Cloning And Prokaryotic Expression Of Pyruvate Dehydrogenase E1 Subunit Genes Of Mycoplasma Bovis And Preparation Of Monoclonal Antibody To α Subunit

Mycoplasma bovis is an important pathogenic mycoplasma of cow, which could result in pneumonia and mammitis et al. Pyruvate dehydrogenase E1 is the key enzyme of glycolysis for organisms, but there are few research related to the biological characteristics of E1 in Mycoplasma bovis and whether it could be used as a diagnostic target to the related diseases. Therefore, to prompt the development of products for prevention, diagnostic and remedy of diseases casused by Mycoplasma bovis, this article amplified the genes of pyruvate dehydrogenase E1α and E1β of Mycoplasma bovis Wuwei isolates and analysed sequences with bioinformatics softwears. Constructed prokaryotic express plasmids p ET-pdhα and p ET-pdhβ. Fusion recombinant proteins were expressed with induction of IPTG. Then the subcellular localization of E1? and E1β in Mycoplasma bovis was studied preliminarily with ELISA and Western-blot. The female BALB/c mice were immunized with purified products r PDHA to prepare spleen cells, which would fusioned with SP2/0. The positive hybridoma cells were tested through in direct ELISA and then subclone screening should be in process, titers and reaction specificity were detected at the same time. NBL-4 cells are infected with E. coli-pdhα-gfp and E. coli-pdhβ-gfp, which both carried GFP label, to observe adhesion situation under fluorescence microscope.Results display that pdhα and pdhβ were similar to other published related genes and antigenic index and surface probability of E1? and E1β subunits are both high.The genes of pdhα and pdhβ were expressed in E. coli BL21(DE3) successfully and both held favorable immunogenicity. Western blotting and ELISA analysis indicated that the E1? and E1β of Mycoplasma bovis equally distributed between in the membrane and the cytoplasm. And titers of monoclonal antibody is 1:3 200 and reaction specificity to Mycoplasma bovis Wuwei strain and Lintao strain is significant. The result also suggested that E. coli-pdhα-gfp and E. coli-pdhβ-gfp had good adhesion ability to NBL-4 cells and it could be restrained by polyclonal antibody serum. E. coli-gfp, however, had no adhesion ability.This study has showed that pyruvate dehydrogenase E1 subunit of Mycoplasma bovis could be submitted on the cytomembrane in Mycoplasma bovis and it has certain adhesion ability, which lays a foundation to explore the pathogenic mechanism further. And preparation of monoclonal antibody aimed to E1α subunit could make a reference to develop the products for diagnosis and remedy of Mycoplasma bovis.