Isolation Of Porcine Circovirus 2 In Henan Province And Production Of Cap Protein

Porcine circovirus type 2 can cause a variety of infectious diseases,such as Postweaning Multisystemic Wasting Syndrome?PMWS?,Porcine Dermatitis and Nephropathy Syndrome?PDNS?,Porcine Congenital Tremor?PCT?and Porcine Respiratory Disease Complex?PRDC?.In recent years,PCV2 has spread rapidly at home and abroad.With the widespread spread of PCV2 and the intermittent burst,this diseases have brought great damage to the pig-raising industry.Therefore,the diagnosis and prevention of PCV2 has become the focus of research among scholars.In order to screen for the excellent PCV2 strains,11 materials suspiciously infected with PMWS disease which come from different regions of Henan province were dealed with grinding processing and amplified by PCR detection.PCV2 positive samples were inoculated into PK-15 cell which no infected by PCV,after then it was further identified by immunoperoxidase mono-layer assay.PCR products of complete genomes of PCV2 isolates were cloned and sequenced.The separation of one individual plant PCV2b was success.This results of this research were contribute to the screening of PCV2 strains and inactivated vaccine's production and selection.In order to get a rapid and accurate detected method,With the template of Positive standards,we built a diagnostic method what use SYBRGreen Real-time PCR to detect porcine circovirus type 2.The results show that the method has high sensitivity,which arrives at 3.0x10² copies/mL.The analysis from the dissolution curve show that the specific primer has a good specificity and no primer-dimers have happened.There is no cross reactions,which declare that the method has good repeatability.Capsid?Cap?protein is the major immunogenic protein of PCV2 and has the ability to self-assemble into virus-like particles?VLPs?in vitro.In this study,we adopt the method of prokaryotic expression system for the soluble expression of recombinant Cap protein.After the optimization of expression conditions,we obtain the best expression condition of Cap-His protein.Through the Ni-NAT affinity chromatography purification,we got the high purity of recombinant protein and laid a solid foundation for the development of new type of PCV2 VLPs subunit vaccine.