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Construction of Outer Membrane Proteins Map and Identification of D15 Immunogencity from Haemophilus Parasuis

Author: GuoYi
Tutor: JinMeiLin
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Parasuis type 5 Reverse vaccinology Immune proteomics Outer membrane protein
CLC: S852.61
Type: Master's thesis
Year: 2009
Downloads: 132
Quote: 2
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Abstract


Upper respiratory tract pathogen Haemophilus parasuis (Haemophilus parasuis, HPS) as a pig pig can cause multiple serositis, arthritis and meningitis, with the development of the pig industry in the world, the disease has become a worldwide an important bacterial diseases affecting the pig industry, causing huge losses to the pig industry. Clinical inactivated vaccine can provide some protection, but due to the diversification of the serotypes, such that its protective immunity difficult to evaluate. Parasuis pathogenic mechanism is vague and unclear protective antigen component, restricting the development of new vaccines. This is the first use of reverse vaccinology parasuis type 5 screened new immunogenic outer membrane protein, and the study of the protective antigen. Laid an important foundation for the development of novel subunit vaccine research. Now the main results of the report are as follows: the experimental parasuis serotype 5 the SH0165 as experimental material, the use of two-dimensional electrophoresis and software analysis established a two-dimensional electrophoresis reference map of the outer membrane protein. PH3-10 gels Spectrum analyzed by ImageMasterv6.01 software display of approximately 85 points, the point of gel digestion the MALDI-TOF-MS analysis identified 60 kinds of proteins, wherein the outer membrane protein 13 species. The by Vice pig hemostasis bacillus antibody-negative pigs artificially infected with SH0165 rehabilitation serum Western Blot tests detect potential immunogenicity of protein on the outer membrane protein profiles of the 2D separation SH0165. A total of 21 positive protein points on the Western blot hybridization patterns, representing 15 species of protein, including HPS 06257 belong to the same bacteria has been identified as D15, PalA, OMP P2, etc., further comprising never been reported, Wza more. All potentially immunogenic protein found in parasuis are found for the first time. Our selection immunogenic protein D15 for the purpose of gene cloning in E. coli expression strain BL21 build the expression and purification of recombinant proteins. Subsequently reverse verified in vitro using convalescent sera, the display D15 Western blot results of recognizing antibody in vitro, confirming the accuracy of the expression of the purified protein. By pre-test. Measured of 5LD 50 (6.5 × 10 9 ), the use of the recombinant protein emulsified with Freund's adjuvant preparation of a vaccine to immunize mice. to detect recombinant protein D15's immune protective effect. The results showed that: 5 LD 50 attack toxic dose, D15 can provide 60% higher protection force. We believe that the D15 as parasuis novel subunit vaccine candidate antigen, has a very broad prospects for development.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria
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