| Porcine circovirus type 2(PCV2) is one of the primary causative agents of postwening multisystemic wasting syndrome(PMWS) and other associsted diseases.After porcine reproductive and respiratory syndrome(PRRS),PMWS is defined as one of the immunological suppression diseases of pigs.It has cased general popularity and made great economy loss.It aroused a great reconstruction in worldwide swine industry.Now,there are no favourable vaccines and drugs to control diseases.In order to preclude and monitor the diseases,a serology method must be established to detect the popular information of diseases by PCV2.According to the differences between antigenicity and genetic constitution,PCV has two types,one is PCV1 and the other is PCV2.PCV1 has no pathogenicity,PCV2 has pathogenicity.Open reading frame(ORF) 1 is contained in PCV1 and PCV2.It encodes virus replication(Rep) protein.Rep protein of PCV1 shares about 86%nucleotide sequence identity with that of PCV2,which is the major reason why PCV1 and PCV2 have cross-reaction of antigenicity.The ORF2 gene of PCV encodes the major constitutive protein.Cap protein of PCV1 shares about 65%nucleotide sequence identity with that of PCV2.There is no cross-reaction of antigenicity between them,and it has good immunogenicity.Therefore,the ORF2 gene could be used to differentiate PCV1 and PCV2.This takes advantage to diagnose PCV2 and vaccine research.The indirect ELISA assay had high sensibility,specificity.The prokaryotic expression protein of ORF2 could be used to establish the indirect ELISA assay as antigen.It is used to detect porcine serum antibodies to PCV2.Total ORF2 gene was low expressed in E.coli,so EcoRI and XhoI were used to excise the nucleic localization region and pMD-18T from pMD-ORF2.The part ORF2 as exogenous gene was cloned and inserted into the prokaryotic expression vector pGEX-6P-1.Restrict enzyme identification confirmed that the recombinant expression plasmids were constructed successfully.The recombinant expression plasmids were transformed into the receptive cells of E.coli BL21 and induced by IPTG with a final concentration 1 mmol/L. The expression protein had a high production and could react with polyclonal antibody against PCV2 by SDS-PAGE and Western-blot,sharing a good reactionogencity.The recombinant expression plasmid pGEX-ORFc was induced by IPTG in E.coli BL21.The recombinant protein was expressed in the form of inclusion bodies in the E.coli.After the bacterium cell walls were disrupted by ultrasonic wave,the inclusion bodies of recombinant proteins were cleaned,dissolved, annealed by urea for different concentration.At last,the recombinant proteins were purified by electrodialysis method.An indirect enzyme-linked immunosorbent assay(ELISA) based on the recombinant protein was developed.Using the purified recombinant protein as coating antigen,the optimal coating conditions were determined.It was shown that the optimal concentration of recombinant protein was 12.85 ug/mL,coating time was 37℃for 2 hours and 4℃overnight,0.5%BSA/PBST was used as the best confining liquid,after being confined for 2 hours at 37℃,the dilution of serum sample was 1:40,reacting time was 37℃for 1 hour, the working concentration of HRP-labeled rabbit anti-swine protein IgG was 1:1 000,the detection should be incubated at 37℃for 45 minutes before terminated with the stopping solution,the threshold for ELISA was 0.39.The 179 serum samples were detected from JiLin Province by this method,the total masculine ratio was 42.5%.The result showed that the developed indirect ELISA had the advantages such as high sensibility,strong specificity and this method could be used to detect the porcine serum antibodies to PCV2.It would found the base of developing a ELISA diagnostic kit of PCV2,and provide a quick scientific method for diagnosing PCV2 diseases in wide scope.
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