Construction of an Infectious Clone of Porcine Circovirus Type 2
Based on the rolling loop replication principle of porcine circovirus type 2(PCV2),two primers were designed for PCR amplification of the PCV2 genome sequence,taking into account the analysis of the genome structure and sequence of representative strains from different gene subtypes of PCV2.Using the DNA extracted from 10 pig serum samples suspected to be infected with PCV2 as templates,the target fragments were amplified with the designed primers and subjected to sequencing analysis.Among them,one strain was PCV2a type,four strains were of the PCV2b type,and five strains were of the PCV2d type.A sample of each genotype strain was selected and PCR amplified with the above two pairs of primers,and the amplification product was cloned into the pEASY-Blunt simple vector,and two PCR fragments were ligated by double digestion to construct a plasmid containing about 1500 bp overlapping sequences of PCV2 genome.To rescue the PCV2 virus,the plasmid containing the complete PCV2 genome was transfected into PK-15 cells using the liposome transfection method.The successful rescue of the PCV2 virus was confirmed through PCR and immunofluorescence assay(IFA),validating the rescue of three PCV2 genotypes.By analyzing the whole genome structure and sequence of PCV2,this method is also suitable for the construction of infectious clones of PCV2g and PCV2h genotypes.In this study,a method of constructing PCV2 infectious clone based on reverse genetics technology was established to provide technique support for the etiology research of PCV2.
porcine circovirus type 2infectious cloningvirus rescuegenetic evolution analysis
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