Screening Of Transposon Mutant Libirary And Studies On Biological Characteristics Of Two Membrane-associated Enzymes In Mycoplasma Gallisepticum

Mycoplasma gallisepticum (MG) is a bacterium belonging to the class Mollicutes and the family Mycoplasma. It is the causative agent of chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys, chickens, game birds, pigeons, and passerine birds of all ages. The first critical step in pathogenesis is the adherence of the invading mycoplasmas to the tracheal epithelium. To understand the role of MG in adhersion and colonization to the host, we focus on two membrane-associated enzymes-TpiA and LicA, to study their biological functions. And in order to select the mutants to the two enzymes, the existed transposition method has been improved and the whole screening methods have been established to identify the deletion of any cytoadhesins of MG.1. Screening methods were set up for transposon mutant libraryA universal mini-Tn4001tetM transposition vector was constructed in our previous study. However, the narrow dosage range of Tetracycline hampered its application in the transposition screening. In this study, the tetM gene encoding Tetracycline resistance protein was substituted by a gentamycin resistance gene, aacA. The random-mutant libraries were then screened by a set of methods, such as the identification of aacA gene by polymerase chain reaction (PCR), adhesion assays to DF1 cells or on glasses, identification of the silent proteins by SDS-PAGE and further screening with specific antibodies by Western-blot analysis and peptide sequencing by Mass spectrometry method.2. Studies on the biological characteristics of TpiATriosephosphate isomerase (TpiA) plays an important role in glycolysis and is essential for efficient energy production. in this study, the tpiA gene of MG strain Rlow was amplified and then subcoloned into a prokaryotic expression vector, pET28a(+). By the induction of IPTG, the fushion protein was solubly expressed in the bacteria BL21 (DE3) by SDS-PAGE analysis. After purified by the His Band Resin kit, the purified protein, designated as rMGTpiA, was used to prepare the polyclonal antibodies against TpiA in mice. The purified protein has a high enzymatic activity with 3928IU/mL. The optimum temperature of this enzyme was 37℃and the optimum pH was between 7 and 8. It was confirmed that MG TpiA related to adherence and invasion of MG to DF1.3. The study of the biological characteristics of LicAThe licA gene encodes Lipooligosaccharide/ Lipopolysaccharide synthase, or choline kinase catalyzing choline to phosphorylcoline; which is the major component of membrane lipid and associated with the pathogenicity of Mycoplasma. In order to study the biological activity, in this study, the licA gene of MG was amplified by overlap PCR method, and then subcoloned into a prokaryotic expression vector pET32a(+). The fusion protein of LicA was induced to expression by IPTG and determind by SDS-PAGE analysis. After purification of the inclusion bodies, the purified and refolded protein was used to prepare the polyclonal antibodies against LicA in mice. By western-blot analysis on the cell extracts of membrane proteins in all the virulent and attenuated strains, LicA showed a stable expression amount and sublocalized on the cell membrane. Overall, the results of our study provide the possibility for the establishment of LicA biological model in vitro.