Establishment Of An ELISA Method For Detection Of Mycoplasma Hyopneumoniae Antibody Based On Mhp336 Protein

At present,the most common method for detecting Mycoplasma hyopneumoniae is to use the Mhp antibody detection kit produced by IDEXX company in the United States.It uses an indirect ELISA(Enzyme linked immunosorbent assay)method,which uses the entire M.hyopneumoniae strain as an antigen coated ELISA plate.However,some proteins in the whole strain cross react with proteins in other Mycoplasma strains,reducing the sensitivity of this method.In clinical testing,there is a significant difference in the change curve of Mhp antibodies compared to expectations.This is very unfavorable for the seroepidemiological investigation of M.hyopneumoniae and the purification of M.hyopneumoniae in pig farms.Therefore,the aim of this study is to establish a highly sensitive ELISA method for detection of M.hyopneumoniae antibody from porcine sera.1.Screening of humoral immune dominant regions of Mhp336,Mhp460,and Mhp677 proteins of M hyopneumoniaeObjective:To screen the optimal humoral immune dominant regions of Mhp336,Mhp460,and Mhp677 proteins of M.hyopneumoniae.Method:Divide the mhp336 gene into 4 segments,the mhp460 gene into 3 segments,and the mhp677 gene into 3 segments.Primer Premier 6.0 software was used to design primers for each segment,and recombinant plasmids pGEX-6P-1-mhp336,pGEX-6P-1-mhp460 and pGEX-6P-1-mhp677 were used as templates to amplify the corresponding segments using different primer combinations.The amplified products were respectively linked to pGEX-4T-3/pGEX-5X-3 and pGEX-6P-1 vector plasmids,and transformed into E.coli BL21(DE3)to construct recombinant bacteria.IPTG induced expression of the target protein,while completing the induction expression of recombinant bacteria E.coli BL21(DE3)-pGEX-6P-1-mhp336,E.coli BL21(DE3)-pGEX-6P-1-mhp460,E.coli BL21(DE3)-pGEX-6P-1-mhp677,and GST engineering bacteria.The supernatant after ultrasonic sterilization was coated with glutathione plates,and the optimal serum humoral immune dominant regions of Mhp336,Mhp460,and Mhp677 proteins of M.hyopneumoniae were screened by ELISA test.Result:Recombinant bacteria connecting 19 different genes of M.hyopneumoniae were constructed using the pGEX vector as the backbone,all of which were able to express recombinant proteins in soluble form.The optimal serum humoral immune dominant regions screened by ELISA test are Mhp336,Mhp460-F4,and Mhp677-F4.Conclusion:The serum humoral immune dominant regions obtained through screening provide candidate coating antigens for the establishment of subsequent ELISA detection methods.2.Prokaryotic expression and purification of Mhp336 proteinObjective:Obtaining purified Mhp336 protein.Methods:Design primers for the mhp336 gene using Primer Premier 6.0,and the recombinant plasmid pGEX-6P-1-mhp336 was used as a template for PCR amplification.The amplification product was linked to the pET-32a(+)vector plasmid,and then transformed into competent cells of E.coli BL21(DE3)-pET-32a(+)-mhp336.The inducer IPTG induces the expression of the target protein at 16℃ for 20 hours.The bacterial supernatant was purified by nickel ion affinity chromatography column.Result:A recombinant bacterium was constructed using the pET-28a(+)vector as the backbone to connect the mhp336 gene.The recombinant bacterium can express the Mhp336 protein in two forms:soluble and inclusion bodies.The target gene size of mhp336 is 1473 bp;The molecular weight of the Mhp336 recombinant protein is 61 kDa.After purification,Mhp336 protein with a purity of up to 90%was obtained.Conclusion:The purified Mhp336 protein provides a coating antigen for the subsequent establishment of an ELISA antibody detection method for M.hyopneumonia.3.Establishment of an ELISA method for detection of M hyopneumoniae antibodyObjective:Establishment of a relatively sensitive ELISA method for detection of M.hyopneumoniae antibody.Method:Firstly,determine the optimal reaction conditions for the detection method,including the determination of antigen coating concentration,selection of blocking solution,determination of blocking time,dilution ratio of primary antibody,incubation time of primary antibody,dilution ratio of secondary antibody,incubation time of secondary antibody,color development time,and determination criteria for the detection method.Then,intra batch repeatability test,inter batch repeatability test,specificity test,and sensitivity test are conducted.Finally,226 clinical pig serum samples were tested using the established method,and compared with the ELISA antibody detection kit based on Mhp366 protein established in our laboratory earlier and the Mhp antibody test kit from IDEXX company.Result:The antigen coating concentration was determined to be 0.05 μg/mL,the blocking solution is 2.5%skimmed milk powder,with a blocking time of 1 hour.The dilution ratio of the first antibody is 1:500,the incubation time of the first antibody is 0.5 hour,the dilution ratio of the second antibody is 1:10 000,the incubation time of the second antibody is 1 hour,the color development time is 5 minutes,and the critical value of the detection method is 0.332.The coefficient of variation of both intra batch and inter batch repeatability tests was less than 7%,indicating good specificity and sensitivity.Conclusion:We have established an ELISA antibody detection method for M.hyopneumoniae based on Mhp336 protein,which has good repeatability,specificity,and sensitivity.