Prokaryotic Expression Of Protein TbpB,GPD,HbpA From Haemophilus Parasuis And Identification Of Its Immunoprotective Efficacy

Haemophilus parasuis,which belongs to Haemophilus genus of the family Pasteurellaceae,is a gram negative,rod like,nicotinamide adenine dinucleotide dependent bacterium.Haemophilus parasuis can cause Glasser’s disease in swines,characterized by severe infection of the upper respiratory tract,meningitis and arthritis.The outbreak rate of Glasser’s disease is increasing worldwide,and the economic losses associated with it are the most concern of swine producers.Nowadays,drug control and vaccination are commonly used to control H.Parasuis.Although antibiotic therapy can control Glasser ’s disease in a sense,the Haemophilus parasuis has a strong resistance due to the long-term use of a large number of antibiotics,and the great differences in drug use in different areas and farms.There are many serotypes of Haemophilus parasuis,and 15 serotypes have been identified.Besides,there are still a large number of strains that can not be established.There is a lack of cross protection between serotypes,and the local characteristics of bacteria are obvious,resulting in very limited protection effect of inactivated vaccines.Therefore,it makes a difference to develop a cross-protective vaccine.Our laboratory preliminary has completed the H.Parasuis standard serovar 4,5 and 12 proteomic analysis.2 sTable proteins expressed in serovar 4,5 and 12 related to the metabolic pathway of Haemophilus parasuis.Those names are glucose-6-phosphate 1-Dehydrogenase(GPD),Heme bind protein A(HbpA).According to the literature recombinant transferring-binding protein B is synthetized artificially according to the literature by the way.We start the research on three proteins to determine whether they have immune protection against Haemophilus parasuis in different serovars by guinea pigs immunization and challenge test.1.Prokaryotic Expression of Protein TbpB,GPD,HbpA from Haemophilus ParasuisBased on the published gene sequence of Haemophilus parasuis serovar 5,the point mutation(Y167A)at the gene level of TbpB protein was synthesized,and the TbpB fragment was synthesized artificially and the purified genes successfully cloned into pColdI expression vector.Three recombinant plasmids,pCold-TbpB,Pet-28a-GDP and pET-32a(m)-HbpA,were transformed into E.coli BL21 competent cells and induced by IPTG.The three proteins were purified by HisTrap HP chromatography column and centrifugation used centrifugation by.molecular sieve to desalt the protein samples.Protein samples were removed endotoxin by Triton X-114 cloud point method,and protein endotoxin content was detected by limulus lysate kit.The results showed that the three recombinant proteins were expressed in soluble form in E.coli;Western-blot showed that all three proteins could react specifically with His monoclonal antibody;the purified three sTable proteins had high concentration,clear target bands and low impurity protein content;The endotoxin content of the three proteins including diluted water was all qualified after the endotoxin removal by Triton X-114 method.The successful preparation of Haemophilus parasuis subunit vaccines is the basis for further screening of vaccine candidates with cross protection by animal experiments.2.Identification of Haemophilus Parasuis Immunoprotective EfficacyIn this study,three sTable proteins expressed Haemophilus parasuis serovar 4,5 and 12 were selected as candidate candidates for subunit vaccine.After expression and purification,endotoxin was removed and the protein was emulsified by Carbopol adjuvant.3 kinds of protein samples were equally mixed and emulsified with Carbopol water adjuvant(antigen:adjuvant=2:1).According to the growth curve of Haemophilus parasuis serotype 4/5/12 and the relationship between OD600nm and CFU,the number of viable bacteria in fresh culture Hps-4/5/12 was calculated,3 serovars bacterium were mixed with 0.3%formaldehyde to inactivate them,and ISA 15A adjuvant was used to emulsify inactivated bacterium(antigen:adjuvant=9:1).The physical properties and stability were observed after the vaccine conFig.uration has been completed.After the protein sample has been prepared as a qualified vaccine,the immunoprotective ability of these three proteins to Haemophilus parasuis of different serovars was determined through guinea pig immunization and challenge test with Haemophilus parasuis.A total of 30 guinea pigs in four groups were randomly divided into subunit vaccine group(9 guinea pigs),inactivated vaccine group(9 guinea pigs),challenge control group(9 guinea pigs)and blank control group(3 guinea pigs).The inactivated vaccines and PBS were emulsified with ISA 15A adjuvant and the recombinant proteins were emulsified with Carbopol adjuvant.Guinea pigs were immunized by subcutaneous injection once every two weeks,twice in all.The serums of guinea pigs were collected from hearts immediately after death of guinea pigs.The titers of serum antibody were determined by ELISA test.Second weeks after the twice immunity,three strains of Haemophilus parasuis serovars 4,5 and 12 were used to attack the guinea pigs.The results showed that After emulsification,the protein samples showed a pale white homogeneous suspension,wtin no stratification at room temperature for nearly 2 months,and could quickly dissolve with water when dripped into the water;the inactivated bacterium showed a white homogeneous emulsion appearance,no stratification and demulsification at room temperature for 2 months,and did not diffuse when dripped into the water as oil droplets;The emulsified inactivated bacterium and protein samples are sTable and can be used as vaccines for the next animal test.The serum antibodies,titers of inactivated vaccine group and subunit vaccine group were significantly higher than control group attacked by Haemophilus parasuis and blank control group.At the same time,six cytokines including IL-2,5 and 8,MCP-1,TNF-α and IFN-γ were detected by SYBR Green real-time PCR.The cytokine levels of the inactivated vaccine group were significantly higher than those of the negative control group.In the subunit vaccine group,the cytokine IFN-γexpression levels of the group attacked by HPS-5 were significantly higher than control group,and the expression levels of IL-8 and IFN-γ of the group attacked by HPS-4 were significantly higher than those of the negative control group.The expression levels of IL-5,IL-8 and IFN-y in the group attacked by HPS-12 increased significantly than control group.In inactivated vaccine group,the survival rates of the three standard Haemophilus parasuis groups were 66.7%respectively,indicating that the inactivated vaccines have protective efficacy against serovars 4,5 and 12 of Haemophilus parasuis infection.In the subunit vaccine group challenged with 3 serovars of Haemophilus parasuis,the survival time of guinea pigs was significantly longer than that of the control group and both of them gradually died 3 days later.Lung pathological sections showed that the subunit vaccine group had widened pulmonary interstitium,thickened alveolar wall and a small amount of alveolar hemorrhage,which was milder than that of the challenge control group,while the inactivated vaccine group had mildly pathological changes.Pathological sections of spleen showed that a certain exten of neutrophils exuded,inflammatory cells infiltrated and white pulp area hemorrhage were observed in subunit vaccine group,which was milder than that in challenge control group.Spleen of inactivated vaccine group had almost no pathological changes.The data showed that the three sTable expressed proteins had protective effect on HPS-4/5/12 infection,but the subunit vaccine showed a low protective rate in the challenge test.In the post period,we should optimize the conditions of protein immune dose,adjuvant selection and bacterium dose used to attack experimental animals.Besides,we ought to explore the cross-protection of subunit vaccine in depth.