Establishment Of Indirect ELISA And Dielectrrophoresis Of Outer Menbrane Proteins On Haemophilus Parasuis

Haemophilus parasuis is a common resident located in upper respiratory tract of swine which can invade their hosts to cause Glasser’s disease in certain situation. The symptoms of this disease are fibrinous polyserositis, meningitis and arthritis. This organism is a gram-negative bacteria belonging to Pasteurellaceae family which is a threat to piglets and sows and it has already brought huge losses to pig industry. There are over15different serotypes of Haemophilus parasuis among which there is lack of cross protection. At present bacterins including serotype4and5are used for prophylaxis. The sequencing of Haemophilus parasuis HS0165genome which makes immunoproteome-based approach possible is well accomplished. The detection of Haemophilus parasuis is mainly depend on IHA, Agar Diffusion Test and IHC which have disadvantages such as cross reaction and low sensitivity. Therefore, an ELISA test approach which is more sensitive, more convenient and less expensive becomes a hot topic in the world. The content of the research are as follows:1. Establishment of2-D map of Haemophilus parasuis HS0165outer membrane proteins and MALDI-TOF-MSFirstly, extracted outer membrane proteins from Haemophilus parasuis HS0165through ultracentrifugation and then process the protein with2-D clean up kit to wipe out impurities and ions. Subsequently quantified the proteins with2-D quantity kit. Secondly optimized the situations such as lysis buffer, the weight of loading sample, the voltage in isoelectric focusing, and the2-D map of Haemophilus parasuis HS0165outer membrane proteins was successfully established. Thirdly blotted the gels onto PVDF membrane and detect the proteins through western blotting. At last identify the proteins with MALDI-TOF-MS.2. Prokaryotic expression of P2, nqrA, rpsB, Plp4, D15genes and analysis of immunogenicity of expressed proteinFive pairs of primers were designed according to the P2, nqrA, rpsB, Plp4, D15genes reported in Gene Bank. Then genes were amplified and subcloned into pET-28a vector. Subsequently the recombinant pET-28a vectors were transformed into E.coli BL21(DE3). Five recombinant proteins were expressed and purified and subsequently used to immune Blab/C mice. The results showed that every protein could provide protection against Haemophilus parasuis.3. Development of indirect ELISA for detecting antibody of Haemophilus parasuis based on D15recombinant protein.The situations including working concentration of antigen and sera, blocking reagents and time, incubation time of serum, anti-pig IgG-HRP and TMB were optimized. The indirect ELISA based on D15recombinant protein was developed. The total agreement to pathogenic diagnosis and serodiagnosis of the ELISA was88.3%...