics and scale of ns1 gene of H5N1 subtype influenza virus strain, NS1 protein was first prepared. The virus was passaged in chicken embryos and RNA was extracted from the allantoic fluid harvested. RT-PCR was used to amplify the full length of influenza virus gene. Sequencing showed that the H5N1 subtype influenza virus NS1 cDNA was 678bp in length and encoded 225 amino acids. BLAST analysis showed that the ns1 gene of Qa / ST / 852/01 (H5N1) strain has high homology with the ns1 gene of avian H5N1 strain isolated in South China in recent years. The cDNA fragment of ns1 gene was amplified by PCR, cloned into pGEX-4T-3 vector and fused with glutathione S-transferase (GST) gene to construct recombinant plasmid pGEX-4T-3 / NS1 cDNA E. coli BL21 (DE3) and induced expression. SDS-PAGE and gel scanning analysis, the GST-NS1 fusion protein was highly expressed in E. coli and existed in a soluble form. The recombinant fusion protein accounted for 28.5% of the total bacterial protein. The expression of the fusion protein was confirmed by affinity After purification, the purity of the protein is more than 96%. Western blotting confirmed that the recombinant fusion protein was recognized by GST-specific antibody. The construction of this expression vector provided the basis for obtaining a large number of NS1 proteins for functional studies and antibody preparation.