摘要苦苣菜黄网病毒(Sonchus yellow net virus，SYNV)是一种细胞核内复制的负链RNA病毒，属于弹状病毒科细胞核弹状病毒属，它侵染菊科植物，在莴苣等农作物上造成病毒病害。SYNV P蛋白能与N蛋白互作来调控病毒复制和转录之间的转换 ，它与细胞核输入和细胞核内病毒发生机制有关。47108
本研究从感染SYNV的本氏烟叶片中提取总RNA，用RT-PCR方法扩增获得了SYNV-P基因，纯化PCR产物，连接pMD-18T克隆载体，转化E.coli DH5a菌株，PCR和双酶切鉴定阳性重组质粒，将已克隆的SYNV-P基因连接到原核表达载体pET30a上，构建重组表达载体pET30a-P，并获得高效表达的P蛋白，为SYNV 单抗的研制提供实验条件，继而为深入研究病毒复制以及与寄主互作等的侵染机制奠定实验基础。
Sonchus yellow net virus (SYNV) is a negative-strand RNA virus that replicates in the nucleus, belonging to the Nucleorhabdovirus genus of Rhabdoviridae family.SYNV infects composite plants and leads to viral diseases in some crops such as lettuce. P protein interacts with the N protein to regulate transitions between transcription and replication, and it contributes to nuclear import and forming complexes in vivo.
The P gene of SYNV was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the total RNA of the infected N. benthamiana leaves, and the purified PCR products were ligated with pMD-18T vector, then transformed into E.coli DH5a. After being identified by PCR and double enzymes digestion, the positive recombinant plasmids were determined, then prokaryotic expression vector pET30a-P was constructed, which effectively expressed the P protein. The experimental results would provide the experimental conditions for monoclonal antibody preparation of SYNV and a foundation to study on viral replication and host interaction.
毕业论文关键词：苦苣菜黄网病毒；P蛋白；克隆；原核表达。
Keywords: Sonchus yellow net virus; P protein ; cloning; prokaryotic expression.
目 录
1.1 大肠杆菌表达系统 1
1.1.1 大肠杆菌表达系统研究现状 1
1.1.2 外源基因高效表达的影响因素 1
1.1.2.1 外源基因本身的特性对表达的影响 2
1.1.2.2 表达系统的特性对表达的影响 2
1.1.3 存在的问题和发展方向 2
1.2 枯草芽孢杆菌表达系统 3
1.2.1 存在的优势 3
1.2.2 存在的问题和发展方向 3
1.3 链霉菌表达系统 4
1.3.1 作为基因工程受体菌的优缺点 4
第二章 苦苣菜黄网病毒P基因的克隆及原核表达 5
2.1 引言 5
2.2 材料 5
2.2.1 植物材料、菌株、质粒 5
2.2.2 生化试剂 5
2.2.3 常用仪器 5
2.3 方法 6
2.3.1 SYNV P基因的克隆 6
2.3.1.1 Trizol方法提取总RNA 6
2.3.1.2 引物的设计与合成 6
2.3.1.3 反转录 苦苣菜黄网病毒P基因的克隆及原核表达:http://www.751com.cn/shengwu/lunwen_49053.html