The objective of this study was to construct the recombinant eukaryotic expression plasmid of ORF2 gene harboring enhanced green fluorescent protein （EGFP） report gene. ORF2 gene of porcine circovirus type 2 cloned ...The objective of this study was to construct the recombinant eukaryotic expression plasmid of ORF2 gene harboring enhanced green fluorescent protein （EGFP） report gene. ORF2 gene of porcine circovirus type 2 cloned by PCR was ligated to the expression vector pEGFP-N1, which contains enhanced green fluorescent protein （EGFP） report gene, the recombinant eukaryotic expression plasmid pEGFP-N1-ORF2 was constructed successfully and was transfected into pre- pared duck myocardial cells （DMCs） by lipofectin. According to the result, the fluorescence expression was directly detected with fluorescence microscope, and the expression of ORF2 were analyzed by RT-PCR and indirect immunofluorescence assay （IFA） respectively. About 48 h after transfection, green fluorescent can be observed on transfected cells ： T-PCR and IFA were positive. This indicated that ORF2 gene of PCV2 was expressed efficiently in transfected duck myocardial cells.展开更多
The PCV-2 positive tissues by PCR-restriction fragment length polymorphism (PCR-RFLP) were inoculated to the PK-15 cells not contaminated by PCV. The viral particles were observed in the infected PK-15 cells by eletro...The PCV-2 positive tissues by PCR-restriction fragment length polymorphism (PCR-RFLP) were inoculated to the PK-15 cells not contaminated by PCV. The viral particles were observed in the infected PK-15 cells by eletronmicroscope .It was concluded that the isolate was PCV-2 and was named SCH-A strain.The capsid protein gene(cp) in ORF2 was amplified from PK-15 cell line infected with SCH-A strain by PCR, and was cloned and sequenced.The nucleotide sequences of ORF2 gene of the SCH-A strain was compared with other porcine circovirus isolates.The results showed that the homologies of nucleotides were between 90.5% and 96.7% in comparision with the PCV-2 reference strains,but the homologies of nucleotides were only about 67% with the PCV-1 reference strains. SCH-A strain displayed the highest nucleotide homology((96.7)%) with the reference strain Hunan .Phylogenetic tree analysis showed ORF2 gene of the SCH-A isolate belongs to PCV-2 .展开更多
基金Supported by Young and Middle-Aged Scientists Research Awards Fund of Shangdong Province(BS2011SW026)
文摘The objective of this study was to construct the recombinant eukaryotic expression plasmid of ORF2 gene harboring enhanced green fluorescent protein （EGFP） report gene. ORF2 gene of porcine circovirus type 2 cloned by PCR was ligated to the expression vector pEGFP-N1, which contains enhanced green fluorescent protein （EGFP） report gene, the recombinant eukaryotic expression plasmid pEGFP-N1-ORF2 was constructed successfully and was transfected into pre- pared duck myocardial cells （DMCs） by lipofectin. According to the result, the fluorescence expression was directly detected with fluorescence microscope, and the expression of ORF2 were analyzed by RT-PCR and indirect immunofluorescence assay （IFA） respectively. About 48 h after transfection, green fluorescent can be observed on transfected cells ： T-PCR and IFA were positive. This indicated that ORF2 gene of PCV2 was expressed efficiently in transfected duck myocardial cells.
文摘The PCV-2 positive tissues by PCR-restriction fragment length polymorphism (PCR-RFLP) were inoculated to the PK-15 cells not contaminated by PCV. The viral particles were observed in the infected PK-15 cells by eletronmicroscope .It was concluded that the isolate was PCV-2 and was named SCH-A strain.The capsid protein gene(cp) in ORF2 was amplified from PK-15 cell line infected with SCH-A strain by PCR, and was cloned and sequenced.The nucleotide sequences of ORF2 gene of the SCH-A strain was compared with other porcine circovirus isolates.The results showed that the homologies of nucleotides were between 90.5% and 96.7% in comparision with the PCV-2 reference strains,but the homologies of nucleotides were only about 67% with the PCV-1 reference strains. SCH-A strain displayed the highest nucleotide homology((96.7)%) with the reference strain Hunan .Phylogenetic tree analysis showed ORF2 gene of the SCH-A isolate belongs to PCV-2 .
