| Porcine pleuropneumonia(PCP)and Swine Pasteurellosis are bacterial respiratory infectious diseases of pigs caused by Actinobacillus pleuropneumoniae(APP)and Pasteurella multocida(Pm),respectively.Both APP and Pm belong to the family Pasteurellaceae,and infection with pigs can cause similar respiratory symptoms.They all have a large number of serotypes,and the cross-protection between serotypes are weak.A safe,effective and low-cost domestic universal vaccine has become an urgent need of breeding industry under the situation of "antibiotics should be prohibited in feed and reduced in breeding".The dual subunit vaccine can achieve the effect of one immunization against multiple diseases.It can not only reduce the times of immunizations and production costs,but also provide strong cross-protection.With the discovery of several strong immunogenic antigen proteins in recent years,the development of APP-Pm dual subunit vaccine has become a hot topic in the industry.However,the bottleneck problem of recombinant expression and purification of bacterial antigen proteins in vitro is that insoluble expression leads to poor protein activity or very low yield,which increases the downstream production cost.Based on this,this study not only achieved efficient and soluble expression of immunogenic proteins from APP and Pm,but also reduced downstream purification and mass production costs by optimizing expression vectors,host bacteria and a series of induction conditions on the basis of retaining important antigen epitopes of APP and Pm immunogenic proteins.Then,APP and Pm protective antigen proteins were screened to evaluate the immune protective effects of different combinations of antigen proteins,and the candidate antigen combinations of APP and Pm subunit vaccine with the best immune efficacy were screened to evaluate their safety and efficacy.The APP-Pm dual subunit vaccine was further developed to evaluate its cross-protection against APP and Pm predominant serotypes(APP serotypes 5 and 7,and Pm capsular serotypes A and D)circulating in Chinese fields.The main contents are listed as following:1.Efficient,soluble expression and purification of important immunogenic proteins from APPBy comparative analysis,it was confirmed that the immunogenic proteins Omp D and Lpp B were highly conserved among different serotypes of APP.Then,on the basis of preserving the important epitopes of each protein,the p ET-SUMO vector and most compatible Escherichia coli(E.coli)engineered strains were used to construct the recombinant strains,and the induced expression conditions were further optimized.The results suggested that the supernatant expression of each recombinant protein was the highest,when the protein expression conditions were 18 ℃,120 r/min and 12 h of induction.Then r ApxⅠAN,r ApxⅡAN,r ApxⅢAN,r Omp D and r Lpp B proteins with high purity and activity were obtained successfully.2.Screening of candidate antigen components and optimal antigen combination of APP subunit vaccineThe immunoprotective effect of r Omp D,r Lpp B,r Oml A and r Tbp B in mice was evaluated to screen protective antigen proteins.The results indicated that the protection rate of r Omp D,r Oml A and r Lpp B was 37.5%,better than that of r Tbp B.Moreover,r Omp D and r Lpp B can effectively reduce the lung pathological injury caused by APP infection in mice.Therefore,r Omp D,r Lpp B and Apx toxin proteins(r ApxⅠAN,r ApxⅡAN,r ApxⅢAN)were selected to prepare APP subunit vaccine containing different antigen combinations.The results showed that the antigen combination containing r ApxⅠAN,r ApxⅡAN,r ApxⅢAN and r Omp D had 90% challenge protection rate against APP serotypes 5 and 7 strains in mice,and effectively reduced the lung tissue damage.3.Evaluation of the safety and immunological efficacy of APP subunit vaccine in pigsEndotoxin of r ApxⅠAN,r ApxⅡAN,r ApxⅢAN and r Omp D were removed and tested to ensure that the endotoxin residues were within the safe range.Then,the recombinant proteins were diluted appropriately and mixed with aluminum hydroxide adhesive adjuvant to prepare APP subunit vaccine.The content of each antigen protein in each 2 m L vaccine was 250 μg.The safety test of the vaccine showed that after immunizing 5 healthy piglets with a double dose(4 m L)of the vaccine,the body temperature of the pigs showed