The Construction Of Infectious Clone Of Porcine Circovirus Typeâ…±and Development Of An ELISA Based On The Multi-epitope

According to the difference of pathogenicity, antigenicity and nucleotide sequence, PCV can be divided into two genotypes PCV1 and PCV2. PCV2 was demonstrated to be a causative agent of post-weaning multisystemic wasting syndrome (PMWS). In addition, PCV2 is very likely to be associated with a number of other porcine diseases, and those diseases that are widely prevalent and have caused huge losses of the swine industry in recent years. Some researches indicated that single PCV2 genome can be infectious, but the infectivity will further enhance while two copies of the complete PCV2 genome ligate in tandem. The Construction of Infectious clone is useful for accurate research of pathogenicity and infection process of virus, which has drew extensive attention.The full-length genome of PCV2 was amplified by polymerase chain reaction (PCR), and the sequence was compared with other published genomes of PCVs by DNAstar. The molecular clones were generated by ligating single copy and two copies of the complete PCV2 genome in tandem into the pcDNA3.1+ vector, respectively. After continuous cell culture of passage 7, the recombinant plasmids which were transfected into the PCV free PK-15 cells were shown to be infectious in vitro, and PCV2 antigen was detected by indirect immunofluorescent. PCV2 specific nucleotide fragment which coded the Cap protein was amplified by RT-PCR with the mRNAs from the cell culture. The virus-like particle could be seen in transfected cells by electronic microscope. BALB/c mice were immunized with the recombinant plasmid p-PCV2 and p-2PCV2 and serums collected from control and vaccinated animals at different days when post-inoculation (dpi) were assayed for anti-PCV2 capsid antibodies by ELISA, the results indicated that the recombinant plasmids induced specific antibodies against the pathogenic PCV2 capsid antigen in mice at 35 dpi.The sequence of EGFP was inserted into 3'terminal of ORF2 of PCV2 using fusion PCR method, constructing a molecular clone with EGFP tag. The PCV2 gene could be detected but the EGFP could not be seen when the PCV free PK-15 cells were transfected by the clone and passed on over 7 generations. It is probably because the complete virus does not allow the heterologous gene to insert. The rebuilt molecular clone was constructed by ligating a gene into the pcDNA3.1+ vector, and this gene was generated by inserting the DNA sequence of a fusion protein which contain several epitopes of PCV2 into 3'terminal of ORF2 of PCV2.A fusion protein was obtained from three antigenic regions of capsid protein. The protein is indicated to have strong immunologic activity by westen-bloting and ELISA assay. An indirect ELISA based on this protein was established for the detection of antibody against PCV2.The successful rescue of PCV2 virus laid the foundation for the further research to PCV2. The detective method of ELISA which is convenient for clinical sample test was established.