Development Of Indirect ELISA For Detection Of Antibodies Against PCV3 And Research Of Subunit Vaccine

Porcine circovirus(PCV)belongs to the genus Circovirus of the family Circoviridae,includs PCV1,PCV2,and PCV3,PCV1 is not pathogenic,PCV2 infection of pig results in porcine circovirus associated disease(PCVAD),causing severe immunosupression and usually secondary infection,leading to severe economic losses to the pig industry.PCV3is an emerging porcine circovirus that is associated with porcine dermatitis and nephropathy syndrome(PDNS)-like clinical signs,reproductive failure,cardiac pathologies and multisystemic inflammation in piglets and sows.The PCV3 genome is about 2.0 kb in length,virions have an icosahedral structure without a capsule,and the diameter is 17-20 nm in size.The viral genome mainly comprises three open reading frames:ORF1,ORF2 and ORF3 genes,wherein the ORF1gene encodes a replicase protein(Rep,297aa);the ORF2 gene encodes a nucleocapsid protein(Cap,214aa),the N-terminale of Cap is rich in arginine and contains the viral nuclear localization signal region,It is associated with the localization of the virus and the induction of a specific immune response in vivo while ORF3 encodes a protein of unknown function(231aa).Because of being the major structural protein gene of PCV3,the ORF2 gene encodes Cap protein,and similarities with PCV1 and PCV2 Cap proteins are 21%and 36%,respectively.The available PCV2 vaccine does not provide protection for PCV3 infection.Therefore,it is very necessary to establish a rapid and specific serological test method to reveal the prevalence of PCV3 and to develop a new subunit vaccine to prevent PCV3related diseases.Therefore,the following study in performed:1.Development and application of indirect ELISA for detection of antibody against PCV3The bioinformatical prediction revealed that most antigen epitopes of PCV3 Cap protein located at the C terminal(carboxyl terminal),while first 33 amino acids at the N terminal of Cap protein are the nuclear localization sequences.In order to increase the protein expression,primers were designed to amplify a part partial ORF2 gene which does not contain first 360 nucleotides of N-terminal.PCV3 positive samples were used as templates for PCR amplification.The recombinant plasmid was constructed by cloning the PCR product into pET-30a vector,the construction of recombinant plasmid was confirmed by enzymatical enzyme digestion and sequencing.The recombinant plasmid was transfected into E.coli BL21 competent cells,expression of the recombinant protein was induced by adding 1mmol/L IPTG,then incubation at 37?for 6 h.Western blot result showed that the recombinant protein possessed good reactivity with positive serum against PCV3.The expressed protein was purified by Ni-NTA affinity chromatography.Finally,by using the Cap protein as a coating protein,an indirect ELISA method for detection of antibody against PCV3 was established.As a result,the optimal antigen concentration,dilution of tested serum,working dilution of enzyme conjugated antibody of ELISA were 1?g/mL,1:20 and 1:5000,respectively.The 60 negative sera were tested to determine positive cut off value that was 0.273 while coefficients of inter-and intra-batches were less than 10%,indicating the good repeatability of the method,positive virus sera against after dignoses negative,no false positive reaction,indicating its'high specificity.Finally,439 clinical serum samples were tested by this ELISA method where60.59%(266/439)of them were identified as positive.Hence,A rapid,sensitive and specific method for detection of antibody against porcine circovirus type 3(PCV3)was established.2.Study on the immunological characteristics of recombinant Cap protein subunit vaccineThe baculovirus expression system has a translation processing modification function,the expressed protein functions a natural protein and is a common carrier for producing VLPs.Based on the codon-optimized ORF2 gene,two different ORF2 genes were amplified by PCR:(1).The secreted ORF2-Cap protein(Sc:(35)33Cap),the first 33amino acids of the ORF2 gene,which in the nuclear localization sequence was truncated,the Honeybee Meltintin signal peptide(Hbm)was cloned and inserted into the pFastBac1vector plasmid.(2).The nuclear localization type ORF2-Cap protein(Pc:Cap),the N-terminus of the optimized full-length ORF2 gene was inserted into the KOZAK sequence,and cloned into the pFastBac1 vector plasmid.The two recombinant plasmids with the correct identification were transformed into DH10Bac competent cells,and the white positive clones were selected by blue-white plaque assay.The recombinant Bacmid was extracted and identified by PCR to confirm the correct recombinant Bacmid.Recombinant Bacmid was transfected into Sf9 cells using Cellfectin transfection reagent to yield recombinant baculoviruses rvBac-(35)33Cap and rvBac-Cap,respectively.The F4generation of MOI=2 recombinant virus was used to inoculate 1.5×10~6/ml Sf9 insect cells,the recombinant proteins Sc and Pc were correctly detected by SDS-PAGE,Western blot and indirect immunofluorescence assay(IFA).The sizes of Sc and Pc proteins were about23kDa and 30kDa,respectively.Each protein was self-assembled into approximately 20nm in diameter virus-like particles(VLPs)detected by transmission electron microscopy(TEM)that revealed two VLPs.The recombinant proteins and oil adjuvant in equal volume were used to prepare vaccines for immunizing mice 50?g/mice,the serum specific antibodies were produced two weeks after immunization,and the antibody level reached peak level at fourth week the booster immunization,the antibody remained positive for one month,after between the experimental group and the control group IFN-?level was extremely significant difference.The results indicated that VLPs could efficiently elicit humoral and cellular immunity in the body.In summary,in this study recombinant PCV3 Cap protein was used as an antigen to establish an indirect ELISA method for detecting PCV3 antibodies,providing a method for epidemiological investigation and providing a basis for the subsequent evaluation of virus-like particle-based subunit vaccines.