transient increase at the 6 h,and recovered within 1 d.All the piglets had normal breathing,mental state,water intake and food intake,and there was no abnormality at the injection site.There was no significant difference in the average daily gain after vaccination compared with the control group.After the safety of the vaccine was confirmed,the effectiveness of the prepared APP subunit vaccine was further evaluated.The prepared APP subunit vaccine was used to immunize 6-week-old pigs,and the control group and the Merck commercial vaccine group were set.Three weeks after the first immunization,booster immunization was administered,and two weeks later,APP serum strains 5 and 7 were used for tracheal injection challenge.The results showed that APP subunit vaccine could induce strong specific antibodies in pigs,stimulate the immune response of Th1 and Th2 cells,and the challenge protection rate against APP serotype 5 and 7 strains was 80% and 60%,respectively.In addition,it could effectively reduce the lung damage of pigs,and the immune protection effect is better than that of commercial Merck vaccine.4.Selection,efficient,soluble expression and purification of important immunogenic proteins from PmBased on previous studies and existing reports,conservative analysis of Pm immunogenic protein was conducted.The results showed that Omp D,Vac J,Omp W and Omp A were strongly conserved in different serotypes of Pm and APP,and Plp E was highly conserved in Pm serotypes A,B and F,but different with that of serotype D.Meanwhile,Omp D,Vac J,Omp W and Omp A showed high amino acid similarity between APP and Pm.Then,the recombinant strains were constructed by using p ET-SUMO vector and selecting the E.coli engineered strains suitable for different proteins.Further optimization of the induction expression conditions(18℃,120 r/min,12 h)achieved the efficient and soluble expression of Pm immunogenic proteins.The r Vac J,r Plp E,r Omp W and r Omp A proteins with good quality and activity were obtained after purification.5.Screening of candidate antigenic components and optimal antigenic combination of Pm subunit vaccineFirstly,the recombinant proteins r Vac J,r Plp E,r Omp W,r Omp A and the antigen component r Omp D in the APP subunit vaccine,as well as the Pm immunogenic protein r Omp H stored in the laboratory,were mixed with aluminum hydroxide adhesive adjuvant,respectively.Then all mice were immunized to evaluate the immune protection of individual proteins.The results declared that the protection rates of r Plp E and r Vac J against Pm A strains were 100% and 50%,respectively.The protective rates of r Vac J,r Omp D and r Omp W against Pm D strains were 62.5%,50% and 50%,respectively.Therefore,r Vac J,r Omp D,r Omp W and r Plp E were selected as candidate antigenic components of Pm subunit vaccine.Then,different antigen combinations were used to evaluate their immune efficacy.The results showed that the antigen combination of r Vac J,r Plp E and r Omp D had the best immune protective effect against the Pm capsular serotype A and D strains prevalent in the wild,which were 60% and 90% respectively.The lung tissue lesions of mice were mild,and the immune protective effect was better than that of Swine Fever,Swine Erysipelas and Swine Pasteurellosis triple live vaccine.6.Preparation of APP-Pm dual subunit vaccine and evaluation of immunological efficacy in miceBased on the antigenic components of the APP subunit vaccine and the Pm subunit vaccine,this study further combined the antigenic proteins of the two vaccines to prepare the APP-Pm subunit vaccine containing r ApxⅠAN,r ApxⅡAN,r ApxⅢAN,r Omp D,r Vac J and r Plp E.Then immunized mice with this vaccine,and APP(serotypes 5 and 7 strains)and Pm(capsular serotypes A and D strains)were used to challenge mice.The results show that: The dual subunit vaccine could stimulate the production of highly specific antibodies against r ApxⅠAN,r ApxⅡAN,r ApxⅢAN,r Omp D,r Plp E and r Vac J in mice,and effectively induce the immune response of Th1 and Th2 cells.The protection rate against APP serotype 5 and 7 strains was up to 100%,and the protection rate against Pm capsular serotype A and D strains was 90%.It was confirmed that the prepared APP-Pm dual subunit vaccine has good cross-protection. |
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