摘要
【目的】通过分离一株猪圆环病毒2型(PCV2)流行毒株,并构建其感染性克隆,为研究PCV2基因功能提供操作平台。【方法】通过PCR方法,从疑似患断奶仔猪多系统衰竭综合症(PMWS)的仔猪淋巴结中鉴定为猪圆环病毒(Porcine circovirus,PCV)2型阳性。把阳性病料接种PK-15细胞传代培养,在培养物中扩增出PCV2的全基因序列。对扩增出的全序列进行序列测定,并与GenBank中公布的5株广东PCV2分离株(GD-pz、GD-gj、GD-jm、GD-ss和GD-sz)进行同源性分析。通过EcoRⅠ和SalⅠ将PCV2全基因组序列克隆进pUC18载体中,获得含PCV2 GD-zq株全基因组单拷贝的重组质粒pPCV2-GD-zq,再通过SalⅠ和HindⅢ把另一个全长拷贝克隆进pPCV2-GD-zq质粒中,使PCV2 GD-zq株基因组DNA以头尾相接的双重复方式克隆进pUC18载体中,获得重组质粒pPCV2-2GD-zq。将pPCV2-2GD-zq DNA纯化和定量后转染PK-15细胞,拯救PCV2 GD-zq病毒。【结果】从PMWS感染的猪淋巴结中分离到了一株PCV2,命名为GD-zq株;序列分析结果显示,GD-zq株全基因组为1 767 bp,与GenBank中公布的5株广东PCV2分离株ORF1核苷酸一致性为97.1%-99.7%,编码氨基酸一致性为98.7%-100%;ORF2核苷酸一致性为93.2%-99.6%,编码氨基酸一致性为92.3%-99.1%;全基因一致性为96.0%-99.6%。pPCV2-2GD-zq质粒转染PK-15细胞后,其通过间接免疫荧光实验(IFA)能从转染细胞及其传代细胞中,检测到拯救出的病毒。【结论】分离了一株PCV2广东株GD-zq,成功构建了PCV2 GD-zq株的感染性克隆。
To identify and construct an infectious clone for an isolation of porcinecircovirus type 2 （PCV2）. [Methods] PCR was used to identify an isolate, PCV2 GD-zq strain, from the tissues of clinical pigs with postweaning multisystemic wasting syndrome （PMWS）. The complete sequence of the isolate was megaligned with other 5 PCV2 Guangdong isolates （GD-pz,GD-gj, GD-jm, GD-ss and GD-sz） from GenBank by DNAstar. Two copies of the whole genomic sequence was amplified and cloned into pUC 18 with the restriction enzymes EcoR Ⅰ, Sal Ⅰ, Sal Ⅰ and Hind Ⅲ, and the positive clone pPCV2-2GD-zq was identified by enzyme analysis. By purificationand quantitation, the pPCV2-2GD-zq DNA was transfected to PK-15 cell lines to rescue the infectious PCV2 GD-zq. [Results] PCV2 GD-zq strain was isolated and identified from the lymphonodus of clinical pigs with PMWS. Sequence analysis shows that the isolate＇s completegenome was composed of 1 767 nucleotides, which shares 96.0%-99.6% homology between other 5 Guangdong reference strains, and shares 97.1%-99.7% homology in ORF1, 93.2%-99.6% in ORF2, respectively. Amino acid homology alignment shows 98.7%-100% in ORF1 and 93.2%-99.6% inOFR2. Seventy two hours post transfection of pPCV2-2GD-zq, GD-zq strain could be detected by immunofluorescence assay （IFA）. [Conclusion] A PCV2 was isolated, and its infectious DNA clone was constructed successfully.
出处
《微生物学通报》
CAS
CSCD
北大核心
2014年第6期1168-1174,共7页
Microbiology China
基金
重大动物疫病新型疫苗的关键技术研究与产业化项目(No.2011A090200117)
关键词
猪2型圆环病毒
全基因
感染性克隆
序列分析
Porcine circovirus type 2 （PCV2）
Complete genome
Infectious clone
Sequenceanalysis
作者简介
通讯作者： ：chenruiaidhn@126．com
