| Porcine circovirus type 2(PCV-2),the main pathogen,leads to Postweanig multisystemic wasting syndrome(PMWS).Besides,this virus also can lead to immunosuppression,multiple concurrent diseases as well as secondary infection,bringing huge economic losses to pig industry.Vaccination is the main measure to prevent PCV-2.However,the existing vaccines can only prevent the happening of the diseases but cannot avoid the infection.Wild virus infected pigs still exist in the immunized swine flocks.To eliminate them from the immunized swine flocks,it is necessary to establish a method for large-scale identification of wild virus infection.The purpose of this study was to establish an ELISA method for detection of antibodies of wild virus infection.In order to identify the antigenicity of PCV-2 ORF3 proteins,firstly this study inserted ORF3 gene into pT7-IRES expression vectors.And then Cell-free protein synthesis system of human cell extracts was used to express proteins.Finally,the author employed swine serums infected by wild virus to identify the antigenicity of expressed proteins by Western blot analysis.For the sake of obtaining a large number of expressed proteins to purify and establishing an ELISA method,we did some attempts.The author Constructed recombinant expression plasmids by inserting ORF3 gene into the pET-28 expression vectors.Then expressed proteins after identification and purification was employed as a coating antigen to establish indirect ELISA.Finally,wild virus infected clinical samples were detected by the newly constructed ELISA method.Test results showed that PCV-2 ORF3 gene was inserted into the pT7-IRES expression vectors and then built pT7 PCV-2-ORF3 expression plasmids.And then using Cell-free protein synthesis system of human cell extracts,the plasmids could express the ORF3 protein specifically.In Western-blot test,expressed protein could specificially react with swine serums infected PCV-2 wild virus.This result showed that ORF3 protein was capable of stimulating the body to produce antibodies and specificially reacting with them.For obtaining numerous recombinant expressed proteins to purify,ORF3 gene was inserted the pET-28 a prokaryotic expression vectors and successfully built a pET-PCV-2-ORF3 prokaryotic expression plasmids.Expression could be efficiently induced after transforming to BL21(DE3)e.coli.Western-blot test suggested that the expressed proteins could react with 6 xhis monoclonal antibodies and swine serums infected by wild virus but they were unable to react with mice serums immunized by PCV-2 CAP protein recombinant vaccines.The ORF3 proteins of prokaryotic expression were purified by affinity chromatography.The indirect ELISA method was preliminary established by using the ORF3 proteins as coating antigens.Results of 40 clinical serums test showed that the method can detect 24 positive serums from 26 known positive serums.Positive coincidence rate is 92%.This study showed that PCV-2 ORF3 had good immunogenicity,and ELISA using PCV-2 ORF3 as antigens can be employed for the detection of PCV-2 wild virus infection.Combined with the application of recombinant protein vaccines,the new method was better able to prevent,control and purify the diseases. |
