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诺如病毒衣壳蛋白的原核表达与抗血清的制备

发布时间:2018-07-29 19:40
【摘要】: 诺如病毒(Norovirus,NV)是目前继轮状病毒后,致使人类非细菌性腹泻的第二大病原。人类主要通过被污染的食物摄入诺如病毒,而在被污染的食物中牡蛎占了主要部分。诺如病毒作为一种食源性腹泻病毒首先在美国被发现后,世界各地相继出现了有关诺如病毒的报道。近些年,世界各地相继爆发了大规模的诺如病毒引起的急性肠胃炎疫情,许多国家已对我国进出口水产品中诺如病毒的检测提出要求,因此,对诺如病毒的检测已不能仅仅局限于原来的实验室复杂、繁琐的检测,快速、经济且适用于大量样品的检测的方法的研究已日趋重要。但目前,世界上还没有一种快速、有效且价格低廉的诺如病毒检测试剂盒。 诺如病毒变异性大,检测困难,但利用原核表达的诺如病毒衣壳蛋白,其特异性保守的11个氨基酸可以很好的展示在外,利用这种蛋白免疫动物,可以获得具有广泛识别能力的抗血清,该抗血清可以从一定程度上解决诺如病毒变异性强、难于检测的缺点。 本实验通过大肠杆菌表达诺如病毒衣壳蛋白,并制备抗血清,为诺如病毒的快速酶联免疫检测试剂盒的建立奠定了一定的基础。 具体研究结果如下: 1、通过Trizol法提取样品中的RNA,通过对诺如病毒RNA聚合酶基因片段(特异性保守区域)的RT-PCR扩增与测序得到该基因片段的核苷酸序列,该核苷酸序列长301个碱基,通过对序列用DNA Star Megline、MEGA软件进行分析和通过NCBI网站进行在线BLASTN分析,确定该病毒株为NV GⅡ型。 2、由于NV GⅡ-3型与NV GⅡ-4型,都是我国流行病毒株型,且具有很高的同源性因此,因此本实验后期通过对GⅡ-4、GⅡ-3型病毒代表株衣壳蛋白基因同源性比较,设计简并引物对分离病毒株衣壳蛋白基因进行PCR扩增,结果显示,只有GⅡ-3简并引物可以扩增出特异性目的带。将PCR扩增产物进行基因测序与序列分析表明该病毒株衣壳蛋白基因共1647个碱基,与GⅡ-3型病毒代表株衣壳蛋白基因长度相同,通过对该病毒株衣壳蛋白基因与GⅡ-3、GⅡ-4代表株的同源区域相似性比较,得出,该病毒株衣壳蛋白基因与NV GⅡ-3型的同源相似性大于NV GⅡ-4型。 3、用DNAtools对衣壳蛋白基因进行分析发现,在衣壳蛋白基因在扩增过程中未产生终止密码子突变,可用于表达,此外,在该衣壳蛋白氨基酸序列N末端,含有YODA提出的11个氨基酸保守区域QQNIIDPWIMN,该区域位于诺如病毒衣壳蛋白抗原表位,为NV GI、GII型共有。 4、将衣壳蛋白基因插入表达载体pQE30,构建重组质粒,转化大肠杆菌M15,IPTG诱导蛋白表达,Ni-NTA亲和层析分离纯化目的蛋白,通过SDS-PAGE电泳判断出该衣壳蛋白大小约60kDa。 5、将纯化的衣壳蛋白免疫新西兰大白兔,制备抗血清,并通过间接ELISA方法对抗血清效价进行测定。结果表明该抗血清效价可达到1:10000,符合酶联免疫检测的要求。
[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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