Development Of PCV2 Cap Protein Monoclonal Antibody And Establishment Of Direct Immunofluorescence Method

Porcine circovirus type 2（PCV2）is a single stranded circular DNA virus without envelope.The genome has five open reading frames.Among them,ORF2 encoding a structural protein（Cap protein）is the main immunogenicity of the virus.Protein is also the only protein associated with PCV2 infection.PCV2 is one of the important pathogens that cause economic losses in the world’s pig industry,and can lead to post-weaning pigs multi-systemic wasting syndrome（PMWS）.Therefore,a fast and accurate method is particularly important for the diagnosis of PCV2 infection.This study intends to establish an immunofluorescence detection method by preparing a monoclonal antibody to the PCV2 Cap protein to lay a foundation for the clinical diagnosis and pathogenic mechanism of PCV2.To prepare Cap protein monoclonal antibody,use PCV2 Cap protein expressed by prokaryotic cells as immunogen to immunize BALB/c mice,use in vitro cell fusion technology,after multiple subcloning and screening,and finally obtain 6 stable secretion strains The antibody hybridoma cell lines were named3G1,4D2,2B7,3G5,4C2,and 3B10.Among the 6 hybridoma cell lines,the subclasses of 3G1,4D2,and3B10 were all subclasses of Ig G2a,2B7,3G5,and 4C2.The class is Ig M;indirect immunofluorescence experiment（IFA）and Western blot results show that all 6 monoclonal antibodies can react specifically with PCV2;at the same time,Western blot analysis shows that the 6 monoclonal antibodies all interact with the Cap protein expressed by the recombinant Reus A specific reaction occurred,confirming that all6 monoclonal antibodies were specific to the PCV2 Cap protein.In vitro culture was continuously passaged to the 45th generation,and the titer of secreted antibodies was basically the same.After neutralizing activity identification,3G1 has neutralizing activity,and the other 5 strains have no neutralizing activity.The hybridoma cells 3G1,4D2,and 3G5 were injected intraperitoneally into BALB/c mice to prepare ascites.The ascites titers could reach 1:204800,1:51200,and 1:51200,respectively;all three monoclonal antibodies ascites could specifically recognize PCV2,and It does not recognize other viruses（PCV3,TGEV and PEDV）,indicating that the prepared ascites has good specificity.In order to confirm that the prepared monoclonal antibody recognizes the epitope region of the Cap protein,the Cap protein was truncated and expressed in this study.After removing the nuclear localization signal of the Cap protein,primers were designed to construct a vector and express three proteins,named Cap A（aa42-103）,Cap B（aa96-139）and Cap C（aa132-234）.The truncated expressed protein was reacted with monoclonal antibodies,and the epitope regions recognized by the two monoclonal antibodies 3G1and 4D2 were preliminarily identified at aa132-234.In view of the good reactivity of 3G1,the ascites of the prepared 3G1 monoclonal antibody cells was purified through a Protein G purification column,and its titer was determined to be 1:204800.The purified ascites was labeled with a phycoerythrin（R-phycoerythrin,R-PE）labeling kit to form the fluorescent antibody R-PE-3G1,and the PCV2 monolayer cells were detected.There was no significant difference compared with the unlabeled antibody test results,indicating the successful establishment of PCV2 direct Immunofluorescence detection method,and optimize the established method;This method can specifically detect porcine circovirus type 2（PCV2）,has no cross-reactivity with PEDV,TGEV and PCV3,and can detect 10⁴ virus dilution(ie 1 TCID₅₀);And the reactivity of the labeled fluorescent antibody remains stable after storage for 4 months.PCV2 was infected with BALB/c mice.The lungs and spleens of the infected mice were identified by PCR to identify the proliferation of the virus,and frozen sections of the lungs and spleen were made.Direct immunofluorescence was used to detect the distribution of the virus in the tissues.The experimental results showed that the lungs and spleen PCV2 virus was detected in the spleen with specific fluorescence,which was consistent with the PCR identification results.In summary,this study obtained 6 monoclonal antibodies that specifically react with PCV2,and initially identified the epitope regions recognized by the monoclonal antibodies 3G1 and 4D2,and established the detection of PCV2 with the phycoerythrin-labeled monoclonal antibody 3G1.The above results lay a foundation for the further development of porcine circovirus type 2 detection technology and the study of the pathogenic mechanism of the virus.