Expression Of Fragment Of ORF2 Of Circovirus Type 2 In E.Coli And The Development Of Indirct ELISA With Recombinant Protein

Porcine circovirus type 2 (PCV2) is the primary causative agent ofpostwening multisystemic wasting syndrome (PMWS) and other associateddiseaes. It has made a great loss in world's swine industry. It will provide PCV2prevent and control step if we know the epidemic condition of PCV2. The ORF2gene of PCV encodes the major immunogenic capisd protein. Sequence analysesrevealed that ORF2 gene of PCV2 shares about 67% nucleotide sequenceidentity with that of PCV1. There are common antigenic determinants in the Capprotein between the two types of PCVs, however, no cross-reaction of antigenicityis observed between them.Therefore, the ORF2 gene can be used to differentiatePCV1 and PCV2.Based on the porcine circovirus type 2 (PCV2)LC strain, 5 pair of primerswere designed according to the Luchuan sequence, the ORF2, ORF2a, ORF2b, ORF2cand ORF2d gene was amplified from the PCV2-LC plasmids by PCR, andinserted into the prokaryotic expression vector PET-32a. After identified byBamHⅠand HindⅢand sequencing, the constructed recombinant expressionplasmids were transformed into the receptive cells of E. coli BL21 and induced by IPTG with a finial concentration lmmol/L,then the recombinant protein wasexpressed in the form of inclusion bodies.The expression protein could reactwith polyclonal antibody against PCV2 by Western-Blot,sharing a goodantigencity.After the inclusion bodies of recombinant proteins were purifiedwith washing buffer consisiting of 2mol/L,4mol/L,8mol/L urea for severaltimes,the recombinant proteins were purified by Ni2+ affinity columns.An indirect enzyme-linked immunostorbent assay (ELISA) based on therecombinant protein was developed.Using the purified recombinant protein ascoating antigen,The optimal coating condition were determined.It was shownthat the optimal concentration of recombinant protein was 0.24μg/ml,coatingtime was 37℃for 2 hours and 4℃overnight,the dilution of serum sample was1:40,the working concentration of HRP-labeled rabbit anti-protein IgG was1:4000,serum sample for detecting and HRP-labeled rabbit anti-protein IgGshould be incubated at 37℃for 30 rain before terminated with the stoppingsolution,the threshold for ELISA was 0.39.160 serum samples were detected by the method and market ELISA kitrespectively. Compared with the market ELISA kit, the sensitivity andspecificity of the ELISA were 90.4％and 97.2％respectively, and the totalcoincident rate between the two methods reached at 95％. No significationdifference was found between the two methods(P＞0.05). The results showedthat the developed indirect ELISA had the advantages such as high sensibility,strong specificity and good repeatability and the method can be used to detect the porcine serum antibodies to PCV2...