Studies On Biological Functions Of Two Membrane-associated Enzymes In Mycoplasma Gallisepticum

Mycoplasma gallisepticum (MG) is one of the most important avian pathogens. It belongs to Mycoplasma, Mycoplasmataceae, Mycoplasmas, causing chronic respiratory disease in chickens and infectious sinusitis in turkeys. The most important step of the mycoplasma infection and parasitism is the firm adherence to the proper coat of the respiratory tract. The adherence capability determines the M. gallisepticum. Here, we chose two membrane-associated enzymes——Enolase and Neuraminidase, which were few reports about biological functions. The study will be helpful to reveal the relationship between the enzymes and pathogenicity of M. gallisepticum.1 Biological functions of Enolase in M. gallisepticumThe enolase gene of M. gallisepticum strain Rlow was amplified, mutated by overlapping PCR method to introduce 2 point mutation sites (960nt and 1158nt). The gene was subcloned into pET32a(+) vector. The results of prokaryotic expression showed that MG Eno was expressed in E. coli BL21 (DE3) induced by IPTG, which was soluble. After purified by His Band Resin kit, the purified protein was designated as rMGEno having 4.2mg.To get the special antibodies against Enolase, BALB/c mice were inoculated with purified rMGEno to prepare antisera and monoclonal antibodies. The results showed that we obtained antisera and 6 specific mAbs against rMGEno, designated as 1A4, 1E5, 2E4, 2F8, 3B4 and 3D8, respectively. By western blot analysis, fluorescence assay, colloidal gold immun-electeon microscope, it showed that MG Enolase could display on the membrane surface.As an endocellular enzyme, Enolase participates in the glycolytic cycle, catalyzing the D-2-glycerophosphoric acid to phosphoenolpyruvic acid. Here, we surveyed the relative enzymatic activity of rMGEno. It results showed that rMGEno was 25.22IU/mg by an indirect detection method to determine mean values at A340 for NADH; And then by direct determination of mean values at A240 for catalyzing the D-2-glycerophosphoric acid to phosphoenolpyruvic acid, in which the Michaelis constant of the reaction was 0.0696 mol/L.In order to investigate the relationship between Enolase and adherence and invasion into avian cells of M. gallisepticum, proproties of the binding of rMGEno and plasminogen or fibronectin, and Inhibition of adherence and invasion were well studied. As a result, rMGEno was identified as a Plg- and Fln-binding protein, and the antisera against rMGEno could significantly inhibit adherence and invasion of M. gallisepticum Rlow to DF1 cell lines (P<0.05), the inhibition ratios were 80.06% and 83.01%, respectively.As a result, it was confirmed that MG Enolase, possessing the enzymatic characteristics of Enolase family, is related to adherence and invasion of M. gallisepticum to avian cells.2 Original research on the M. gallisepticum neuraminidaseTo investigate the neuraminidase activity, M. gallisepticum virulent or avirulent strains were determined by the neuraminidase detection kit. The results showed that the virulent stains were with a highly activities, while the low virulent or avirulent stains had low activities. Moreover, along with the passage of the high virulent strains, the NA activities decreased obviously. Accordingly, NA may relate to the virulence of M. gallisepticum, which might be related to the biofilm formation.In order to research the biological functions of NA, multiple mutation technique by overlapping PCR was introduced to mutate 21 points of UGA codon in the NA gene to UGG codon. The result indicated that the gene was successfully mutated and subcloned for expression. It is helpful for us to study the biological function of MG NA in future.