甲型H1N1流感病毒NS1蛋白核仁定位的研究

为研究2009年甲型H1N1流感病毒的NS1蛋白的核仁定位情况,采用RT-PCR对其NS1基因进行了扩增,将其克隆至PEGX-KG载体,构建重组质粒KG-NS1,转化大肠杆菌BL21,IPTG诱导表达重组蛋白.然后采用GST柱亲和层析方法纯化NS1重组蛋白,免疫家兔来制备多抗,Western blot检测抗体.通过间接免疫荧光对表达不同长度NS1 (NS1-219、NS1-230、NS1-237)的3种重组流感病毒进行了核仁定位的研究,3种重组毒的NS1蛋白存在于细胞核和细胞质,但都不能定位于核仁,说明NS1蛋白的截短与否并不影响其核仁定位,其生物学意义有待于进一步研究.

LOCALIZATION OF NS1 PROTEIN OF H1N1 INFLUENZA A VIRUS IN NUCLEI

In order to investigate the distribution of NS1 protein of H1N1 Influenza A virus of 2009 in nuclei,the NS1 gene was amplified in RT-PCR and cloned into PEGX-KG vector.The resulting plasmid KG-NS1 was then transformed into E.coli BL-21 and the recombinant protein NS1 was expressed with induction of IPTG.NS1 protein was purified using GST affinity chromatography and injected into rabbits for the preparation of anti-NS1 polyclonal antibodies.The production of rabbit anti-NS1 antibodies was determined in Western blotting.Then,the nucleic localization of NS1 protein with three different lengths(219,230 and 237 amino acids) was examined in indirect immunofluorescence using rabbit antibodies.The NS1 protein with three different lengths accumulated in the nucleus and cytoplasm but not in the nucleolus.The results showed that truncation of NS1 protein did not affect its nucleolus localization.