Expression Of The ORF2 Gene Of Porcine Circovirus Type 2 And Development Of ELISA Based On Recombinant Protein

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) that has become an emerging and important disease in swine. The ORF2 gene of PCV2 is the main region encoding the structural protein that can be used to differentiate PCV1 and PCV2. In the present study, ORF2 gene of the PCV2 BF strain was amplified and modified by PCR and successfully expressed in Escherichia coli. An indirect ELISA method based on the purified rcombinant protein was developed for detecting antibodies against porcine circovirus type 2. In addition, the epidemiological disease associated with PCV2 infection was investigated and PCR was applied for the detection of PCV2 in tissues from diseased or dead pigs in 12 intensive swine farms. Six PCV2 strains were isolated from the clinical samples. The complete genome sequence for the isolates were determined and compared with other PCV2 strains.1. A pair of primers was designed according to the PCV2 sequence in Genbank (AF381175). The ORF2 gene, a fragment of 720 bp, was amplified by polymerase chain reaction (PCR), then cloned into pGEM-T Easy vector and identified by BamHl and Xhol digestion. The ORF2 gene was modified by PCR using another pair of primers. A fragment of 593 bp of ORF2 gene was obtained and cloned into pET-32a expression vector.2. The recombinant pET-ORF2 plasmid was transformed into BL21 (DE3) comptent cell of E.coli and induced by IPTG with a finial concentration of 2mmol/L. SDS-PAGE analysis showed that the recombinant protein expressed could reach 20 percent of the whole, bacterial protein. The protein expressed could react with polyclonal antibody against PCV2 by Western-Blot, sharing a good antigencity.3.An indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant protein was developed. Using the purified recombinant protein as coating antigen, the optimal conditions including the coating concentration of the protein, serum sample dilution etc,were determined for the ELISA. The recombinant protein showed no reaction with antiserum to classical swine fevervirus, pseudorabies virus, Japanese epidemic encephalitis virus B, porcine reproductive and respiratory syndrome virus. It was shown that the ELISA was specific and suitable for large-scale sero-epidemiological investigation for PCV2 infection.4. The epidemiological disease associated with PCV2 infection was investigated in 12 intensive swine farms in Beijing, Tianjin, Guangdong, Shenzhen, Shandong and Shanxi Provence. The results showed that postweaning multisystemic wasting syndrome caused by PCV2 occurred in 11 swine farms and porcine dermatis and nephropathy syndrome was present in one swine farm. Ninety-six percent of tissue samples was positive for PCV2. It was indicated that the infection of PCV type 2 existed widely in swine farms in China. The complete genome of six PCV2 isolates were sequenced and compaired. The results showed that the complete genome of the SZ strain was 1768bp in size, the others were 1767bp. For the complete genome sequence, these PCV2 isolates shared higher homology with the isolates from Europe and Amercian isolates.