Prokaryotic Expression Of PCV2-ORF2 Protein And The Establishment And Preliminary Application Of Indirect ELISA Method

ObjectiveThe recombinant expression vector containting the ORF2 gene of porcine circovirus PCV2 was constructed and expressed in E.coli.After purified,the expressed protein was analyzed.The indirect ELISA method was established to detect human anti-PCV2-Ig G,which was initially applied to clinical screening for possible PCV2 infection.Methods1.Prokaryotic expression of ORF2 gene in PCV2The PCV2 gene sequence of PCV2 was optimized,and the optimized gene sequence was synthesised and subcloned into the prokaryotic expression vector p ET-30 a.The recombinant plasmid p ET-30a-PCV2-ORF2 was constructed.The recombinant plasmid was transferred into E.coli BL21(DE3)and induced by IPTG for expression.The expressed product was analyzed by SDS-PAGE,verified by Western blot and purified by Ni-chelating affinity chromatography.2.An indirect ELISA for detection of human anti-PCV2-Ig GAn indirect ELISA was developed by coating microwell plates with the purified protein,then its reliability test and stability test were carried out after optimization.1400 serum samples were collected from September 2018 to February 2021 in a hospital in Fujian province to detect the serum-specific PCV2-Ig G antibodies to estimate PCV2 infection in the population of Fujian Province.3.Detection of PCV2 Infection in serum samples by PCRA pair of primers were designed to amplify a partial fragment of PCV2-ORF2,referring to PCV-2 complete genome published in Genbank.The PCR product was about 780 bp in size detected by gelose gel electrophoresis.All the possible infected serum samples were detected by the PCR for PCV2.Results1.p ET-30a-PCV2-ORF2 vector was successfully constructed and the recombinant protein was obtained.The ORF2 gene of PCV2 was synthesised and subcloned into the prokaryotic expression vector p ET-30 a.Then a 27 KD protein was expressed in recombinant strain BL21 after induced by IPTG at 37℃,which was confirmed by SDS-PAGE and Western blot analysis.The recombinant protein was purified through Ni-chelating affinity hromatography.2.PCV2 antibody was detected by indirect ELISA.Indirect ELISA was developed by coating microwell plates with the purified protein.The conditions for the ELISA were optimized as following: 11.9 μg/ml as the final coating concentration of the recombinant protein,overnight at 4℃for the recombinant protein coating,1:100 as the dilution fold for serum samples.Among 1400 serum samples tested,PCV2 specific antibody positive cases were detected,and the positive rate(crude rate)of PCV2 antibody was 16 in1400,indicating that there were some cases of occult infection of PCV2 in adult population.The positive rate of serum PCV2-Ig G was 1.09% in healthy subjects and 1.33% in inpatients,there was no significant difference between them.3.A specific PCR detecting method for PCV2 was constructed.All the possible infected serum samples were detected by the PCR,no specimens were positive for PCV2.Conclusions1.The recombinant vector p ET-30a-PCV2-ORF2 was successfully constructed and could be expressed efficiently in the expression system of E.coli.The target protein with high purity was obtained by Ni-chelating affinity chromatography.An indirect ELISA was developed by coating microwell plates with the purified protein.2.The detection of antibodies against PCV2 in the sera of humans in Fujian province indicates that there is latent infection of PCV2 in humans.There was no significant difference between the positive rate of serum PCV2-Ig G in healthy subjects and the positive rate of serum PCV2-Ig G in hospitalized patients,which indicated that there was no significant correlation between the positive rate of human PCV2-Ig G and the current disease of hospitalized patients to a certain extent.3.All the possible infected serum samples were detected by the PCR specific for PCV2,there were no specimens were positive.