Construction Of PCV3 Infectious Clone And Preparation Of PCV3 Capsid Protein Monoclonal Antibody And Preliminary Application

Porcine circovirus（PCV）has long threatened the health of pig industry.The PCV3 that has emerged in recent years has brought huge economic losses to the pig industry.The use of vaccines and early rapid diagnosis of pathogens can help prevent the spread of the virus.Since the PCV3 infection and replication mechanism has not yet been clarified,the separation of the PCV3 virus remains stagnant.Therefore,obtaining PCV3 pathogen and establishing PCV3 early diagnosis method are of great significance for prevention and control of PCV3 infection.At present,many laboratory detection methods have been used for early diagnosis of PCV3,but few studies have reported on the rapid detection methods of PCV3.The acquisition of good antibodies and the corresponding standard pathogens are the basis for the development of rapid detection kitss.In this study,based on the biological characteristics and gene structure characteristics of PCV virus,the specific monoclonal antibody of PCV3 Cap protein was prepared and the infectious clone of PCV3 was obtained.On this basis,the PCV3 fluorescence immunochromatographic test strip was prepared based on the obtained antibody,and the preliminary detection of PCV3 infected materials in Guangdong was carried out at the same time.Finally,the application value of PCV3fluorescence immunochromatographic test strip was evaluated comprehensively.Obtaining good antibodies is the basis of establishing relevant immunological diagnostic kitss.According to the biological characteristics of single capsid protein of PCV virus,PCV3,PCV2 and PCV1 Cap proteins were prepared by prokaryotic expression method,and monoclonal antibodies were prepared based on PCV3 Cap protein.It was confirmed by antibody specific detection that the B6 and D17 monoclonal antibodies only specifically reacted with PCV3 Cap,but did not cross-react with PCV2 and PCV1 Cap proteins.The results of subtype analysis of the antibody indicated that the B6 antibody subtype was Ig G1,while the D17 antibody subtype was Ig G2a.The titer of B6 antibody was 2×10⁵and that of D17 antibody was 6×10⁵by indirect ELISA test.In addition,the antibodies obtained by immunoblotting assay were used to detect PCV3 Cap protein and PCV3.The results showed that both B6 and D17 monoclonal antibodies could react with PCV3 Cap protein and PCV3 tissue by immunoblotting.Although it has been preliminarily confirmed that monoclonal antibodies can interact with PCV3 virus,in order to finally verify the obtained monoclonal antibodies,it is necessary to obtain the standard strain of PCV3 virus.However,there are few reports on the isolation of PCV3 virus.In this study,according to the characteristics of PCV genomic DNA as a circular structure,the existing linearPCV3 gene sequences were rearranged so that there was a single restriction site at both ends of the rearranged linear DNA sequence.Finally,PCV3 circular DNA was obtained in vitro by conventional molecular biological manipulation.Because PCV has the characteristics of infecting lymphocytes and macrophages,the obtained PCV3 circular DNA was transfected into porcine alveolar macrophage cell line to save PCV3 virus.In addition,the reverse amplification primers were designed according to the sequence of PCV3 linear DNA.In the process of PCR reaction,the primers could only bind to the linear DNA template,but could not be amplified.However,for circular DNA,this primer can be amplified by PCR.In view of this,this study used this primer and PCV3Cap gene specific detection primers to identify PCV3 virus in continuous culture for 6generations.The results showed that PCV3 circular DNA and its Cap gene could be detected in each generation of venom.In order to further verify the obtained virus strains,the titer of F3 generation PCV3 virus was detected in this study.The results showed that the TCID₅₀of F3 generation PCV3 was 10^-4.8/m L.In order to evaluate the pathogenicity of the constructed virus,Kunming mice were used as the infection model.However,compared with the negative control group,the viscera of mice in the infected group and the control group showed no obvious pathological changes.We speculated that mice may only be carriers of PCV3 virus,so this study further detected the heart,liver,spleen,lung and kidney of mice by PCR and immunological methods.The results showed that the Cap gene of PCV3 virus was detected in the myocardium and lungs of infected mice.Immunoblotting and immunohistochemistry confirmed the existence of PCV3 virus in the myocardium and alveoli of mice.This is consistent with the current reports that PCV3 can infect mice.The above studies verified the monoclonal antibody of PCV3 Cap protein and PCV3virus strain,and finally proved that the monoclonal antibody can react with PCV3 virus.Based on the acquisition of antibodies,the corresponding detection kitss can be developed.However,in order to comprehensively evaluate the application value of the detection kits,a large number of clinical cases need to be tested and verified.In order to obtain valuable clinical samples,fluorescence quantitative detection methods of PCV2 and PCV3 were established in this study.The results show that the lowest copy number of PCV2fluorescence quantitative PCR detection is 9.35×10¹copies/μL,while the lowest copy number of PCV3 detection is 1.05×10¹copies/μL.Based on this,a dual fluorescence quantitative PCR detection method for simultaneous detection of PCV2 and PCV3 was established.The collected samples were detected by this method,and the results showed that 160 samples were detected.45 samples of PCV2 and PCV3 were not detected.Among the samples detected with PCV2 and PCV3 virus,80 samples were PCV2 positive,the positive rate was 69.5%,and 35 samples were PCV3 positive,the positive rate was 30.4%.28 samples of PCV2 and PCV3 were detected,and the total infection rate was 24.3%.The fluorescence quantitative PCR detection method is more sensitive,but its detection cost is higher,the detection time is longer,and the sample needs to be processed.The establishment of rapid detection reagent of PCV3 is helpful for clinical front-line staff to prevent and control PCV3 in the early stage.In this study,on the basis of obtaining PCV3 Cap protein specific monoclonal antibody,PCV3 virus strain and clinical materials,the rapid detection reagent of PCV3 was studied.The appearance of fluorescence immunochromatographic test strip provided a rapid detection method for rapid detection of pathogens.Therefore,in this study,the obtained antibodies were coated,assembled and optimized for the activation dose of fluorescent microspheres and the dosage of antibodies.The results showed that the optimum volume of EDC（10 mg/m L）of activated fluorescent microspheres was 35μL,and the optimum quality of Cap antibody was 80μg.By detecting the sensitivity of the test strip,the results showed that the minimum concentration of PCV3 Cap protein detected by the established PCV3 fluorescence test strip was 10 ng/μL.The preliminary detection results of clinical samples showed that the sensitivity of the fluorescent immunochromatographic test strip detection method was lower than that of the PCR detection method.However,considering the factors such as cost detection and detection time,the test strip method still has important application value.This study provided a basis for the follow-up study on the virulence of PCV3,related pathogenic factors and the interaction between virus and host.At the same time,this study provided an auxiliary means for clinical detection of PCV3 virus.Finally,it lays the foundation for the prevention and control of PCV3 infection.