【摘要】：目的研究諾如病毒的基因特征和變化規(guī)律,預(yù)測(cè)諾如病毒的進(jìn)化方向及流行趨勢(shì),為預(yù)防諾如病毒病的暴發(fā)打下良好基礎(chǔ)。方法采用RNA提取試劑Trizol提取諾如病毒毒株RNA,采用試劑盒TaKaRa Ex TAP進(jìn)行PCR擴(kuò)增,利用Bioedit軟件對(duì)基因序列進(jìn)行拼接,然后對(duì)基因序列分析。將PCR擴(kuò)增出的P區(qū)域克隆到表達(dá)載體,構(gòu)建原核表達(dá)質(zhì)粒。在大腸埃希菌中表達(dá)P蛋白,重組蛋白經(jīng)SDS-PAGE凝膠分離檢測(cè)。以ABO血型健康人唾液為受體研究P粒子與唾液HBGA結(jié)合能力,檢測(cè)方法采用間接ELISA法。結(jié)果實(shí)驗(yàn)用諾如病毒毒株與GⅡ.4流行株在RdRp區(qū)的核苷酸序列同源性達(dá)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%。實(shí)驗(yàn)株與GⅡ.4流行株在衣殼蛋白區(qū)的核苷酸序列同源性達(dá)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%;氨基酸同源性達(dá)到96%以上,其中US95/96株為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%。實(shí)驗(yàn)株在RdRp區(qū)核糖核苷酸序列的與New_orleans2009和Sydney_2012同源性較高,實(shí)驗(yàn)株中P2區(qū)氨基酸位點(diǎn)發(fā)生多點(diǎn)位定向突變,如P2區(qū)第95位由天冬酰胺(N)突變?yōu)榻M氨酸(H)。重組蛋白經(jīng)SDS-PAGE凝膠分離檢測(cè)產(chǎn)物大小為35ku的目的蛋白表達(dá)。檢測(cè)P粒子與唾液HBGA結(jié)合能力(A450值)血型A為3.81~5.12;血型B為3.12~4.05;血型O為2.85~3.51。結(jié)論諾如病毒在遺傳上具有多樣性,變異快的特點(diǎn),因而很難有疫苗對(duì)它長(zhǎng)期安全有效。通過(guò)對(duì)GII.4型基因序列的研究,有助于尋找其關(guān)鍵位點(diǎn)變化規(guī)律和流行株進(jìn)化機(jī)理,有助于預(yù)測(cè)諾如病毒的進(jìn)化方向及流行趨勢(shì)。
[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，

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