Identification Of B-cell Epitope Of Porcine Circovirus Type 3 And Preliminary Establishment Of RPA Detection Method

Porcine circovirus 3（PCV3）is a single-stranded circular DNA virus belonging to the circovirus of the family Circoviridae.Since the United States first reported PCV3 in 2016,PCV3 infection has subsequently appeared in pigs in different regions of Europe,Asia and other countries.Its clinical characteristics are porcine dermatitis and nephrotic syndrome（PDNS）,myocarditis,piglet diarrhea,sow reproductive disorders,abortion and multi-system diseases,which have brought new threats to the world’s swine industry.However,there is currently a lack of specific rapid diagnostic reagents for PCV3.Analysis of the specific epitopes of PCV3 Cap will lay the foundation for the development of PCV3 specific detection reagents.In addition,clinical frontline personnel urgently need a simple and rapid detection method.In this study,PCV3 Cap specific monoclonal antibodies were prepared based on the biological characteristics and genetic structural characteristics of porcine circovirus.The antigenic characteristics of the PCV3 Cap protein were analyzed,and B-cell epitopes were identified.In addition,Recombinase Polymerase Amplification（RPA）for PCV3 detection was initially established.In this study,truncated PCV3-Cap were synthesized and cloned into prokaryotic expression vector p ET-32a by removing nuclear localization signal sequence.Recombinant vectors of p ET-32a-PCV3-Cap was constructed successfully,which was transformed into E.coli BL21（DE3）.SDS-PAGE analysis after induction of expression revealed that PCV3recombinant Cap protein was expressed in the form of insoluble inclusion body.Furthermore,after optimized expression conditions,the PCV3 recombinant Cap protein was fully expressed at 37℃for 4 h induced by a final concentration of 1 mmol/L IPTG.The PCV3Cap fusion protein（about 35 k Da）was purified by using the Ni⁺protein purification column and the purified PCV3 Cap was used as the immunogen to immunize BALB/c mice.Finally,two positive hybridoma cells（B10D,C2B）were successfully obtained from fused hybridoma cell,by indirect ELISA screening and subcloning.The results of IFA and Western-blot showed that m Abs B10D and C2B could react with PCV3 Cap fusion protein.The analysis of antibody subtypes showed that m Ab B10D was heavy chain Ig G1,while m Ab C2B was heavy chain Ig G2a,and all light chains were K chains.The antibody specificity test was verified by PCV1 and PCV2 Cap.It was confirmed that m Abs B10D and C2B only had specific immune reaction with PCV3 Cap protein,and no cross reaction occurred.In addition,the titers of B10D and C2B were 1:204800 and 1:409600 respectively.The above results showed that PCV3 Cap had good immunogenicity,and the two m Abs produced by PCV3Cap had immunoactivity.In this study,the antigenic epitopes of PCV3 Cap protein B-cells were identified by the prediction analysis of antigenic epitopes theory and peptide scanning technology.First,we used software to predict the epitope of Cap protein B-cells.Then we designed and synthesized 20 polypeptides with 16 amino acid residues that overlap each other according to the whole amino acid sequence.The results showed that two linear epitopes,Cap⁴⁷NVISVGTPQNNKPWHA⁶²and Cap¹⁴¹HSRYFTPKPLLAGTTS¹⁵⁶,were identified by m Abs B10D and C2B respectively.The reverse verification results of the peptide mouse immunoassay show that only Cap47-62 can induce the body to produce antibodies that react with PCV3 Cap.Then using this method to further truncate the identified epitope of polypeptide ⁴⁷NVISVGTPQNNKPWHA⁶²,and finally determined that the precise epitope recognized by m Ab B10D was Cap⁵⁵QNNKPW⁶⁰.In addition,a PCV3 Recombinase Polymerase Amplification（RPA）detection method was established in this study.Based on multiple PCV3 gene sequences in Gen Bank,design six sets of basic RPA primers for the conserved region of the Cap gene,optimize the preliminary reaction conditions and perform specific tests.Moreover,sensitivity comparison was performed by classical PCR detection method and RPA detection method,and clinical samples were detected.The results showed that the optimal reaction conditions for this method were constant temperature amplification at 39℃for 30 min.The lowest sensitivity of this method is 4.56×10²copies/μL,which is consistent with the classical PCR method.And there were no cross-reactions with PCV2,PCV1,PRRSV,PRV and SVA in the specific test.The results of detected 35 clinical samples showed that 16 positive samples had a positive detection rate of 45.71%,which was consistent with the PCR detection rate,indicating that the detection method has certain clinical application value.In conclusion,PCV3 Cap specific monoclonal antibody was prepared in this study.Two B cell epitopes of PCV3 Cap protein were preliminarily screened using a short peptide synthesis method,and one B cell precise epitope Cap⁵⁵QNNKPW⁶⁰ was further identified.In addition,based on PCV3 Cap gene sequence,a PCV3 Recombinase Polymerase Amplification（RPA）detection method was established in this study,and provide certain technical support for subsequent early diagnosis of PCV3 infection.