猪圆环病毒Ⅱ型Rep基因在PK15细胞中的表达及特性

Expression and Characterization of PCV2 Rep in PK15 Cells

为研究猪圆环病毒型(PCV2)Rep基因在PK15细胞中的表达特性,通过PCR方法克隆了PCV2杭州株(HZ0201)Rep基因全长945bp片段,与真核表达载体pCI-neo构建为重组质粒pCI-PCV2-Rep。pCI-PCV2-Rep质粒转染PK15细胞后48h,通过RT-PCR可检测到PCV2RepmRNA的转录;用猪PCV2多抗血清作间接免疫荧光试验,可检测到Rep基因表达产物。在表达量低的细胞中,PCV2Rep蛋白主要位于PK15的细胞浆,在表达量高的细胞中,细胞浆和细胞核中均含有大量的Rep蛋白,表明Rep对PK15细胞的细胞浆和细胞核的亲嗜性没有明显差别。

To study the expression of porcine circovirus type 2 (PCV2) rep gene in PK15 cells, the full-length Rep gene of 945 bp was amplified by PCR from the genome of PCV2 isolate (HZ020). The cloned gene was inserted into the eukaryotic expression vector pCI-neo, a recombinant plasmid pCI-PCV2-Rep, which was then transfected into PK15 cells for PCV2 Rep expression. The PCV2 Rep mRNA transcript was detected by RT-PCR analysis in the transfected PK15 cells 48 h posttransfection, meanwhile, PCV2 Rep protein was detected by an indirect immunofluorescent assay(IFA) with swine polyclonal antiserum to PCV2. The expressed PCV2 Rep protein predominantly localized in the cytoplasm of transtected PK15 cells in case of lower expression, but abundant amount of PCV2 Rep protein was also observed in the nuclei in case of higher expression, indicating that the expressed PCV2 Rep protein does not have definite localization pattern limited to either the cytoplasm or the nuclei of PK15 cells.