| A pair of specific primers were designed and synthesized according to S1 genesequences of Avian reovirus(ARV) from GenBank, which included the whole open readingframe(ORF) of P10 and P17 nonstructral protein genes.Both P10 and P17 protein genes of 13ARV strains were amplified,cloned and sequenced.The whole ORF of P10 and P17 proteingenes of those 13 ARV strains were 297bp and 441bp in length,respectively.And they werepredicted to encode P10 and P17 nonstructral proteins of 98 and 146 amino acids,respectively.The percent ages of nucleotide and aminoacid sequence identities weredetermined.The nucleotide and aminoacid sequence identities of P10 protein gene amongthose 13 ARV strains exhibited 96.6％-100％and 91.9％-99.0％,respectively.The identities ofP17 protein gene showed 98.2％-100％and 95.2％-99.3％, respectively. Comparison of thenucleotide and predicted aminoacid sequences and phylogenetic analysis between ARV andother orthoreoviruses (DRV, NBV, RAM21,SOM24) showed that Reovirus had the divergenceamong regions and species.RT-PCR amplify the Whole Reading Frame (ORF) of P17 protein gene from ARV S1133strains. The product ligated with PGEX-4T-1 Prokaryotic expression Vector, and transformedinto Escherichia BL21 competent cell. The recombination was induced with 0.2mmol/L ofIPTG. The results of SDS-PAGE analysis revealed that the molecular weight of the expressedproduct was 42.4kD. Western-blotting analysis showed that the Prokaryotic expression P17recombinant protein could be specifically recognized by the positive sera of avian infectedwith ARV.In additional,two structural proteinδ3 andδ2,and two nonstructural proteinδNS andP17 of ARV were Prokaryotic expressed.The inclusion bodies of four proteins were purifiedby urea and Triton X-100.The concentration of these four proteins were 38mg/mL,48mg/mL,28mg/mL,46mg/mL, respectively. The purity of urea-purified fusion proteins were 68.6％, 75.7％,84.0％and 85.0％, respectively. Achieves a higher density and purity, which showedthat this experimental exploration can effective purify the four recombinant proteins.Finally, These four proteins were used for coating antigen of indirect enzyme-linkedimmunosorbent assay (ELISA) after purified.The optimal conditions was determined.It wasshown that the optimal of the coating concentration of these proteins were 9.5μg/mL, 12.0μg/mL,9.3μg/mL and 11.5μg/mL respectively, all the optimal dilution of serum sample were1:200, the optimal dilution of enzyme-labeled goat-chicken IgG were 1:5000. These fourrecombinant proteins showed no reaction with antiserum to NDV, AI,IBDV.Which indicated that these indirect ELISA had good specifity for detetion of ARVantibody in serum. Our research had developmented ofδ3-ELISA,δ2-ELISA andδ3-δ2ELISAmethods,and use these methods to detected the serums which were infected with ARV orinjected with died vaccine.The result showed thatδ3-δ2-ELISA was the most sensitivity inARV antibody detection.We used the same method to development ofδNS-ELISA,P17-ELISA andδNS-P17-ELISA, It show that detection of antibodies of ARVnonstructural proteinδNS and P17 can distinguish between vaccinated and infected chickens.Compared to the sensitivity ofδNS-ELISA,P17-ELISA andδNS-P17-ELISA,and relationwith statistics analysis,we can indicated that theδNS-P17-ELISA which two nonstructuralproteins as coating antigen simultaneity has advantages over theδNS-ELISA and P17-ELISAin distinguish between ARV vaccinated and infected serum samples.
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