Purification And Characterization Of Urease From Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumoniae is a respiratory system disease caused by actinobacillus pleuropneumoniae. It is a worldwide disease causing tremendous economic loss to the swine industry especially in Europe and America.It can infect every stage of the pigs and infect mixed with Psterurella multocida, The clinical symptoms of infected pigs breathe hardly.The pathological changes are that the lung has diferent degree of hyperaemia , haemorrhage and fibroid pneumonia.The incidence and mortality of the disease are usually above 20%,and the mortality of the most acute cases can reach 80～100.%.If the desease develops to chronic or latency in the hogpens,the growth of the pigs will slow down,the breeding cost will increase and the resistance of the pigs will descend.The occurance tendence of porcine contagious pleuropneumoniae in our country is rising now,and it has brought serious econimic damages to mordern pig industry.The virulence factors of APP include Haemolysins , Capsular Polysaccharide,lipopolysaccharide, outer membrane proteins, transferrin binding proteins, urease and factor of adhesion,et al.Study the function of the virulence factors can help us to develop new subunit vaccines.This is important to prevent and control porcine contagious pleuropneumonia.The following two parts were included in the studies:Part one : Purification and Characterization of Actinobacillus pleuropneumoniae urease.The serotype 7 of Actinobacillus pleuropneumoniae was sonicated and centrifuged at 10000r/min for 20 minutes at 4℃to extract urease. And (NH4)2S04 fractionation and Sephadex G-200 gel filtration were used to purify APP urease. After purified, the molecular weight were determined by SDS-PAGE ,and the characterizations of the enzyme were studied. The results showed that the urease had three subunits for about 11,25 and 60KD by SDS-PAGE , the optimum pH and optimum temperature were 8.5 and 45℃ and the specific activity of urease under this situation was 96.253 U/mg .Part two:The immunological study of Actinobacillus pleuropneumoniae ureaseThe extracted APP urease were used as antigens to immune the mice, and then they were challenged with the same serotype bacteria serotype 7 of APP, the results showed that the mouse were well protected under the dose of 25μg. When challenged with serotype7 of APP, the mice of control group were all dead,and the lung were hemorrhage seriously and the liver tumefied and hemorrhage too.Exept the group immuned with 5μg urease died all and had the pathological changes of the lung hemorrhaged and the liver tumefied and hemorrhaged,the mice of other immuned groups only had the pathological changes of the lung hemorrhaged sightly compared with the control group. The histology changes of the control group were that the liver were nerosis and metaboly;the interstitial tissue of the lung was hyperplasia and haemorrhaged. The immuned groups had the simple changes but had different degrees.These results revealed that APP urease would have certain functions in the progress of Actinobacillus pleuropneumoniae immunization.This research provided the foundation for development and application of the subunit vaccine on Actinobacillus pleuropneumoniae.