Extraction And Biological Effects Study About The Outer Membrane Protein Of Aggregatibacter Actinomycetemcomitans

Objective:To separate outer membrane proteins from Aggregatibacter actinomycetemcomitans rough strains and using the different concentration outer membrane proteins solutions. to act on the mouse macrophage R264.7. To compare the difference in the different serotype of Aggregatibacter actinomycetemcomitans outer membrane protein electrophoresis results and to see if there was any difference in IL-1βproduction.Methods:1 Three kinds of different serotype of Aggregatibacter actinomycetemcomitans rough strains(D7S,SA716,SA1151) were cultured, and the rough colony state was observed at the third day.2 Bacterial cells were collected and extraced their outer membrane proteins separately, and disposed the cell suspension. The extracts were recorded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the form of bands.3 Outer membrane proteins from different serotypes of Aggregatibacter actinomycetemcomitans rough strains were extraced by super centrifugation and sonicate, The extracts were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.4 Logarithmic phase Mouse macrophage R264.7 were stimulated by 3 different concentrations of outer membrane protein solutions, to set blank group and 3 experiment groups which were added in 3 different outer membrane proteins. The contents of IL-1βwere detected in the 12th and 24th acting time by ELISA. The experiment was repeated 3 times. The difference in the group of different concentrations and group of different stimulating times were analyzed by SPSS 11.5 statistical software.Results:1 The rough colonies of Aggregatibacter actinomycetemcomitans growed singly, in pairs, or in small clumps on solid medium, were translucent, irregular edge and adhered to the agar surface tightly. 2 There was no significant difference in the whole cell electrophoresis of three strains.3 Six common outer membrane proteins from three strains were identified and they were 100Ku,75Ku,64Ku,45Ku,34Ku,31Ku, the common membrane protein in SA716 and SA1151 was 29Ku, the common outer membrane protein in SA716 and D7S was 27Ku, and the protein of 110Ku only was found in SA1151.4 The contents of IL-1βstimulated by three different concentrations omp solution in 12 hours were different (P<0.05), they were 120±4.36pg/ml,110±13.11pg/ml, 150±17.69pg/ml respectively; and in 24 hours were different (P<0.01), they were 80±12.12pg/ml,280±19.31pg/ml,320±20.07pg/ml respectively;There were no different in the IL-1βcontents between 12 and 24 hours in the same Omp concentrations (P>0.05).Conclusion:1 Aggregatibacter actinomycetemcomitans can grow well on solid medium with blood at 37℃, and the colony will be approximately lmm diameter after 2-3 days.2 In this study, no significant differences can be observed in the whole cell electrophoresis of different serotype Aggregatibacter actinomycetemcomitans rough strains.3 There may be some differences in the electrophoresis results of outer membrane proteins of different serotype Aggregatibacter actinomycetemcomitans rough strains.4 In a certain range, the contents of IL-1βproduced by the Raw264.7 stimulated by different concentrations of Aggregatibacter actinomycetemcomitans Omp may be different.