猪圆环病毒2型双拷贝感染性DNA的构建及体外拯救

Rescue of porcine circovirus type 2 from cloned DNA

摘要:

感染性分子克隆是研究病毒复制和致病机制的有力工具。本研究应用PCR诱变技术解决了外源片段易于自连的难题, 成功将2个PCV2 SD1株全基因组(DQ346683)头尾相接插入到真核生物表达载体pSK的多克隆位点中, 构建重组质粒pSK-2PCV2; 另外课题组成功构建含单个PCV2全基因组的pSK-PCV2和自身环化质粒ds-PCV2。将所得3种质粒分别转染无PCV污染的PK-15细胞系, 经10次连续传代后, 间接免疫荧光试验检测显示三者均在细胞核中聚集大量的病毒抗原; 经RT-PCR检测都有PCV2特异性基因转录; 透射电镜下可观察到直径约为17~20 nm的典型PCV2病毒粒子; 经测序鉴定所拯救出的病毒与亲本病毒核苷酸同源性为100%。拯救出的PCV2与亲本病毒具有相同的病毒学及分子生物学特性。本研究应用PCR诱变技术成功构建PCV2双拷贝感染性克隆, 并经体外拯救证实其具有感染性, 为进行PCV2分子特性及致病机理研究打下了基础。

Abstract:

Infectious clone is a useful tool in exploring viral replication and pathogenesis. In order to prevent linear PCV2 cyclization, PCR mutagenesis was used to construct the first molecular clone (pSK-2PCV2) by ligating two copies of the complete PCV2 genome with the pBluescript SK (pSK) vector. In addition, pSK-PCV2 and ds-PCV2 were constructed. PK-15 cells were transfected with above three infectious clones. Indirect immunofluorescence assay (IFA) revealed that the virus antigen mainly localized in infected cell nucleolus and cytoplasm. PCV2 specific nucleotide fragment in cell culture was amplified by RT-PCR. Typical porcine circovirus particles with diameter about 17 nm were also observed by transmission electron microscope (TEM) in the infected cells. The rescued virus sequences from the cultures had 100% homology with the inserting PCV2 genome. The rescued virus shared similar properties with that of the parental virus. The study establishes a platform for further research on the virus molecular biology and pathogenicity.