Characteristic And Immunogenicity Of Neuraminidase From Haemophilus Parasuis

Haemophilus parasuis disease was caused by Haemophilus parasuis (Hps), which is a systemic disease characterized with fibrinous serositis, arthritis and meningitis. The disease has become a major factor of death for piglets, caused severe economic losses. Neuraminidase is a important virulence factors of Hps, in our study, neuraminidase of Hps was extracted and purified, and the enzyme activities, physical and chemical characteristics and immunogenicity were studied, as follows:1 Physical and chemical characteristic study of neuraminidase from HpsExtraction and purification of neuraminidase from Hps:Hps-5 and Hps-12 strains were cultured and collected respectively, crude enzyme was extracted by breaking cells with Ultrasonic cell crusher, crude enzyme were purified with ammonium sulfate precipitation and anion exchange chromatograp Q Sepharose FF. the purity of extracts was detected by the SDS-PAGE and Detection Kit for neuraminidase. The result shows that, neuraminidase of Hps is a monomer form, molecular weight is 100kD; enzyme activity assay shows purified Hps neuraminidase possesses high activity, neuraminidase activity from Hps-5 and Hps-12 are 375.9 unit/mg,377.9 unit/mg respectively. The results prove that highly purified and active neuraminidase was extracted from Hps-5 and Hps-12 strains successfully.Physical and chemical characteristics of neuraminidase from Hps:The neuraminidase activity under the conditions of different PH, temperature and metal ions was detected by Detection Kit for neuraminidase. The results showed that, physical and chemical characteristics of neuraminidase from Hps-5 and Hps-12 strains was similar, the optimum pH value is 8.0, optimum temperature is 30℃, Under such conditions, the enzyme activity is the maximum. With 0.05mol/L concentration of Fe2+, Cu2+ and SDS all show the strongest inhibition on the enzyme, neuraminidase activity disappeared almost, With 0.05mol/L concentration, Ca2+, Mg2+ and EDTA have influence on the enzyme activity also, activity is inhibited partly.2 Immunogenicity of Haemophilus parasuis neuraminidaseThe extracted neuraminidase was detected by Western Blot, the results show that, the two occur a strong positive reaction with positive serum of Hps. Mice were immunized every two weeks in total of three with inactivated Hps-5, Hps-12 whole cell and neuraminidase (NA-5, NA-12) with Freund's adjuvant by 1:1 volume emulsified. Blood serum of immunized mice were Collected on the od,14d,28d and 42d from first immunization, serum antibody was measured by indirect ELISA, results show that, antibody, appears on the 14d, the OD450 of ELISA is 1.2-2.0 on the day 28d to 42d. Lymphocyte proliferation of neuraminidase was tested by MTT, conversion efficiency of the two enzymes was 15%-22%. Two weeks after last immunization, the mice were Inoculated with Strains and strains with heterotypic, the results show that, protection rate of NA-12 immunized mice on Hps-5 strain and Hps-12 were 66.7% both, NA-5 on Hps-5 was 50%, NA-5 on Hps-12 was 66.7%. The result show that, neuraminidase is one of the main immune protective antigen for the Hps, which provides a theoretical basis for further study of the Hps subunit vaccine.