Soluble Expression Of Hexon Hypervariable Region And Establishment Of Indirect ELISA Of Porcine Adenovirus Serotype 3

Porcine adenovirus serotype 3 is a common virus in clinic.Infected pigs often have mild diarrhea symptoms and and often mixed with other viruses that cause diarrhea in pigs.The current methods of PADV-3 detection are common PCR or fluorescence quantitative PCR,and there are few related researches on serological detection.Hexon protein is the main protein in the capsid of PADV-3,which can stimulate the production of neutralizing antibody,so it is generally as a major concern for PADV-3 detection.Hexon full-length genes have expressed in prokaryotic expression systems,but the expressed protein are in the form of inclusions.It is reported that SUMO could promote the proper folding of the target protein,and increase the protein expression and shield the toxicity.Thus SUMO tag was used to improve the solubility of Hexon in E.coli.and a serological detection method of ELISA was established based on hypervariable region protein of Hexon in the study.The hypervariable region HVR1-6 of Hexon was amplified and the yeast-derived SUMO gene or pig-derived SUMO1 gene or SUMO3 gene were added at N-terminal,then they were inserted into pET28a.The recombinant plasmids were transformed into BL21(DE3)for induction expression.The fusion protein with highest expression was selected to optimize its induction condition.A large number of fusion proteins were obtained according to the optimal conditions,and then purified by nickel column.The fusion protein was detected by SDS-PAGE analysis and western-blot.The result showed that pig-derived SUMO3 had the best effect of improving the solubility of hexon protein.The optimized temperature of the fusion protein SUMO3-Hexon-HVR1-6 was 37℃ and the optimized concentration of IPTG was 0.1 mM.The fusion protein concentration was 6mg/mL after purifying by nickel column.According to SDS-PAGE analysis and western-blot detection,the fusion protein showed good purification effect and specificity.In order to effectively detect the positive rate of PADV-3 in pigs,the fusion pretein SUMO3-HVR1-6 expression by prokaryotic was used as coating antigen of indirect ELISA detection methods and the sensitivity and specificity of the method were evaluated.The result showed that the optimized concentration of coating antigen was 6 μg/mL;the optimal dilution and condition of serum sample were 1:80 and 37℃;the optimal dilution and condition of enzyme-labled second antibody was no dilution and 37℃ 1h;the critical value of the positive sample and negative sample was 0.362.There is no serological cross-reaction when detecting several common porcine viruses positive serums using this ELISA method.Positive serum could be detected when serum was diluted at 1:320.The coefficients of variation(CV)of inter-batch repetitive was between 1.0%and 3.9%,the coefficients of variation of intra-batch repetitive was between 0.5%and 8.3%.The coincidence rate between this ELISA method and PCR nucleotide detection was 88%.241 serum samples collected from different pig farm were detected by the established indirect ELISA method,and 48 positive serum samples were detected,with a positive rate of 19.9%.The indirect ELISA established in this experiment is a rapid and sensitive method for detecting PADV-3 or investigating its epidemiology.