【摘要】： 諾如病毒(Norovirus,NV)是目前繼輪狀病毒后,致使人類非細(xì)菌性腹瀉的第二大病原。人類主要通過(guò)被污染的食物攝入諾如病毒,而在被污染的食物中牡蠣占了主要部分。諾如病毒作為一種食源性腹瀉病毒首先在美國(guó)被發(fā)現(xiàn)后,世界各地相繼出現(xiàn)了有關(guān)諾如病毒的報(bào)道。近些年,世界各地相繼爆發(fā)了大規(guī)模的諾如病毒引起的急性腸胃炎疫情,許多國(guó)家已對(duì)我國(guó)進(jìn)出口水產(chǎn)品中諾如病毒的檢測(cè)提出要求,因此,對(duì)諾如病毒的檢測(cè)已不能僅僅局限于原來(lái)的實(shí)驗(yàn)室復(fù)雜、繁瑣的檢測(cè),快速、經(jīng)濟(jì)且適用于大量樣品的檢測(cè)的方法的研究已日趨重要。但目前,世界上還沒(méi)有一種快速、有效且價(jià)格低廉的諾如病毒檢測(cè)試劑盒。 諾如病毒變異性大,檢測(cè)困難,但利用原核表達(dá)的諾如病毒衣殼蛋白,其特異性保守的11個(gè)氨基酸可以很好的展示在外,利用這種蛋白免疫動(dòng)物,可以獲得具有廣泛識(shí)別能力的抗血清,該抗血清可以從一定程度上解決諾如病毒變異性強(qiáng)、難于檢測(cè)的缺點(diǎn)。 本實(shí)驗(yàn)通過(guò)大腸桿菌表達(dá)諾如病毒衣殼蛋白,并制備抗血清,為諾如病毒的快速酶聯(lián)免疫檢測(cè)試劑盒的建立奠定了一定的基礎(chǔ)。 具體研究結(jié)果如下: 1、通過(guò)Trizol法提取樣品中的RNA,通過(guò)對(duì)諾如病毒RNA聚合酶基因片段(特異性保守區(qū)域)的RT-PCR擴(kuò)增與測(cè)序得到該基因片段的核苷酸序列,該核苷酸序列長(zhǎng)301個(gè)堿基,通過(guò)對(duì)序列用DNA Star Megline、MEGA軟件進(jìn)行分析和通過(guò)NCBI網(wǎng)站進(jìn)行在線BLASTN分析,確定該病毒株為NV GⅡ型。 2、由于NV GⅡ-3型與NV GⅡ-4型,都是我國(guó)流行病毒株型,且具有很高的同源性因此,因此本實(shí)驗(yàn)后期通過(guò)對(duì)GⅡ-4、GⅡ-3型病毒代表株衣殼蛋白基因同源性比較,設(shè)計(jì)簡(jiǎn)并引物對(duì)分離病毒株衣殼蛋白基因進(jìn)行PCR擴(kuò)增,結(jié)果顯示,只有GⅡ-3簡(jiǎn)并引物可以擴(kuò)增出特異性目的帶。將PCR擴(kuò)增產(chǎn)物進(jìn)行基因測(cè)序與序列分析表明該病毒株衣殼蛋白基因共1647個(gè)堿基,與GⅡ-3型病毒代表株衣殼蛋白基因長(zhǎng)度相同,通過(guò)對(duì)該病毒株衣殼蛋白基因與GⅡ-3、GⅡ-4代表株的同源區(qū)域相似性比較,得出,該病毒株衣殼蛋白基因與NV GⅡ-3型的同源相似性大于NV GⅡ-4型。 3、用DNAtools對(duì)衣殼蛋白基因進(jìn)行分析發(fā)現(xiàn),在衣殼蛋白基因在擴(kuò)增過(guò)程中未產(chǎn)生終止密碼子突變,可用于表達(dá),此外,在該衣殼蛋白氨基酸序列N末端,含有YODA提出的11個(gè)氨基酸保守區(qū)域QQNIIDPWIMN,該區(qū)域位于諾如病毒衣殼蛋白抗原表位,為NV GI、GII型共有。 4、將衣殼蛋白基因插入表達(dá)載體pQE30,構(gòu)建重組質(zhì)粒,轉(zhuǎn)化大腸桿菌M15,IPTG誘導(dǎo)蛋白表達(dá),Ni-NTA親和層析分離純化目的蛋白,通過(guò)SDS-PAGE電泳判斷出該衣殼蛋白大小約60kDa。 5、將純化的衣殼蛋白免疫新西蘭大白兔,制備抗血清,并通過(guò)間接ELISA方法對(duì)抗血清效價(jià)進(jìn)行測(cè)定。結(jié)果表明該抗血清效價(jià)可達(dá)到1:10000,符合酶聯(lián)免疫檢測(cè)的要求。
[Abstract]:Norovirus-NV (NV) is the second leading cause of human non-bacterial diarrhea after rotavirus. Humans ingest Norovirus mainly through contaminated food, and oysters make up the bulk of contaminated food. Norovirus was first found in the United States as a foodborne diarrhea virus, and there have been reports of Norovirus all over the world. In recent years, there has been a large-scale outbreak of acute gastroenteritis caused by Norovirus in various parts of the world. Many countries have put forward requirements for the detection of Norovirus in our country's import and export aquatic products. The detection of Norovirus can not only be limited to the original laboratory complex, tedious detection, rapid, economical and suitable for a large number of samples detection methods have become increasingly important. But at present, there is not a rapid, effective and low-cost Noruvirus detection kit in the world. Norovirus is highly variable and difficult to detect, but with the prokaryotic expression of norovirus capsid protein, its specific and conservative 11 amino acids can be well displayed, using this protein to immunize animals. An antiserum with wide recognition ability can be obtained. To some extent, the antiserum can solve the disadvantages of high variability and difficult detection of Norovirus. In this study, E. coli was used to express norovirus capsid protein and to prepare antiserum, which laid a foundation for the establishment of rapid enzyme linked immunosorbent assay (Elisa) kit for norovirus. The specific results are as follows: 1. Trizol method was used to extract the RNA from the sample. The nucleotide sequence of the RNA polymerase gene fragment (specific conserved region) was amplified and sequenced by RT-PCR. The nucleotide sequence was 301 bases long. The nucleotide sequence was analyzed by DNA Star Meglineomega software and by on-line BLASTN analysis through NCBI website. It was confirmed that the virus strain was NV G 鈪，

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