猪圆环病毒2型流行株的分离及其感染性克隆的构建

Isolation and identification of porcine circovirus type 2 of GD-zq strain and construction of its infectious clone

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Hind Ⅲ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。

Abstract:

[objective] To identify and construct an infectious clone for an isolation of porcine circovirus 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 pUC18 with the restriction enzymes EcoRⅠ, SalⅠ, SalⅠand Hind Ⅲ, and the positive clone pPCV2-2GD-zq was identified by enzyme analysis. By purification and 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 complete genome 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% in OFR2. 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.