【摘要】 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。更多还原

【Abstract】 To understand the characteristics of the ns1 gene of Avian influenza A virus H5N1 and to prepare NS1 protein in scale, viruses were cultivated in embryonated hen eggs and virion RNA was extracted from allantoic fluid. The complete ns gene was amplified by RT-PCR and analyzed. Sequence analysis demonstrated that the NS1 cDNA contained one open reading frame of 678 bp, which encoded a polypeptide of 225 amino acids. The ns1 gene of Qa/ST/852/01 (H5N1) isolate was closer to that of some H5N1 strains prevalent in southern China in recent years, the homologies of nucleotide and amino acid sequences were 99.0%～97.5% and 99.1%～97.8%, respectively. Subsequently, ns1 cDNA was obtained by PCR and cloned into pGEX-4T-3 vector to construct recombinant plasmid pGEX-4T-3/NS1 cDNA, and expressed in BL21(DE3) E.coli strain. SDS-PAGE and scan analysis showed recombinant fusion protein GST-NS1 was expressed in soluble form, and fusion protein amounted to 28.5% of the total soluble bacterial protein. The protein was purified by affinity chromatography and the purity was greater than 96%. Western blot analysis of recombinant fusion protein confirmed it could be specifically recognized by antibody of GST. Construction of the prokaryotic expression vector provided a foundation for further studying the function of NS1 protein and preparation of NS1 antibody.更多还原