Identification Of Novel Immunogenic Proteins Of Pasteurella Multocida HN06and Study On The Immune Function Of PGAM

Pasteurella mulotcida is an important multi-host pathogens that causes many diseases, such as Atrophic rhinitis, Swine plague, Fowl cholera, Haemorrhagic septicaemia. The kind of bacteria is widely popular in the world, and to the aquaculture industry caused a great economic loss. At present, the vaccines of the prevention of Pasteurella mulocida are:strong inactivated virus vaccine, live attenuated virus vaccine, subunit vaccine. To a certain extent, all kinds of vaccines can prevent or treat the diseases of Pasteurella multocida, but different serotypes are lack of cross protection, so the effect of immune is not ideal. Inactivated virus vaccine could lead to important immune antigen inactivated; Attenuated vaccine is potential to virulence, not suitable for large-scale application; Subunit vaccine is currently a hot research point to the vaccine, the ideal subunit antigen should be:highly antigenicity, immunogenicity, highly security. But in the past the ideal antigen is difficult to screening. With the development of proteomics technology:2-DE electrophoresis and Mass spectrometry. Especially, Screening new protective antigens for novel subunit vaccine by immunoblot techniques. The development of proteomics technology provides a fast and convenient method for finding new drug targets, microbial virulence, subunit vaccine candidates etc. Therefore, we focus on the bacterial surface proteins that generates PMT serotype D P.multocida HN06to immunogenic screening, hoping to find the important immunogenic function protein, paving the way for the development of new Pasteurella multocida vaccine or the research of immune mechanism, the main results are as follows:1. Identification of novel surface immunogenic proteins of HN06The extraction of surface proteins of Pasteurella multocida HN06were separated by two-dimensional electrophoresis, and using the infectious serum and control serum made Western blot, the positive reaction point would be identified by Mass spectrometry. The study that4new immune proteins identified:1). trimethylamine-N-oxide reductase;2). Elongation factor Ts;3). Phosphoglyceromutase;4). Peroxiredoxin. The4proteins are first reported in Pm immunoproteomics.2. Expression and purification of PGAM proteinPGAM is a key enzyme of energy metabolism of glycolysis. A variety of metabolic pathways regulate the formation of pyruvate, so as to control Tricaboxylic acid cycle, and control the growth and reproduction of bacterial. Using the prokaryotic expression system of E.coli in the study:the expression plasmid pET-30a and E.coli BL21. After prokaryotic expression. the purification of PGAM by forceps column. Detecting the biological activity of purified protein by Westerin blot, showed that the prokaryotic expression of PGAM protein own immunogenic activity.3. The study of the immunity and function of PGAMThe study of PGAM was determined to the influence on the growth of HN06, found that PGAM protein can promote the growth of bacteria. ELISA tested the level of antibody showed that PGAM could produce high specific antibodies in mice. After second immunization,10LD50of HN06attacked mices in test group, PGAM protein can provide70%protection, while the control group and the blank group almost died, showed that PGAM protein has a certain protective immunity, can become a new candidate vaccine and to mat the study of the immune theory.