Prokaryotic Expression Of Cap Protein Of Porcine Circovirus Serotype3 And Development Of An Indirect ELISA Diagnostic Method

Porcine circonvirus serotype 3(PCV3)was first discovered on a farm in North Carolina in the United States in 2015.The virus mainly causes reproductive disorders and respiratory disorders in pigs,Clinical manifestations were weight loss,dermatitis and nephrotic syndrome and other reactions.At present,the United States,Canada,Poland,and South Korea,through epidemiological surveys,have confirmed the prevalence of PCV3;In our country,the virus has been reported to be endemic in the central and eastern provinces such as Fujian,Guangzhou,Jiangxi,Shandong,and Henan.Its widespread prevalence and damage to the breeding and growth performance of pigs have caused great concern in the pig industry.Cap protein of PCV3 and PCV2,both of which have no cross-immunoprotective properties.Existing commercial PCV2 vaccines and PCV2 serum antibody ELISA test kits cannot provide effective immune protection and corresponding detection for potential PCV3 infected patients.Therefore,it is necessary to establish a serological detection method for detecting PCV3 in order to grasp its epidemic situation in time and lay a foundation for the serological investigation and etiological study of the virus.As a single-stranded circular DNA construct of PCV3,Cap protein encoded by its ORF2 gene is its major antigenic structural protein,and is associated with the neutralizing antibody.Based on the sequence of PCV3 ORF2 gene provided on NCBI GenBank,the main antigenic sites were analyzed and specific primers were designed.BamHI and SalI restriction sites were added to the primers at the upstream and downstream primers,and the target gene was amplified by PCR.The prokaryotic expression vector pET-32a was used to clone the target gene into pET-32a using T4 DNA ligase,and the recombinant expression vector pET-32a-PCV3 cap was constructed and transfected into DH5?competent cells.The plasmid was extracted and identified by PCR,restriction enzyme digestion and sequencing,which confirmed that the structure of the recombinant plasmid was complete.And the recombinant plasmid was transfected into transl-tl competent cells.The inducing conditions were optimized,and the expression product was identified by SDS-PAGE analysis and Western-blot analysis.The PCR results showed that the size of the PCR product was 1204 bp,641 bp,1094 bp,and 531 bp,respectively,using the universal primer T7 and the target gene primers.The size of the PCR product was consistent with the expected size,indicating that the Cap gene was inserted into the expression plasmid.Corresponding position;Identification by enzyme digestion and sequencing using BamHI and SalI showed that the recombinant expression vector pET-32a-PCV3cap was constructed.The optimal inducing conditions were as follows:At 37?,shaking approximately 4-6 h,the optimal concentration was IPTG 1.5 mmol/L,induction 8 h.The expressed bacteria were collected by ultrasonication and purified by Ni-NTA affinity chromatography column.The effluent was analyzed by SDS-PAGE.The results showed that the eluent of 400 mmoL/L imidazole concentration had the highest elution efficiency.Further Western-blot identification was performed.It was proved that the PCV3 Cap protein was expressed,and the Cap protein had a molecular weight of about 40 ku and had good reactogenicity.The prokaryotic expression product Cap protein was used as an antigen to coat the enzyme label and explore the establishment of an indirect ELISA for the detection of PCV3.The optimum coating antigen concentration,primary antibody concentration,secondary antibody concentration,and blocking solution were optimized for optimal working conditions.The principle of scientific statistics was used to calculate the critical value of the indirect ELISA method,and the specificity and reproducibility of the established method were verified.Finally,317 serum samples from pig farms in Fujian Province were tested using this method.The results showed that the optimal reaction conditions were as follows:the optimal coating antigen concentration was 0.009mg/mL,and the overnight coating was performed at 4?.The optimal dilution of the tested serum was 1:400,and 5%BSA was the best blocking solution.37 After blocking for 1h at 37?,the optimal dilution of enzyme-labeled antibody was 1:4000,the optimal reaction time of the enzyme-labeled secondary antibody was 0.5h,and the optimal time for the substrate fluid was 15min.The critical value was 0.24.The results of the reproducibility test showed that the coefficient of variation of the intra-and intra-item repeatability tests was less than 10%.The specific test results showed that this method only reacts with PCV3 positive serum,but it is also associated with the pigs foot-and-mouth disease(PMDV),pig jaundice(JEV),porcine pseudorabies(PRV)and porcine circovirus type 2(PCV2)virus positive serum no cross reaction.The indirect ELISA was used to detect PCV3 antibodies in pig farms in Fuzhou,Nanping,Longyan,Quzhou and Putian.The results showed that the number of positive serum was 91,and the overall positive rate was 28.71%.In summary,this study used the prokaryotic expression system to successfully construct the recombinant expression vector pET-32 a-PCV3 cap,and used the recombinant protein to establish an indirect ELISA method for clinical detection of PCV3 antibodies,and this method has high sensitivity and specificity,strong characteristics.It provides a certain technical basis for the investigation of the epidemiology of swine adenovirus type 3 and the development of serological test kits.