Expression Of Procine Circovirus Type 2 Cap Protein And Its Immunogenicity Research

Porcine circovirus type 2(PCV2)is one of the main infectious disease that threats the pig industry and causes great financial losses.It can cause post-weaning multisystemic wasting syndrome,porcine dermatitis and nepHropathy syndrome,porcine respiratory syndrome,and a series of diseases.Cap protein encoded by ORF2 is a major protective antigen,can induce pigs to produce neutralizing antibodies to form an effective protective force.Therefore,using yeast expression system and E.coli expression system respectively to express PCV2 Cap protein for the preparation of efficient and inexpensive subunit vaccine has a broad application prospect.The main research contents are as follows: 1 The expression of procinre circovirus type Cap protein.1.1 The expression of procinre circovirus type2 b Cap protein in yeast expression system.The ORF2 gene without the nuclear localization signal(NLS)was cloned into the expression plasmid pPICZ?A to obtain recombinant plasmid pPICZ?A-ORF2 b.Transform linearized pPICZ?A-ORF2 b to Pichia pastoris strain X-33 competent cells and cultivate in YPD plates containing 1000?g/mL Zeocin to obtain six high resistance recombined strains containing the target gene.The recombinant strains was induced and detected by SDS-PAGE and Western blot.Optimizate the induce conditions in shake flask,the best conditions: pH=7.0,induction time 144 h,methanol concentration of 1%.1.2 The expression of procinre circovirus type2 b Cap protein and type2 d Cap protein in E.coli expression system.The ORF2 gene of PCV2 b without the nuclear localization signal(NLS)and the ORF2 gene of PCV2 d with the nuclear localization signal was cloned into the expression plasmid pET28 a to obtaine recombinant plasmid pET28a-ORF2.Transform pET28aORF2 to E.coli strain BL21 competent cells transformed pGro7.The recombinant strains was induced and detected by SDS-PAGE and Western blot.Optimizate the induce conditions,the best conditions: induction time 16 h,the temperature 16?.2 The evalution of the immune effect of PCV2 Cap subunit vaccine.The expressed Cap proteins were purified by nickel column respectively.PCV2 b Cap protein expressed in E.coli is 203?g/mL,PCV2 d Cap protein expressed in E.coli is 80?g/mL and PCV2 b Cap protein expressed in Pichia is 108?g/mL.Prepared Cap subunit vaccine with adjuvant of ISA201 VG,then immunized 6-8 weeks old BALB/c mice at doses of 4.0?g,same with the control group.The results showed:Each group could produce ELISA antibodies against PCV2.The antibody level after the first immunization has tended to increase and antibody level peaked after the second immunization two weeks later.Antibody level of Boheringer group was higher than PCV2b(Pichia)group,PCV2d(E.coli)group was higher than Boheringer group,PCV2b(E.coli)group was higher than PCV2d(E.coli)group,PCV2b(BEVS)group was higher than PCV2b(E.coli)group.Compared to the PBS group,the vaccine groups can produce neutralizing antibodies against PCV2.Detect serum cyokines IL-4,IFN-?,IFN-? by ELISA Kit.Compared with PBS group content of IFN-?,2b(E.coli)group,2b(BEVS)group,Boehringer group had significant differences(P <0.05),the difference between the other groups was not significant.Compared with PBS group content of IL-4,2b(E.coli)group were significantly different(P <0.05);the other groups were extremely significant(P <0.01).Compared with PBS group content of IFN-?,the vaccinated groups were extremely significant(P<0.01).To further assess the efficacy of PCV2 immunity subunit vaccine,all the groups were inoculated at the 21 d after the second immune.Three weeks after challenge,lung was collected for the detection of viral load by RT-PCR.Detection of virus load in lung: Compared with PBS group,PCV2b(E.coli)group,PCV2b(Pichia)group and 2b(BEVS)had significant differences(P<0.05),PCV2d(E.coli)group were extremely significant(P<0.01).Tissue sections showed that the vaccine groups have no serious pathological changes.