Establishment Of Indirect ELISA For Mycoplasma Synoviae And Preparation Of Monoclonal Antibody

Mycoplasma synoviae（MS）is an important pathogen in poultry,which mainly infects chickens and turkeys.There is no effective vaccine in the country.At present,the detection and diagnosis of MS mainly use imported antibody detection ELISA kits.Its high price limits its large-scale clinical use.Therefore,it is necessary to develop economical and practical MS antibody detection kits to replace imported products.1.Preparation of recombinant MSPB,PYK,and P80 proteinsAccording to the MS sequence published by NCBI,MSPB,PYK,and P80 protein coding genes were designed,synthesized,and cloned into p ColdⅠvector,respectively,to obtain recombinant plasmids p Cold-MSPB,p Cold-PYK,and p Cold-P80.The three recombinant plasmids were transformed into BL21（DE3）engineering strain,respectively.Following the induction of IPTG,extraction and purification by nickel column,and identified by SDS-PAGE and Western blot,the recombinant proteins MSPB,PYK,and P80were obtained.2.Establishment of indirect ELISA method for MS antibody detectionPurified recombinant proteins MSPB,PYK,and P80 were used as coating antigens,respectively.MSPB protein was selected as the best coating antigen with MS positive serum as control.Then an indirect ELISA method of MS antibody was established using MSPB as the coated antigen.The reaction conditions were optimized.The results show that the optimal concentration of MSPB coating is 2μg/m L.The packing condition is 4℃overnight.The sealing solution was 0.4%gelatin.The color developing time was 8 min.The lowest relative titer of MS positive serum was 1:12800 indicating good sensitivity.Specificity test results showed that MSPB protein did not react with MG,AIV（H₅,H₇,and H₉）,NDV,and IBDV positive serum.The coefficient of variation within batches was less than 5%,and the coefficient of variation between batches was less than 10%,indicating good repeatability.The method and IDEXX ELISA（MS）kit were used to detect 204clinical samples.The results showed that the coincidence rate of positive sample was96.55%（140/145）.The coincidence rate of negative sample was 92.18%（59/64）.The total coincidence rate of all samples was 97.54%（199/204）.3.Preparation of monoclonal antibody against MSBALB/C mice were immunized with inactivated whole MS cells.After 4 times of immunization,spleen cells of mice were fused with myeloma cells（SP2/0）.The positive cell lines were screened by r MSPB ELISA.After four subclones,a hybridoma cell,named C6,which can secrete recombinant MSPB antibody,was screened.Ascites were prepared with a titer of 1:2×10⁶.In conclusion,in this study,the prokaryotic expression of MS outer membrane protein MSPB was used as the envelope antigen to establish an ELISA method for detection of MS antibody,which has good specificity,sensitivity and repeatability.A hybridoma cell that could secrete MSPB monoclonal antibody was screened by r MSPB ELISA.This study provided technical support for the epidemiological surveillance and investigation of MS in chicken farms in China.