| HaemopHilus parasuis is the etiological agent of Glasser’s disease,which is characterized as fibrinous polyerositis,arthritia and meningitis in veterinary clinics. As the development of the world industry,the disease has become one of the most important bacterial diseases of pigs worldwide.Attenuated vaccine of HPS abtained by gene knockout was developed to prevent the Glasser’s disease. However, H. parasuis is a fastidious bacterium and can only survive7to10days conserved in4℃. The problem with lyopHilizing bacteria and especially HPS, is that they tend to die due to the harsh processes of freezing and drying. Pigs immuned dead bacteria will induce an allergic response in individuals,which can seriously influence the application of attenuated vaccine of HPS.The goal of this study was to find the best excipient solution to stabilize HPS strain in lyopHilization and then provide technical support for the development of HPS attenuated vaccine.OmpA, a major outer membrane protein of Gram-negative bacteria, is very highly conserved and participates in biofilm formation, bacterial conjugation, bacteriopHage binding, cell growth, and the invasion of mammalian cells. Several OmpA-like proteins have been identified in other Gram-negative bacteria. MAb, raised against OmpA of HaemopHilus parasuis, demonstrated neutralizing and protective activities in vitro and in vivo, suggesting that OmpA may be a desirable immunogen to stimulate immune protection or as a diagnose tool.The main research included as below:1. HPS Stabilizer of Thermo-tolerant Cryoprotectant ResearchDifferent culture is compared to determine the best medium suitable for HPS growth. BHI is better than TSB.15serotype reference strains of HaemopHilus parasuis inoculate7days of age chicken embryo and the bacteria liquid was freeze dried and srored at-40℃as working seed lot.Different excipient solutions were tested, varying by protective ingredient they contained. The excipient solutions were formulated, HPS was added, and the solutions were lyopHilized. The samples were tested with plate count. In order to obtain the optimal vacuum freeze-drying protectant formula for HaemopHilus parasuis, single-factor deletion test was used to estimate the importance of the protective components. Trehalose, sodium glutamate, pH, amino acid, herps and BHI were tested to confirm whether they can improve the survival of freeze-drying cells. The result showed that the survival rate reached56.68%with1%trehalose added, it reached65～70%with3%arginine monohydrochloride added,and if1/3BHI was added,it reached73.69～80%.The protective agent was analyzed by Plackett-Burman design and Box-Behnken response surface methodology, the result showed that D-sorbitol, trehalose and sodium glutamate were significant impact factor.2. Prokaryotic expression of ompA genes and analysis of immunogenicity of the expressed proteinsA pairs of primer was designed according to the ompP5gene reported in Gene-Bank. The gene was amplified and subcloned into the prokaryotic expression plasmid pET-32a(+). The recombinant plasmid was transformated into E.coil BL21. With espressing by induced and purified, recombinant protein were got. The guinea pig were immunized after emulsified by Freund adjuvant two times and challenged at the dose of MLD HPS nagasika stain. The recombinant proteins had certain immunogenic. |
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