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目的:对2013年3月发生的感染人的新型H7N9亚型禽流感病毒的非结构蛋白1(NS1)基因序列进行同源性分析,构建NS1重组质粒并表达。方法:从GenBank获得2006~2013年不同来源的H7N9亚型病毒NS1序列,并进行同源性比较;利用PCR方法从H7N9亚型禽流感病毒株A/Shanghai/4664T/2013(H7N9)基因组cDNA中扩增得到全长NS1基因,并将该片段定向克隆到原核表达载体pET28a上,构建重组质粒pET28a-NS1,经酶切鉴定,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞后,IPTG诱导表达,且进行Western印迹分析。结果:经序列分析,2013年暴发的H7N9型禽流感病毒的NS1基因核苷酸序列同源性为95%~100%,与之前暴发的H7N9型流感病毒NS1基因序列的同源性为86.4%~90.7%,表明2次暴发的该型流感分离株属于不同的进化分支;PCR扩增得到约680 bp的NS1基因序列,所克隆的NS1基因在原核细胞中的表达产物主要以包涵体形式存在,SDS-PAGE检测结果表明重组蛋白相对分子质量为25×103,Western印迹分析证实表达产物为H7N9禽流感病毒NS1蛋白。结论:为进一步研究H7N9亚型流感病毒NS1蛋白功能及基于NS1蛋白的抗病毒药物奠定了基础。
OBJECTIVE: To analyze the homology of the non-structural protein 1 (NS1) gene sequence of the new H7N9 subtype of avian influenza virus that infected humans in March 2013 to construct and express NS1 recombinant plasmid. Methods: NS1 sequences of H7N9 subtype virus from different origins from 2006 to 2013 were obtained from GenBank and their homologies were compared. Genomic DNA of H7N9 subtype avian influenza A / Shanghai / 4664T / 2013 (H7N9) The full-length NS1 gene was amplified and cloned into the prokaryotic expression vector pET28a. The recombinant plasmid pET28a-NS1 was constructed and identified by restriction enzyme digestion. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and induced by IPTG Expression, and Western blot analysis. Results: The nucleotide sequence homology of NS1 gene of H7N9 avian influenza virus outbreak in 2013 was 95% -100%, which was 86.4% compared with the NS1 gene sequence of H7N9 influenza virus previously outbreak. ~ 90.7%, indicating that the two outbreaks of the influenza strains belong to different evolutionary branches; PCR amplified about 680 bp of the NS1 gene sequence, the cloned NS1 gene in prokaryotic expression mainly in the form of inclusion bodies exist The results of SDS-PAGE showed that the relative molecular weight of the recombinant protein was 25 × 103. Western blot analysis confirmed that the expressed product was NS1 protein of H7N9 bird flu virus. Conclusion: This study laid the foundation for the further study of NS1 protein function of H7N9 subtype influenza virus and antiviral drugs based on NS1 protein.