| Strangles caused by the bacterium Streptococcus equi subspecies equi(S.equi),which belongs to Lancefield group C streptococcus,continues to be the most frequently diagnosed infectious disease of horses worldwide.According to the encoding gene sequences of M-like protein published on Genbank,a pair of primers was designed and synthesized.Then target gene was amplified from genome DNA of S.equi strain(field isaolate)by PCR and was inserted into the expression vector p GEX-6p-1.The successful construction of recombinant plasmid was confirmed by PCR and restriction endonuclease enzyme indentification,and the recombinant plasmid was transformed into competent cell Rosetta.Positive clones were picked out,cultured and induced by IPTG at 37?.The result of SDS-PAGE showed that the purified protein was produced.Western-blotting showed that the fusion protein could be recongnized by the positive serum,These results showed that,the target protein had good immunogencity and corresponding antigen epitopes.The recombinanted protein had a excellent antigenicity and had potential value in the development of serological detection.The purified recombinant protein was used as the coated antigen,the optimal concentration of antigen and the optimal dilution of serum were determined by chessboard method.An i ELISA method was successfully established by optimizing concentration of the enzyme-labelled antibody and the condition for substitute.Besides,the average value and standard deviation were concluded by the ELISA test for the standard negative serum.The sensitivity,specificity and coincidence rate were 70.59%,74.14%,and 73.68% by test 133 serum.In this study,a simple and convenient serological detection method for S.equi was established,and this method could be used to construct a commodifical kit for Strangles detection in the future. |
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