摘要
以临床分离的鸭圆环病毒(duck circovirus)(GenBank登录号:GU168779)阳性病料为材料,根据GenBank中所登录的鸭圆环病毒基困序列设计引物并对设计的引物5′末端进行磷酸化处理,通过引物设计替换碱基,以突变形成EcoRⅠ酶切位点。利用PCR方法扩增鸭圆环病毒的基因,经胶回收后,用T4 DNA连接酶进行环化,以获得鸭圆环病毒具有感染性的核酸。在含有分子标记的两端设计引物,进行PCR扩增,对PCR产物进行胶回收,连接T载体后测序,对胶回收产物进行EcoRⅠ酶切鉴定,均证明在第587位成功插入EcoRⅠ酶切位点。结果表明,本试验已成功构建带有分子标记的鸭圆环病毒的感染性核酸,为进一步开展该病毒的分子调控机制、致病性和开发基因工程疫苗研究奠定基础。
Duck circovirus（DuCV）,a potential immunosuppressive virus in ducks.To generate the infectious genomic DNA of duck circovirus with a genetic marker,the full-length genome of the virus was amplified using PCR method.A EcoR Ⅰ restriction enzyme site was inserted into the clone as a genetic marker by site-directed mutagenesis.T4 ligase was used to cyclized the DuCV genome by phosphorylation at the 5′ terminal,in order to construct an infectious molecular clone.The viral genome could be differentiated from the wild-type parent by gene fragment sequence analyzed and EcoR Ⅰ restriction digestion.The results indicated that the infectious molecular genomic DNA of duck circovirus with a genetic marker were constructed.This study layed foundation to on pathogenesis,vaccination,molecular diagnosis and development of vaccine candidates of DuCV.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第9期91-94,共4页
China Animal Husbandry & Veterinary Medicine
基金
现代农业产业技术体系建设专项资金
福建省财政专项-福建省农科院科技创新团队建设基金(STIF-Y02)
福建省农科院青年人才创新基金(2008QB-6)
关键词
鸭圆环病毒
分子标记
感染性核酸
duck circovirus
genetic marker
the infectious genomic
