Purification And Characterization Of Protease From Actinobacillus Pleuropneumoniae Serotype 7

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumoniae, a worldwide disease causing tremendous economic loss to the swine industry. It is a common infectious disease which infe- cts the respiratory tract of pigs. The symptoms of infected pigs induced depression, difficult respiratory and pneumonia. With the development of the raising cosmically, porcine pleuropneumoniae becomes one of the five primary diseases that threaten mordern pig industries.Actinobacillus pleuropneumoniae expresses several virulence fact- ors that participate in its pathogenicity; for example, haemolysins, capsular polysaccharide, lipopolysaccharide, outer membrane proteins, transferrin binding proteins, factor of adhesion, ureases and protea- ses,et al. However, many questions regarding colonization and tissue damage remain unanswered. Colonization of respiratory mucous surfa- ce and further invasion could be promoted by proteases that degrade different host substrates including IgA and IgG. Clinical isolates of Actinobacillus pleuropneumoniae that secreted proteases cleaving imm- unoglobulins were demonstrated by Negrete Abascal E and cowor- kers(1994).In this work we describe some biochemical charact eristics of Actinobacillus pleuropneumoniae proteases secreted in vitro. They also could be sereted in the host tissues and contribute to damadge and colonization(Negrete Abascal E et al. , 2000; Negrete Abascal E et al. , 1998). Studying the biochemical characteristics of proteases can help us to develop new subunit vaccines. This is important to efficiently prevent and control porcine contagious pleuropneumonia, to protect and promote the health of development for raising pigs.The following two parts were included in the studies:Part I: Purification and Characterization of Protease from Actino- bacillus Pleuropneumoniae Serotype 7 The Actinobacillus pleuropneumoniae serotype 7 strain which was isolated from Shandong province had been cultivated in the lauria broth medium(containing 0.2%NAD)for 48 hours. The extracellular protease was purified by precipitation with 65% ammonium sulfate, iron-ex- change chromatography on DEAE-cellulose G-100 and chromatography on Sephadex G-200. The result of SDS-PAGE of the purified protease indicated that the molecular mass of its subunit was 45 kD. The optimum pH was 7.5 and the optimum temperature was 45℃. Its activity could be inhibited by EDTA, but not by PMSF or partly by heating at 80℃for 30min.Part II :The Immunological Study of Actinobacillus pleuropneum- oniae ProteaseThe extracted APP protease 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 mice were well protected under the dose of 100μg. When challenged with serotype 7 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 10μg protease 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. These results revealed that APP protease would have certain functions in the progress of Actinobacillus pleuropneumoniae immunization.Protease was purified from Actinobacilluse pleuropneumoniae ser- otype 7 isolated from Shandong province, which is prevalent in China, then emulsified with Freund's incomplete adjuvant in equal volumes to get a sort of subunit vaccine. Groups of white mice were immuned twice at 30 days and 45 days, then challenged intraperitoneally with serotype 5 and serotype 7 at 65 days. Antibodies were detected with ELISA. After second immunization, antibody level increased obviously and the immune protection rate against serotype 5 and serotype 7 reached 37.5% and 62.5%, challenge strains were re-isolated from dead mice. These preliminary results showed that the protease must be of immunogenicity and protection, but it can only confer partial protection against the challenge with the homologous strains or the heterologous strains. This research provided the foundation for development and application of the subunit vaccine on Actinobacillus pleuropneumoniae.