Recombinant Baculovirus Expressing Cap Protein Of Porcine Circorvirus Type2and Preparation Of Monoclonal Antibody And Their Primary Application

Porcine circovirus type2(PCV2), a member of the Circoviridae family, it is the smallest animal virus yet described. There are two recognized PCV genotypes:PCV1and PCV2. PCV1is the contaminant of the PK15cell line and is accepted to be non-pathogenic; PCV2was demonstrated to be a main causative agent of post-weaning multisystemic wasting syndrome (PMWS). Since1996, PCV2infectious diseases had spread all over the world and emerged as an economically important disease in swine herds. Thus, it is an important problem to prevent porcine circovirus associated diseases (PCVAD).The genome of PCV1and PCV2are similar, have two major open reading framed (ORF). ORF1encodes replication-related proteins, ORF2encodes the capsid protein. The ORF1nucleotide sequence identity between two genotypes is about86%, the ORF2is about66%, they don’t produce cross reaction between PCV1and PCV2. ORF2encodes a major capsid protein of approximately28kDa, which can stimulate the host to produce protective antibody, so ORF2gene become the target of PCV2diagnosis and vaccine research. Baculovirus expression system has become one of the four main genetic engineering expression systems, this system has many advantages:no infection to vertebrates, large foreign gene to be cloned, the screening rate of recombinant virus and the activity of express protein is high. It is widely used in microbiology, medicine etc. On this study, ORF2of PCV2was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. Using the recombinant baculovirus as immunogen, by the hybridoma technology, we prepared the PCV2specific monoclonal antibody, to lay the foundation of establishing an accurate PCV2diagnosis method.The PCV2-ORF2genes was subcloned into baculovirus transfer vector pFastBacTM1, E. coli DH10Bac containing baculovirus shutter vector was transformed with recombinant plasmid, The colonies of E. coli containing recombinant bacmid (reBacmid-ORF2) were collected by the resistance, blue and white selection. Through the lipofectamine2000, the reBacmid-ORF2was transfected into Sf9cells, after incubation for72hours, infected Sf9cells appear obvious cytopathic effect (CPE), then the DNA of Sf9was extracted for PCR identification, the recombinant baculovirus P1generation was obtained successfully. The ORF2protein expression was confirmed and the recombinant baculovirus titer was determined, and the recombinant baculovirus was concentrated through the Vivaflow200, immuned into6-8-week-old female BALB/c mice, then the spleen cells of immunized mice were collected and fused with Sp2/0myeloma cells, hybridoma were screened by IF A. The positive clones were subjected to limited dilution for subcloing, three hybridomas consistantly secreting monoclonal antibodies (mAbs) were developed after three subcloning, named as5H6,4E2,5F7respectively. By IFA, IPMA and Western-blot analysis showed that three mAbs were able to react with PCV2-Cap, the mAbs were not confirmed to have the neutralizing ability to PCV2in the neutralization test. In this study the flow cytometry based on mAbs developed to detect PCV2in PK15, it is effective to detect PCV2virus TCID50determination and improve the laboratory monitoring capabilities.