Construction Of A Recombinant Eukaryotic Expression Vector Carrying ORF2 Of PCV2 And Detection Of Immune Antibody

Designed a pair of primers according to the genomic DNA of PCV2_Whn. and ORF2 gene was amplified by polymerase chain reaction (PCR), then cloned into pUCm-T vector. The ORF2 sequence had 705bp and encorded 234 amino acids.pUCm-PCV-0RF2 and pcDNA3.1(+) were digested by restriction enzyme Kpn I and BamH I .then ligated, in the end ORF2 inserted in pcDNA3.1 (+). The recombinant pcDNA-PCV-ORF2 plasmid was transformed into E.coli. JM109. Restrictive enrymes disgestion and PCR analysis results revealed that the combinant expression vector pcDNA-PCV-0RF2 had been constructed successfully.The plasmid pcDNA-PCV-ORF2 was extracted cosmically by alkaline lysis and purified, then injected into the mice. Based on indirect ELISA in different time after the first immunization, the immune antibody emerged in the 1 st week and rised quickly after second immunization at the 15^th day, reached maximum at the 4^th week, then declined slowly, last for over 11 weeks.Detected the distribution of pcDNA-PCV-0RF2 in mice by PCR in different time after the first immunization. The fragment was amplificated from various kinds of tissues and organs from 3 hour to 28 day. The results showed that recombinant plasmid pcDNA-PCV-ORF2 distributed in most tissues of mice 3h after the first immunization. At the 44^th day the PCR result about brain was negative, and the fragment was amplificated only from blood, heart and muscle tissue at the 58^th day, but the plasmid could exist at least 88 days in muscle tissue.The fragment was not amplificated from the DNAs of various kinds of tissues and organs which were purified. The security detection indicated that pcDNA-PCV-ORF2 did not integrate with the mice chromosome, and manifested immuno-safety of pcDNA-PCV-ORF2.This study provided scientific foundation for further study of nucleic acid vaccine of PCV2.