摘要
禽流感(Avian Influenza, AI)是由正粘病毒科,流感病毒属,A型流感病毒引起的禽类感染和/或疾病的综合征。国际兽疫局(OIE)将禽流感定为A类传染病。H9N2亚型低致病性禽流感病毒在世界范围的禽群中广泛流行,感染鸡表现轻度的呼吸道症状和产蛋量下降,有报道称此病毒可以感染人引起流感样疾病。NS1是禽流感病毒唯一的非结构蛋白,在病毒复制及与宿主细胞蛋白的相互作用中发挥着重要作用,同时在鉴别诊断方面有较高的应用价值。本研究克隆并表达了一株H9N2亚型禽流感病毒陕西分离株(WN)的NS1基因,同时初步研究了NS1蛋白转染后对MDCK、Vero和293细胞凋亡的影响,为进一步研究NS1蛋白的功能及其与细胞相互作用的关系提供参考。
1.根据GenBank上已发表的H9N2亚型AIV的NS1基因序列,设计特异的引物,应用RT-PCR技术扩增H9N2亚型AIV陕西分离株(WN)的NS1基因,将其定向插入到原核表达载体pET-32a(+),构建重组质粒pET-NS1,经IPTG诱导,在大肠杆菌中成功表达了分子质量约44 KD的NS1重组蛋白,Western-blotting分析显示,NS1基因表达产物能够与H9N2亚型禽流感多抗血清IgG反应,具有较好的抗原性和特异性。
2.应用PCR技术扩增出H9N2亚型AIV陕西分离株(WN)的NS1基因,将其与真核表达载体pEGFP-C1相连,构建重组质粒pEGFP-NS1,采用LipofectamineTM 2000脂质体转染法将pEGFP-NS1导入293细胞,并应用Western-blotting检测,得到55 KD的特异性条带,与预期结果相符,表明转染细胞中NS1蛋白得到成功表达,且具有良好的免疫学活性。
3.利用脂质体介导法将构建好的重组质粒pEGFP-NS1瞬时转染MDCK、Vero和293细胞。显微镜下观察可见转染NS1基因的细胞与正常细胞相比出现皱缩、体积变小、变形,脱落等凋亡形态。流式细胞仪检测发现NS1蛋白对MDCK和293细胞有促早期凋亡的作用,但不能促进Vero细胞的早期凋亡,实验表明,NS1蛋白是否诱导细胞凋亡与细胞的种类有关。
Avian Influenza (AI) is an infection or syndrome caused by influenza virus type A, a member of Orthomyxoviridae. Highly pathogenic avian influenza (HPAI) is an infectious diseases by World Organization for Animal Health (OIE). Avian Influenza H9N2 virus continues to threaten the poultry population worldwide. H9N2 virus infections in chickens result in mild respiratory signs and egg production losses. The virus has been reported to cause flu-like disease in humans. The NS1 protein of AIV plays an important role in virus replication and the interaction between virus and cells. Meanwhile, because of its antigenicity and conservatism NS1 protein has very important uses in some fields such as differential diagnosis between vaccine immune and natural infection, epidemic survey and forecast of influenza. Therefore, The study used molecular biology methods to express NS1 gene in the prokaryotic and eucaryotic expression systems, which provided base materials to research the function of NS1 gene. This study including:
1. The NS1 gene of H9N2 AIV isolates (WN) was amplified by PCR with specific primers, and cloned into pMD18-T simple vector and sequenced. Then, the gene were inserted between BamHI and HindIII sites of pET-32a(+) after cleavage by corresponding enzymes. The recombinant plasmid was named pET-NS1. SDS-PAGE and western-blotting showed an expected protein of 44 ku in size was successfully expressed in E. coli BL21(DE3) induced by IPTG.
2. The NS1 gene of H9N2 AIV isolates (WN) was amplified by PCR with specific primers, and cloned into pMD18-T simple vector and sequenced. It was, then, subcloned into eukaryotic vector pEGFP-C1. The recombinant plasmid pEGFP-NS1 was transfected into 293 cells using LipofectamineTM 2000. The H9N2 AIV NS1 protein expression in 293 cells was detected by Western-blotting and The special band of NS1 protein expressed in 293 cells were identified. The result showed that the NS1 gene was successfully expressed in 293 cells and had very good antigenicity.
3. The recombinant plasmid pEGFP-NS1 were transfected into MDCK、Vero and 293 cells with the liposome mediated method.The typical morphological changes such as decreased cell size, cytomorphosis, cell shrinkage and detaching were observed by inverted microscope. Apoptosis was observed with flow cytometer. Results showed that NS1 protein could induce the early apoptosis of MDCK and 293 cells, but NS1 couldn’t induce the early aopotosis of Vero cells.
引文
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