猪嵴病毒全长cDNA感染性克隆的构建及拯救

密级：论文编号：

中国农业科学院

学位论文

猪嵴病毒全长cDNA感染性克隆的构建及拯救Construction and rescue of Porcine Kobuvirus full-length cDNA

infectious clone

硕士研究生：李长龙

指导教师：冯力研究员

申请学位类别：农学硕士

专业：预防兽医学

研究方向：兽医微生物及分子生物学

培养单位：哈尔滨兽医研究所

中国农业科学院研究生院

2014年5月

Secrecy:No.

Chinese Academy of Agricultural

Sciences

Master Dissertation

Construction and rescue of Porcine Kobuvirus full-length cDNA infectious clone

Ms. Candidate：Li Changlong

Advisor：Prof. Feng Li

Major：Preventive Veterinary Medicine

Specialty：Molecular Biology and Veterinary

Microbiology

May 2014

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中国农业科学院

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摘要

猪嵴病毒（Porcine Kobuvirus，PKV)是小RNA病毒科嵴病毒属成员，是近年在腹泻猪体内发现的新型病毒。同属成员还有爱知病毒(Aichi virus，AiV)和牛嵴病毒(Bovine kobuvirus，BKV)，目前，爱知病毒和牛嵴病毒都已成功分离，而猪嵴病毒尚没有成功分离的报道。

近年来我国大部分地区规模化猪场暴发仔猪腹泻，导致仔猪大量死亡，造成严重的经济损失。研究人员推测这可能与新发现的小RNA病毒如猪嵴病毒等有关。有研究表明，猪嵴病毒在在腹泻猪和健康猪群内广泛存在，猪群感染率非常高，而且有研究表明猪嵴病毒感染很可能与猪腹泻有关。获得纯化的病毒是研究其致病性及其理化特征的基础，由于猪嵴病毒尚不能成功分离，本研究采用反向遗传操作技术，在获得病毒全基因组序列的基础上，人工构建出囊括PKV全基因组的全长cDNA感染性克隆，通过转染特定细胞系，人工拯救出完整的PKV病毒。

为便于在蛋白水平上检测PKV，我们表达PKV-DX株的VP3结构蛋白，免疫Balb/c小鼠，通过常规杂交瘤技术获得一株能够稳定分泌抗VP3结构蛋白的杂交瘤细胞，为下游PKV拯救病毒的鉴定及深入研究奠定了基础。

通过分析本实验室测得的PKV-DX株全基因组序列及pBeloBAC11载体、CMV启动子、HDV-BGH 终止元件的序列，选择合适的酶切位点将PKV全基因组进行分段，同时设计引物引入无意突变分子标签。使用高保真酶从病料中扩增出囊括PKV基因组的各片段，依次插入含有CMV启动子、HDV-BGH终止元件的pBeloBAC11载体，构建出PKV全长质粒命名为pBAC-PKV-DX。全长质粒转染293T细胞，72h后通过RT-PCR、IFA、WB和电镜等技术对拯救病毒进行验证。结果表明，PKV成功实现了人工拯救。

在全基因组序列比对中发现PKV 2B区在不同基因组中存在缺失现象，而DX株为基因缺失株。为探究2B区域缺失对病毒拯救的影响，在原有质粒pBAC-PKV-DX的基础上，人工合成PKV XM株2B区域不缺失的部分，构建不缺失的重组质粒pBAC-PKV-2B，转染293T细胞，72h后进行鉴定。结果表明，2B区域缺失与否对病毒拯救没有影响。

本研究构建了两个囊括PKV-DX株全基因组的全长质粒，在此基础上实现了对PKV的人工拯救。建立了PKV基因组的操作平台，为PKV的后续研究打下基础。

关键词：猪嵴病毒；反向遗传系统；感染性克隆；仔猪腹泻

Abstract

Porcine Kobuvirus (PKV) is a new member of kobuvirus, Picornavirus family and it is new virus found in the diarrhea pigs in recent years. Aichi virus (AiV) and Bovine kobuvirus (BKV) are both members of kobuvirus and have been successfully separated, while no report indicated that PKV has been separated.

During recent years, many large-scale pig farms in most parts of China encountered the outbreak of piglets epidemic diarrhea, which causing massive death piglets and serious economic losses. The researchers speculated that it may be related to the new emerge of pig intestinal virus such as porcine kobuvirus. Research has shown that porcine kobuvirus is widespread in diarrhea and healthy pigs, and the porcine kobuvirus infection associated with swine diarrhea. As porcine kobuvirus can not be successfully separated and the purified virus is the basis of study the pathogenic and its physical and chemical characteristics, we decide to use reverse genetics technology operation, on the base of the PKV genome sequence, construct the PKV genome-wide cDNA infectious clone artificially and save PKV by transfecting special cell line.

For detection of porcine kobuvirus protein, we express VP3 structure protein of PKV-DX strain, immune Balb/c mice and obtain a hybridoma which can secrete monoclonal antibody against VP3 through routine hybridoma technology. Laid a solid foundation for the identification and depth-study of PKV.

Depend on the sequence of PKV-DX strain and pBeloBAC11 vector, CMV promoter and HDV-BGH stop element, we selected the appropriate enzyme sites to separate the PKV genome. Then we designed primers to introduce nonsense mutations as molecular tag. We amplified fragments of PKV genome and insect in the pBeloBAC11 vector includinng CMV promoter and HDV-BGH stop element to construct the full-length plasmid named pBAC-PKV-DX. The full-length plasmid was transfected into 293T cell and detected the rescue PKV by RT-PCR, IFA, WB and electron microscope after 72h transfection. The results indicated that the PKV was successfully rescued.

Gene deletion in 2B gene of PKV was found during the analysis of PKV sequence. As the PKV-DX strain was gene deletion strain, we decided to explore the influence of 2B gene deletion for the PKV rescue. Based on the plasmid pBAC-PKV-DX, we amplified the full part of 2B gene and construct the 2B gene no-deletion plasmid pBAC-PKV-2B. After transfection and identification, the results indicated that the deletion in 2B gene don’t hinder the rescue of PKV.

The research constructed two full-length plasmids containing PKV-DX strain genome, and rescued PKV successfully. The full-length PKV cDNA clone lay the foundation for PKV follow-up study.