Sequence analysis of a new norovirus(NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase(RdRp) and complete capsid protein(VP1) gene.The isolated strain CHN02...Sequence analysis of a new norovirus(NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase(RdRp) and complete capsid protein(VP1) gene.The isolated strain CHN02/LZ35666 shared high sequence homology with GII-4 NVs.Nucleotide homologies of RdRp region and encoded capsid protein region were 90.4%-98.6% and 89.8%-95.7%,respectively,while amino acid homology of capsid protein region was 94.4%-97.4%.The analysis of GDD motif in RdRp region indicated this GDD motif of Lanzhou strain differed from those of the GII-4 predominant epidemic strains.Lanzhou strain formed an independent branch in GII-4 cluster in the phylogenetic tree based on nucleotide sequence of RdRp region and amino acid sequence of capsid protein.Sequence alignment revealed a mutation at the fourth key site of the receptor-binding interface in the strains isolated after 2002 compared with those of previous strains suggesting a possible change of binding pattern to HBGAs receptors.展开更多
目的为获得大量的单核细胞增生性李斯特氏菌(Listeria monocytogenes,Lmo)溶血素(Hemolysin,hly)蛋白,以便研制Lmo诊断试剂及其在疫苗研制方面的作用。方法本文应用Primer Premier 5.00设计引物,引入BamHⅠ和XhoⅠ酶切位点,以前期合成...目的为获得大量的单核细胞增生性李斯特氏菌(Listeria monocytogenes,Lmo)溶血素(Hemolysin,hly)蛋白,以便研制Lmo诊断试剂及其在疫苗研制方面的作用。方法本文应用Primer Premier 5.00设计引物,引入BamHⅠ和XhoⅠ酶切位点,以前期合成构建的pMD18-T-hly质粒为模板,通过PCR方法扩增出Lmo 0586株溶血素基因。相应酶切后,克隆到原核表达载体pGEX-6p-1中,构建pGEX-6p-hly重组质粒,转化入大肠杆菌BL21(DE3)进行表达。带有重组质粒pGEX-6p-hly的大肠杆菌BL21(DE3)经IPTG诱导后,进行SDS-PAGE及免疫印记分析。结果PCR体外扩增hly基因产物大小约为1 624bp,成功构建了重组表达质粒pGEX-6p-hly;SDS-PAGE显示蛋白表达带的分子量约为72ku,重组蛋白主要以包涵体形式表达,表达量占菌体总蛋白的20.8%;Western免疫印记表明具有良好的反应原性。结论在国内首次构建重组质粒pGEX-6p-hly,并以融合蛋白的形式进行了高效表达,同时该蛋白具有特异的抗原反应性,为研制Lmo诊断试剂及其在疫苗研制中的作用奠定了基础。展开更多
文摘Sequence analysis of a new norovirus(NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase(RdRp) and complete capsid protein(VP1) gene.The isolated strain CHN02/LZ35666 shared high sequence homology with GII-4 NVs.Nucleotide homologies of RdRp region and encoded capsid protein region were 90.4%-98.6% and 89.8%-95.7%,respectively,while amino acid homology of capsid protein region was 94.4%-97.4%.The analysis of GDD motif in RdRp region indicated this GDD motif of Lanzhou strain differed from those of the GII-4 predominant epidemic strains.Lanzhou strain formed an independent branch in GII-4 cluster in the phylogenetic tree based on nucleotide sequence of RdRp region and amino acid sequence of capsid protein.Sequence alignment revealed a mutation at the fourth key site of the receptor-binding interface in the strains isolated after 2002 compared with those of previous strains suggesting a possible change of binding pattern to HBGAs receptors.
