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摘要：诺如病毒（NoV)是一种能引起非细菌性急性肠胃炎的病毒。近年来，由于诺如病毒引起的流行性腹泻频繁暴发，其研究越来越受到人们的重视。目前主要通过基因工程方法进行诺如病毒的研究工作。本论文试图对诺如病毒病毒GII.4型ORF2的衣壳蛋白VP1进行克隆以及序列分析。以感染诺如病毒患者的粪便中提取出诺如病毒的cDNA为模板，Genbank数据库中VP1基因序列设计引物，通过PCR扩增获得VP1编码基因。VP1长度大小为1623bp。将VP1编码基因插入pTrcHisB载体上，并构建出表达VP1的重组质粒pTrcHisB-VP1。通过NCBI网站以及DNAMAN软件对本实验中VP1的序列以及登录号为KF060042、KF060136A、AAK84679、GU991355、DQ456824、AAB97768的诺如病毒的VP1分别进行同源性分析，同源性分别为99.44%、97.78%、91.67%、63.72%、62.41%、63.65%。综上所述，本实验成功克隆出诺如病毒GII.4型衣壳蛋白VP1编码基因。20579
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毕业论文关键词： 诺如病毒；克隆；序列分析；PCR
Cloning and sequence analysis of Norovirus capsid protein encoding gene
Abstract: Norovirus is a kind of virus that can cause non-bacterial acute gastroenteritis. Because norovirus epidemic diarrhea caused frequent outbreaks all over world, it has been paid more and more attention. At present, researches were carried out by genetic engineering methods. In this study, capsid protein VP1 of ORF2 from norovirus GII.4was cloned and sequenced. PCR was carried out using cDNA of norovirus extracted from the infection of norovirus in the faeces of patients, as template. PCR primers were designed by VP1 sequences obtained from Genbank database. The PCR result showed that length of VP1 is 1623bp. Cloned VP1 gene was further inserted into pTrcHisB vector and recombinant expression vector pTrcHisB-VP1 was constructed, VP1 sequence was compared with genes registered from NCBI website (Genbank numbers are KF060042、KF060136A、AAK84679、GU991355、DQ456824、AAB97768) by DNAMAN software. Homology analysis showed 99.44%, 97.78%, 91.67%, 63.72%, 62.41%, 63.65% similarities, respectively. In conclusion, a capsid protein encoding gene named VP1 from norovirus GII.4 was successfully cloned.
Key Words: Norovirus; clone; sequence analysis ; PCR
目 录
1前言 1
1.1 诺如病毒概述 1
1.1.1 诺如病毒的国内研究现状 2
1.1.2 诺如病毒的国外研究现状 3
1.1.3 诺如病毒传播途径 3
1.1.4 诺如病毒临床表现及诊断 4
1.1.5 诺如病毒预防及治疗 4
1.2 pTrcHis载体 4
1.2.1 pTrcHis Vector 简介 4
1.2.2 pTrcHis Vector 功能 5
1.3 pMD-19T 载体 6
1.4 PCR技术 7
1.4.1 PCR技术原理 7
1.4.2 引物设计原理 8
1.5 本课题的主要内容及研究意义 8
2材料与方法 9
2.1 材料与仪器设备 9
2.1.1 菌种及载体 9
2.1.2 实验材料 9
2.1.3 主要仪器和设备 10
2.1.4 培养基及试剂的制备 11
2. 2 实验方法 12
2.2.1 引物设计 12
2.2.2 PCR反应体系及扩增程序 12
2.2.3 PCR产物回收、纯化 12
2.2.4 与TA克隆载体的连接反应及扩增 13
2.2.5 感受态细胞的制备及转化 13
2.2.6 阳性克隆筛选 14
2.2.7 DNA序列测定 15
2.2.7 与pTrcHisB载体的连接反应 15
2.2.8 转化子的验证 16