Cloning And Expression Of Dihydrolipoamide Dehydrogenase Gene And Construction Of Pdhd Gene Knock-out Mutant Of Mycoplasma Bovis

Mycoplasma bovis is one of the main pathogenic mycoplasma of bovine respiratory, which is characterized by pneumonia, arthritis, mastitis and keratoconjunctivitis. Dihydrolipoamide dehydrogenase is one of the important components of pyruvate dehydrogenase complex, which play crucial role in metabolism of Mycoplasma bovis. At present, there is rare documents which related to study on dihydrolipoamide dehydrogenase of Mycoplasma bovis. Therefore, the carrying out relevant research of dihydrolipoamide dehydrogenase of Mycoplasma bovis are bound to lay a solid theoretical foundation for the research of vaccine and diagnostic reagents and development of therapeutic drugs against Mycoplasma bovis.1. Prokaryotic expression and purification of Mycoplasma bovis DLD protein and preparation of polyclonal antibody.Apair of specific primers was designed referring to the sequence of Mycoplasma bovis PG45 strains in Gene Bank, then the pdhd gene of Mycoplasma bovis WUWEI strains was amplified and cloned into the p MD19-T vector. After finished the sequencing and point mutation, the gene was subcloned into p ET-28a(+), the recombinant prokaryotic expression plasmid p ETpdhd was expressed in E.coli BL21(DE3) induced by IPTG, and the recombinant protein His-DLD existed in supernatant.The expressed production(His-DLD) was used to immunize the New Zealand white rabbits after purified and the the polyclonal antibody was collected. The antibody titer was analyzed by ELISA. The result show that the antiserum can specific bind to purified His-DLD.2.Determination of enzymatic activity of the recombinant protein His-DLD.The enzymatic activity of the recombinant protein His-DLD was detected by using continuous monitoring assay. The results showed that the His-DLD has the dihydrolipoamide dehydrogenase enzyme activity, and can catalyze lipoamide to produce dihydrolipoamide, the optimal reaction condition was as following: temperature was at 25 ℃ and p H value was 7.5. Besides, The Km(NADH) of His-DLD was 9.72 μmol/L and the V max was 25.6 μmol/(L·min), which was deduced from Lineweaver-Burk plot.3.Construction of pdhd gene knock-out mutant of Mycoplasma bovis.The upstream and downstream of pdhd gene of Mycoplasma bovis WUWEI strains was amplified, which was fused with gentamicin resistance gene(Genr) by Overlap PCR, then cloned into the p UC19 vector to construct the recombinant plasmid p UC△pdhd. M. bovis was transformed by electroporation with the recombinant plasmid p UC△pdhd, Genr instead of pdhd gene through the homologous recombination between p UC△pdhd and chromosomal DNA of wild type with resistance selection, due to the recombinant plasmid p UC△pdhd can’t survive in Mycoplasma bovis, then construct the pdhd gene knock-out mutant of Mycoplasma bovis WUWEI strains.In this study, the carrying out relevant research of Mycoplasma bovis are bound to lay a solid theoretical foundation for the energy metabolism and the other biological function of dihydrolipoamide dehydrogenase, and also provide the reference for further exploration of Mycoplasma bovis infection mechanism.