The Establishment And Application Of A Colloidal Gold Immunochromatographic Method For Porcine Circovirus Type 3

PCV3 has been widely distributed in pigs in recent years,but so far,no eff ective vaccine has been developed to immunize pigs.It is not only necessary to strengthen feeding management to control the disease,but also to establish a m ethod for rapid diagnosis of PCV3,so that sick pigs can be diagnosed and treat ed as early as possible.A novel immunochromatographic colloidal gold method was established to test the antigen preparation,bioinformatics analysis,and prepa ration of immunochromatographic colloidal gold test strips for PCV3 in three par ts as follows:1.To obtain porcine circovirus type 3 capsid protein（PCV3-Cap protein）,th e PCV3 full base from Gen Bank accession number:MK105924 was used to stud y the bioinformatic characteristics of PCV3 structural protein Cap protein and ant igenic epitope prediction.The molecular formula,molecular size,relative molecul ar mass,protein isoelectric point,half-life,secondary structure,tertiary structure,hydrophilicity,accessibility,plasticity,linear epitope,and B-cell antigenic epitope of recombinant PCV3-Cap amino acid sequence were analyzed by using DNAsatr software to screen out the regions that are not easy to be antigenic epitopes,an d finally screen out the high-quality antigenic sites for Protein Blast analysis was performed.The results showed that PCV3-Cap has a molecular formula C₁₁₅₉H₁₇₈₇N₃₃₇O₃₀₃S₆,an isoelectric point of 10.92 and a secondary structure withα-helix,β-fold,β-turn and irregular curl.There is no transmembrane region in PCV3-Ca p protein,no signal peptide,and the nuclear localization signal is located at 4-32:RAIFRRRRPRPRRRRRRRHRRRRYARRRRLFIRR.7 antigenic sites were deri ved after excluding regions that are not easily formed into antigenic epitopes and were validated by Protein Blast to be compatible with PCV3-Cap.Protein Blast verified 100%homology with PCV3.2.To obtain the porcine circovirus type 3 capsid protein（PCV3-Cap protei n）,the PCV3-Cap protein sequence was obtained from the PCV3 full gene seque nce with Gen Bank accession number:MK105924 as the target gene,and was int roduced into the gene expression vector.After verification,the plasmid was intro duced into Rosetta expression bacteria to construct a prokaryotic expression syste m,and the optimal IPTG induction concentration and induction expression time were screened.Bacteriolytic treatment was performed to detect the existence form of the target protein after expression.Purification conditions were screened.Afte r dialysis and refolding,the murine anti-PCV3 monoclonal antibody was used to perform Western Blot to verify the reactogenicity of the target protein.The resu lts showed that the 42 k Da PCV3-Cap protein was obtained through the construc ted expression system;the target protein was mainly present in the precipitate in the form of inclusion bodies after the bacteriostatic treatment;the protein expres sion was induced after 8 hours of culture with 0.2 mmol/L IPTG at 37℃The a mount is higher;the purification effect is better when eluted with 300m M imidaz ole;Western Blot verified that the target protein can be captured by the mouse a nti-PCV3 monoclonal antibody and has good reactogenicity.3.In order to establish a colloidal gold immunochromatographic technique f or the detection of PCV3 antibodies,the optimal labeling PH and optimal SPA l abeling amount of colloidal gold were screened,the colloidal gold complex soluti on was screened,and the optimal scribing concentration was screened.After sele cting the optimal conditions,chicken anti-SPA antibody was immobilized in the q uality control line and recombinant PCV3-Cap protein was immobilized in the de tection line,the test strips were assembled,and their stability and The test strips were assembled,and their stability and specificity were verified.38 clinical seru m samples were randomly taken for testing.The results showed that the optimal labeling p H of colloidal gold was 8.0,the optimal SPA labeling concentration was 2.5μg/m L,and the optimal T C line concentration was 0.4 mg/m L,and the test strips had good stability and specificity with good sensitivity even after sea led and dry storage for 2 months,and the results of random clinical testing sho wed that the colloidal gold immunochromatographic test strips were consistent wi th ELISA results.This experiment successfully constructs the PCV3-Cap protein expression car rier,successfully expresses the PCV3-Cap protein,optimizes the expression and p urification conditions,provides scientific basis for the development of subunit vac cine and the development and research of PCV3 test reagent box,and provides a scientific basis for the establishment of PCV3-Cap rapid detection method.