Prokaryotic Expression Of Neu,Lipo And OMP5 Protein Of Haemophilus Parasuis And Analysis Of Immunogenicity Of The Expressed Protein

Haemophilus parasuis (H.parasuis) is a Gram-negative bacterium belonging to the Pasteurellaceae family. The H. parasuis can cause the Glasser's disease which is mainly characterzed by polyserositis,arthritis and meningitis. H. parasuis usually affects young pigs from 2 weeks to 4 months in the nursery.The incidence rate of H. parasuis is generally from 10% to 15%.when serious illness come the mortality of pigs are up to 50%. H. parasuis leads to increasing economic losses in the swine industry worldwide.Control of H. parasuis infections can be achieved by use of vaccination using bacterins,but a major variability has been reported when testing the development of cross-protection,depending on H. parasuis strains and serotypes.With the rapid development of molecular biology techniques,vaccine reseach has turn to genetic engineering vaccine.Prokaryotic expression of Neu,Lipo and OMP5 gene of H.parasuis and analysis of immunogenicity of the expressed protein.According to GenBank in Haemophilus parasuis shotgun sequence, the part of Lipo gene was amplified from pMT18-Lipo by PCR with a pair of specific primers which primers designed in the upstream EcoRⅠsites upstream and downstream primers SalⅠsites. By EcoRⅠand SalⅠrestriction enzyme digestion treatment Lipo PCR product and expression vector recovery pBV220, the Lipo gene connected to the pBV220. SDS-PAGE electrophoresis analysis showed that the recombinant plasmid pBV-Lipo in E. coli Rosetta (DE3) to achieve high expression, lipo soluble protein expression and purification of lipo protein by ammonium sulfate precipitation.According to GenBank in Haemophilus parasuis shotgun sequence, the part of Neu gene was amplified from pMT18-Neu by PCR with a pair of specific primers which primers designed in the upstream EcoRⅠsites upstream and downstream primers SalⅠsites. By EcoRⅠand SalⅠrestriction enzyme digestion treatment Neu PCR product and expression vector recovery pET-28a (+), the Neu gene connected to the pET-28a(+).SDS-PAGE analysis showed that pET-Neu in E. coli Rosetta (DE3) to achieve high expression, Neu protein expressed in the form of inclusion bodies and purified according to literature Neu protein. Through exploring the digestion conditions, with EcoRⅠand SalⅠwill pMT18-OMP plasmid expression vector pET-28a (+) for restriction enzyme digestion, the OMP5 gene connected to the plasmid pET-28a (+). The products were transferred to E. coli DH5a by PCR and restriction enzyme selection plasmid. Then positive plasmids were transformed into Rosetta (DE3),37℃shaking culture to OD600 of about 0.5 to 0.6 plus IPTG to a final concentration of 1mmol/L to shaking culture 4h. SDS-PAGE analysis showed that pET-OMP5 in E. coli Rosetta (DE3) to achieve high expression, OMP5 protein expressed in the form of inclusion bodies and purified according to literature OMP5 protein.The recombinant purified Lipo, Neu,Omp5 proteins were divided into two groups, one group with Freund's adjuvant; another group using aluminum hydroxide adjuvant.14 days after the first immunization 21 days to strengthen the immune, immune 3 times in total, each dose of 50μg/only; control group were not immune to any protein, using saline. To lethal HPs and a control group of mice immunized with mouse poison attack. Virus challenge in each experimental group protection was as follows:Neu-Freund's adjuvant group protection was 60%(6/10), Neu-protected aluminum hydroxide adjuvant group was 10%(1/10); OMP5-Freund's adjuvant, the protection rate 30%(3/10), OMP5-protected aluminum hydroxide adjuvant was 11.1%(1/9); Lipo-protection Freund adjuvant was 86.6%(8/9), immune Lipo and the protection of aluminum hydroxide adjuvant group was 50%(5/10); not lethal attack by the immune protein drug control group mice all died in the attack drug; and Immunity protein is not inactivated HPs of the control group injected mice, in mice injected with some of the spirit of malaise within 4h, but soon returned to normal after 4h, the spirit of better. The results showed that these three recombinant proteins can play a protective effect in mice, that Lipo, Neu, OMP5 has certain immunogenic, which Lipo protein was significantly higher than Neu, OMP5, but the results were inferior to inactivated group.