Porcine Circovirus 2b And 2d Genotypes Cap Protein Recombinant Expression And Its Immunogenicity

Porcine circovirus type 2(PCV2)mainly causes many diseases such as weaned piglets multi-systemic wasting syndrome(PMWS),porcine dermatitis and nephrotic syndrome(PDNS),reproductive disorders and respiratory diseases of 6-16 weeks old piglet.According to the international classification method,PCV2 was divided into five genotypes:PCV2a,PCV2b,PCV2c,PCV2d and PCV2e.In recent years,according to epidemiological investigations and genetic variation analysis,the trend of PCV2 mutant prevalence has been mainly in PCV2d genotype in China.There is no effective treatment to PCV2,and vaccine immunization is considered to be the most effective prevention and control measure at present.Cap protein is the main immunogenic protein of PCV2 and has become the best antigen for studying PCV2-related subunit vaccines in recent years.Commercial vaccines on the market mainly target to PCV2a and PCV2b genotype.Studies have shown that PCV2a vaccine can provide better protection against PCV2d strains.Our study focused on whether PCV2b vaccine has a good protective effect on PCV2d genotype strains and so we constructed the recombinant baculovirus expressing PCV2 Cap protein and studied its immunogenicity.The main contents as follows:1.PCV2 was isolated and identified from the pig tissues suspected PCV2 infection.The isolate was tested pathogenicity in mice.Two PCV2 strains 5123 and X46,which are PCV2b and PCV2d subtypes,were isolated,and the virus TCID50 of X46 and 5123 were 1×105.5 TCID50/0.1 ml and 1×104.5 TCID50/0.1 ml,respectively.PCV2 has strong invasiveness and high viral load in liver,spleen,lung and kidney.The liver,kidney,spleen and lung viral load of the mice challenged with X46 strain were higher,the weight gain of the mice were significantly reduced.However the viral load of each organs of the strain 5123 in mice was low.2.The recombinant plasmid pFastBacDual-ORF2b,pFastBacDual-ORF2d,multi-copy recombinant plasmid pFastBacDual-ORF2dl,pFastBacDual-ORF2d1d2d2,pFastBacDual-ORF2dld2d3d4 were successfully constructed with the baculovirus-insect cell expression system pFastBacDual expression vector.The recombinant proteins PCV2b-Cap,PCV2d-Cap,PCV2dl-Cap,PCV2d-2Cap,PCV2d-3Cap and PCV2d-4Cap were obtained by transfection in SF9 cells.Results of SDS-PAGE and Western-Blot showed that the multi-copy PCV2d-3Cap protein was higher expressed,which indicated a better vaccine candidate3.The subunit vaccine was prepared using the recombinant proteins PCV2d-3Cap,PCV2b-Cap and PCV2d-Cap.The effecience of the vaccine was evaluated in mice challenged with strains X46 and P58 PCV2.The results showed that the viral load of the mice in each immunized group was significantly lower than that in the control group.There was no significant difference between the immunized groups.The mice immunized by PCV2b-Cap and PCV2d-Cap subunit vaccines were challenged with PCV2d strain.It still showed a good protective effect.All three subunit vaccines had a good protective effect on the mice challenged by the PCV2d strain,although there are certain differences in antigenicity between different genotype of PCV2b and PCV2d.The interesting was there was no protection difference between PCV2b-Cap and PCV2d-Cap subunit vaccines in mice challenged with PCV2d strain;PCV2d-3Cap protein was the best candidate for PCV2 vaccine although three groups of vaccines could provide better protection in the same immune dose.