Preparation And Identification Of Monoclonal Antibodies Against LP78 Protein Of Mycoplasma Synoviae

Mycoplasma synoviae(MS)infection in chicken can cause acute or chronic infectious disease characterized by joint exudative synovitis,which also causes airsacculitis and systemic infection.It usually co-infects with Newcastle disease virus(NDV)infectious bronchitis virus(IBV)and Escherichia coli.Mycoplasma has characteristics of multiple serotypes and the wide distribution,can infect animals,insects,human and plants.Cytomembrane of Mycoplasma possesses rich lipoproteins,known as lipid associated membrane proteins(LAMPs)which have strong antigenicity and are prone to antigenic variation,and usually result in surface diversity of Mycoplasma.Although the pathogenic mechanism of Mycoplasma is not yet clear,more and more studies show that the lipoproteins play important roles in its pathogenesis.LP78 was known as a lipoprotein on the cytomembrane of Mycoplasma synoviae,has been reported as high immunogenic protein.The LP78 gene sequence of MS strain WVU1853 was extracted from GenBank.According to which,we designed 7 pairs of primers for overlap PCR to obtain the mutated MS LP78 gene.Then LP78 gene was then cloned into the prokaryotic expression vector pCold ?.The expression plasmid pcold I-LP78 was then transformed to the Escherichia coli BL21(DE3).After induced by IPTG,the LP78 protein was expressed and then purified by His tagged magnetic beads.Finally,the purified LP78 protein was obtained,which provides materials for the preparation of monoclonal antibodies.To obtain monoclonal antibodies against MS LP78 protein,BALB/c mice were immunized with purified LP78 protein and the immunized spleen cells were fused with mouse myeloma cells SP2/0.Finally,two hybridoma cell lines which stably secrete specific monoclonal antibodies were successfully obtained by screening.The specificity of the obtained monoclonal antibodies was detected by indirect ELISA and Western-blot assays.The obtained hybridoma cells were repeatedly freeze-thawed and passaged,and the stability of the hybridoma cells was detected.Two hybridoma cell lines H5 and C9 were successfully prepared,and were identified to secrete specific monoclonal antibodies stably.The results of indirect ELISA and Western-blot assays showed that the two monoclonal antibodies specifically bind to Mycoplasma synoviae and did not react with other avian pathogens.The stability test showed that the two hybridoma cell lines stably secreted monoclonal antibodies after serial subcultivation.The monoclonal antibody subtype identification showed that two monoclonal antibodies were both IgG 1 and the light chains of antibodies were both ? type.Peritoneal fluid was prepared from 6-8 week old female BALB/c mice and purification of mouse ascites was performed using HiTrapTM IgG Purification HP.The purified mouse ascites were analyzed by SDS-PAGE gel electrophoresis.Heavy chain of about 53 kD and light chain of 23 kD were observed.The left and right light chains showed two distinct protein bands.The results showed that the mouse ascites were purified better.The concentration of the monoclonal antibody was determined to be 1.019 mg/m L and 1.1 mg/m L by the BCA protein concentration detection kit method.In this study,the full-length of LP78 gene was obtained by overlap PCR,and the recombinant LP78 protein was successfully expressed and purified.Furthermore,the hybridoma cells were prepared by immunization of BALB/c mice with LP78 proteins and two hybridoma cells strains stably secreting specific monoclonal antibodies were obtained.This study provides materials for the establishment of diagnostic methods for MS which gives foundation for further research on the biological function of lipoprotein LP78.