To establish a method for serological investigation and evaluation of vaccination against Mycoplasma hyopneumoniae(Mhp),the present study utilized DNAStar biological software to analyze the epitopes of Mhp-P46 and Mhp-P65.The P46-P65 fusion gene was obtained by overlapping extended PCR(SOE-PCR),and the expression plasmid pETP46-P65 was constructed and transformed into Escherichia coli BL21(DE3)receptor cells.Soluble expression of recombinant P46(aa33-aa419)-P65(aa307-aa627)(rP46-P65)protein was obtained after induction.Using the purified rP46-P65 protein as coated antigen,an indirect ELISA method was established for the detection of Mhp antibody after optimizing the reaction conditions.The specific experimental results showed that the proposed method demonstrated high specificity,showing no cross-reaction with sera positive for other pathogens,including CSFV,FMDV,PEDV,PCV2,PRV,PRRSV,APP.The assay was able to detect Mhp antibodies when serum samples were diluted up to 6400 times,and the coefficient of variation within and between batches was less than 5%.Moreover,two hundred and ninety-eight clinical serum samples from farm herds were detected simultaneously by IDEXX kit and the iELISA.The positive rate of IDEXX kit was 62.4%(186/298)and the positive rate of iELISA was 73.8%(220/298),and the concordance rate of the two methods was 88.6%.All IDEXX-positive samples were also positive in the newly established iELISA and IDEXX-negative samples were tested negative with the iELISA.However,some positive serums from the same herds in the assay of the iELISA were negative by means of IDEXX assay,and 79.4%(27/34)of the serums with different results were also confirmed positive by the Mhp metabolic inhibition test.The results indicates that the sensitivity of the iELISA is significantly higher than that of IDEXX method.The above results show that the iELISA established in this study has significant advantages over currently available methods,and would be useful in the future for clinical investigations of serum antibodies and vaccine efficacy assessments.
维普
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