Study On Biological Characteristics Of The Pyruvate Dehydrogenase And Construction Of Random Mutagenesis Vector For Mycoplasma Gallisepticum

Mycoplasma Gallisepticum(MG) belongs to Mycoplasmatales,Mycoplasmataceae. It is causative agent of chronic respiratory disease in chicken, ducks, parrots, goose, turkey. The clinical signs of chicken infected with M.gallisepticum was cough, sneezing, tears and dyspnoea.Furthermore, M.gallisepticum infection can cause the susceptibility in infection with other avian pathogen, such as Escherichia coli, avian influenza disease, Newcastle disease virus and infective bronchitis virus, thus cause great economic loss. Pyruvate dehydrogenase(PDH) is one of the critical enzyme in the pyruvate dehydrogenase complex. The function of pyruvate dehydrogenase in M.gallisepticum is still not clear. Many studies have demonstrated that many enzyme related to biochemical function in M.gallisepticum, such as enolase, GAPDH, PK, Besides their catalytic activity, also participate in adhesion and virulence. In this assay, the biological function of will be studied. In addition, the random insertion vector has been successfully constructed, and the random insertion base has been established.1. Biological function of pyruvate dehydrogenase in M.gallisepticumAccording to the published gene sequence of M.gallisepticum in NCBI, two pairs specific primers were designed and used for the amplification of pdha and pdhb, respectively. The PCR product was ligated into pGEM-T easy vector, then sequenced. The genetic condons of TGA was tranfered to TGG by site mutagenesis method. The mutated sequence was ligated with the expression vector pET-28a(+). The recombinant expression vector was named as pET-pdha and pET-pdhb.The constructed recombinant plasmids were then induced into Escherichia coli strain BL21(DE3) competent cells for IPTG inducible expression of His-PDHA and His-PDHB, respectively. Molecular weights were 34KDa and 41kDa, respectively. Purified His-PDHA did not exhibit any PDH activity, purified His-PDHB exhibited weak activity. When the two subunit was mixed at a molecular ratio of 1:1, a strong activity can be observed. The purified His-PDHA and His-PDHB was used to immune BALB/c mouse and the anti-serum was collected. The immunicity of recombinant protein His-PDHA and His-PDHB were analyzed by western blotting, the result showed that the antiserum can specific bind to PDHA and PDHB on the membrane of M gallisepticum, respectively. Adherence inhibition assay on chicken fibroblast(DF1) cells reveled more than 30.8% and 45%. Bactericidal assay showed that anti-PDHA and anti-PDHB serum has a well ability to kill M. gallisepticum, suggesting the protective function of PDHA and PDHB.2. Construction of random mutagenesis vector mini-Tn4001 SpGmIn order to discover proteins which are assaciated with virulence in M. gallisepticum. The random insertion transposon vectors has been constructed, named as mini-Tn4001SpGm. This transposon has the following characteristics:an gentamycin resistance gene driven by the spiralin promoter. IS256 inner and outer insertion repeat region on two sides of the resistance gene and Staphylococcus aureus transposon gene outside the repeat region. Using resistance selection and PCR detection, a total of 738 random mutants has been screened.Using sequencing method, the insertion site has been identified. The construction of transpson vector makes the study of virulence protein in M.gallisepticum become possible, and provide a genetic tools for the expression of heterlogous protein in M.gallisepticum become possible.