诺如病毒GⅡ.4型流行株基因序列分析和流行病学研究
发布时间：2019-03-21 17:54
【摘要】：目的研究诺如病毒的基因特征和变化规律,预测诺如病毒的进化方向及流行趋势,为预防诺如病毒病的暴发打下良好基础。方法采用RNA提取试剂Trizol提取诺如病毒毒株RNA,采用试剂盒TaKaRa Ex TAP进行PCR扩增,利用Bioedit软件对基因序列进行拼接,然后对基因序列分析。将PCR扩增出的P区域克隆到表达载体,构建原核表达质粒。在大肠埃希菌中表达P蛋白,重组蛋白经SDS-PAGE凝胶分离检测。以ABO血型健康人唾液为受体研究P粒子与唾液HBGA结合能力,检测方法采用间接ELISA法。结果实验用诺如病毒毒株与GⅡ.4流行株在RdRp区的核苷酸序列同源性达到94%以上,其中US95/96株94%,Farmington Hills株94.2%,Hunter株94.7%,Den_Haag_2006b株95.8%,Osaka_2007株96.5%,New_Orleans_2009株97.2%,Sydney_2012株98.4%。实验株与GⅡ.4流行株在衣壳蛋白区的核苷酸序列同源性达到94%以上,其中US95/96株94.3%,Farmington Hills株94.1%,Hunter株95.2%,Den_Haag_2006b株96.3%,Osaka_2007株96.8%,New_Orleans_2009株97.8%,Sydney_2012株98.6%;氨基酸同源性达到96%以上,其中US95/96株为96.1%,Farmington Hills株96.3%,Hunter株97.1%,Den_Haag_2006b株96.8%,Osaka_2007株97.3%,New_Orleans_2009株97.8%,Sydney_2012株98.9%。实验株在RdRp区核糖核苷酸序列的与New_orleans2009和Sydney_2012同源性较高,实验株中P2区氨基酸位点发生多点位定向突变,如P2区第95位由天冬酰胺(N)突变为组氨酸(H)。重组蛋白经SDS-PAGE凝胶分离检测产物大小为35ku的目的蛋白表达。检测P粒子与唾液HBGA结合能力(A450值)血型A为3.81~5.12;血型B为3.12~4.05;血型O为2.85~3.51。结论诺如病毒在遗传上具有多样性,变异快的特点,因而很难有疫苗对它长期安全有效。通过对GII.4型基因序列的研究,有助于寻找其关键位点变化规律和流行株进化机理,有助于预测诺如病毒的进化方向及流行趋势。
[Abstract]:Aim to study the gene characteristics and variation rules of norovirus, predict the evolution direction and epidemic trend of norovirus, and lay a good foundation for preventing the outbreak of norovirus disease. Methods the RNA, of norovirus strain RNA, was extracted by RNA extraction reagent Trizol and amplified by PCR with kit TaKaRa Ex TAP. The gene sequence was spliced by Bioedit software, and then the gene sequence was analyzed. The P region amplified by PCR was cloned into the expression vector, and the prokaryotic expression plasmid was constructed. P protein was expressed in E. coli and the recombinant protein was separated and detected by SDS-PAGE gel. The ability of P particles to bind to saliva HBGA was studied by using ABO blood group healthy saliva as receptor. Indirect ELISA method was used to detect the binding ability of P particles to saliva DNA. Results the homology of nucleotide sequence between norovirus strain and G 鈪，
本文编号：2445179
[Abstract]:Aim to study the gene characteristics and variation rules of norovirus, predict the evolution direction and epidemic trend of norovirus, and lay a good foundation for preventing the outbreak of norovirus disease. Methods the RNA, of norovirus strain RNA, was extracted by RNA extraction reagent Trizol and amplified by PCR with kit TaKaRa Ex TAP. The gene sequence was spliced by Bioedit software, and then the gene sequence was analyzed. The P region amplified by PCR was cloned into the expression vector, and the prokaryotic expression plasmid was constructed. P protein was expressed in E. coli and the recombinant protein was separated and detected by SDS-PAGE gel. The ability of P particles to bind to saliva HBGA was studied by using ABO blood group healthy saliva as receptor. Indirect ELISA method was used to detect the binding ability of P particles to saliva DNA. Results the homology of nucleotide sequence between norovirus strain and G 鈪，
本文编号：2445179
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