Expression Of The Gene Encoding The σ 3 Protein Of Avian Reovirus In Escherichia Coli And Development Of Elisa Based On Antigen Of Recombinant Protein

ARV infections cause Arthritis/tenosynovitis of chickens in clinical. It is also cause an immunological suppression in chicken both clinical and subclinical. ARV genomes encoded 15 proteins and the Protein σ 3, which is encoded by the S1 genome segment, has been identified as the cell-attchment protein because it is the only viral protein in extracts of infected cells that is able to bind specifically to avian cells. Furthermore, the fact that protein σ 3 completely blocks the binding of purified reovirions to avian cells indicates that the attachment of σ 3 to host cells is specific and saturable.The protein σ 3,as a antigen, can induced host cells release the antibody of neutralization. The present study expressed the gene of σ 3 protein in E.coli and using the expression as antigen,developed ELISA test of ARV.A pair of primers which have the emzyme-digestion was designed according to the S1 sequence of S1133 strain in Genbank (L39002).The S1 gene,a fragment of 1014 bp,was amplified by polymerase chain reation(PCR),and then was cloned into PGEX-4T-1 exprssion vector.The recombinant PGEX-σ₃ plasmd was transformed into E.coli DH₅a comptent cell of E.coli and induced by IPTG with a finial concentration of 1mmol/L.SDS-PAGE analysis show that the reombinant protein expressed could reach 25 percent of the whole bacterial protein. The protein expressed could react with ARV antibodies by Western-blot, sharing a good antigencity.An indirect enzyme-linked immunosorbent assay(ELISA) base on the recombinant protein was developed.Using the purified recombinant protein as coating antigen,the optimal conditions was determined.It was shown that the optimal of the coating concentration of the protein was 3.98 μg/ml,the optimal dilution of serum sample was l:200,the optimal reaction time of serum was 1.5 hours at 37℃,the optimal reaction enzyme-labeled rabbit-chicken IgG was 1 hour,the optimal time of substrate was 25 minutes.The recombinant protein showed no reaction with antiserum to NDV, AI , IBDV, ORT (Ornithobacterium rhinotracheal),indicated that the indirect ELISA had good specifity for detetion of ARV antibody in serum.The positive coincident rate of the developed ELISA was 91.3% according to the results examining 69 positive sera and the negative coincident rat of the ELISA was 100% according to the results examining 18 negative sera. It indicated that the developed ELISA had a good value in clinical.The sdudy have cloned the gene of σ 3 protein to PGEX-4T-1 expression vector , transformed into E.coli DH₅a and expressed the σ 3 protein. Usingthe expression protein as antigen, developed ELISA of ARV.It was shown that the ELISA was specific and suitable for large-scale sero-epidemiological investigation for ARV infection.