Isolation And Purification Of Flagellin,and Simply Discussing Its Roles In ALI Induced By Sepsis

The flagellin from salmonella paratyti A through Acid-splitting was purified by applying AKTA Explorer purified system.Then some mice being immunized by the flagellin ,the anti-serum would be obtained after 7-8 weeks. After the ALI of rat induced by sepsis was obtained through cecal ligation and puncture (CLP),we investigated ①the concentrations of flagellin in serum and BALF;②the partial pressure of oxygen in artery(PaO2);③the wet/dry ratio(W/D) of lung;④the correlation between the concentrations of flagellin and the levels of PaO2 and W/D;⑤the levels of TNF-αand IL-1βin serum and BALF.Contrasting the ALI being given the anti-serum of flagellin,we observed ①the levels of PaO2 and W/D ; ②the concentrations of TNF-αand IL-1βin serum and BALF. The result included (1)molecular quantity of the flagellin was about 52kD by SDS-PAGE analysis.(2)Western blotting analysis showed the purified flagellin strip was located at 52kD location,the protein that negatively stained with phosliolungstic appeared filaceous in scanning electron microscope(SEM).(3)the serum of anti-flagellin antibody that was identified by Western Blotting was at 52kD location,and its valence that calculated by double immunizing inffusion was 1:64.(4)the sepsis inducing ALI of rat was successfully established by CLP.In the prosess,①the concentration of flagellin gradually increased in serum and BALF,and the concentration of flagellin in serum was significantly higher than control group after 6hours(P<0.01),and the concentration of flagellin in BALF was significantly higher than control group after 12hours (P<0.01); ②PaO2 gradually decreased,and PaO2 was significantly lower than control group after 12hours (P<0.01);③W/D gradually increased, and W/D was significantly higher than control group after 12hours (P<0.01);④there was a statistically significant negative correlation between the concentration of flagellin and the level of PaO2 ,and there was a statistically significant positive correlation between the concentration of flagellin and the level of W/D⑤the concentrations of TNF-αand IL-1βgradually increased in serum and BALF,and the concentration of TNF-αis significantly higher than control group after 6hours(P<0.01),and the concentration of IL-1βwas significantly higher than control group after 12hours (P<0.01);⑥there were some obvious pathological changes of lung observed in shape and under microscope after 12hours,and the changes were worse and worse. (5) the ALI induced by sepsis was observed later.The result included①PaO2 was significantly lower than control group(P<0.01),and W/D was significantly higher than control group after 12hours(P<0.01). PaO2 was significantly higher than injury group(P<0.01) ,and W/D was significantly lower than injury group at 12hours and 24hours(P<0.01). ②concentration of TNF-αin serum and BALF was significantly higher than control group after 12hours(P<0.01).The concentration of TNF-αwas significantly lower than injury group at 6hours, 12hours and 24hours(P<0.01). ③concentration of IL-1βin serum and BALF was significantly higher than control group after 24hours(P<0.01). The concentration of IL-1βwas significantly lower than injury group at 12hours and 24hours(P<0.01). ④typical ALI was observed after 24hours. Our results suggest that(1)the purified flagellin is successfully obtained in the test.The protein that is identified by SDS-PAGE,Western Blotting,and SEM is the flagellin,and its molecular quantity is about 52kD.(2)by immunizing rats with the flagellin purified,the valence of anti-flagellin serum obtained gets to 1:64. (3)the ALI of rat induced by sepsis is successfully obtained through CLP. (4)the serum of anti-flagellin antibody has a good role in the ALI .