A porcine contagious pleuropneumonia-swine pasteurella multocida bivalent subunit vaccine

Abstract

The present invention discloses a bivalent subunit vaccine for porcine contagious pleuropneumonia and porcine pasteurellosis, belonging to the field of veterinary vaccines. The bivalent subunit vaccine of the present invention contains truncated porcine Actinobacillus pleuropneumoniae antigenic proteins ApxⅠAN, ApxⅡAN, ApxⅢAN and OmpD and porcine Pasteurella multocida antigenic proteins PlpE and VacJ, which can provide cross-protection to porcine contagious Actinobacillus pleuropneumoniae and porcine Pasteurella multocida of different serotypes. The truncated proteins selected in the antigen protein combination of the present invention can all be efficiently soluble and expressed, and are easy to mass produce with low cost. The present invention provides an effective technical means for the prevention and treatment of porcine contagious pleuropneumonia and porcine pasteurellosis.

Description

Technical Field

[0001] The invention relates to the field of veterinary vaccines, and in particular to a porcine contagious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine. Background Art

[0002] Porcine contagious pleuropneumonia (PCP) is an acute, contagious disease caused by Actinobacillus pleuropneumoniae (APP), characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic fibrinous necrotizing pleuropneumonia (Chen Puyan. Veterinary Infectious Diseases [M]. Fifth Edition. Beijing: China Agriculture Press: 2011: 262-265.). Pigs of all ages can be infected with this pathogen, and breeding pigs and nursery pigs are the most susceptible. The mortality rate of acute APP infection is extremely high, often causing sudden death of pigs without warning. Chronic infection causes growth stunting of pigs, reduced feed conversion rate, and easy secondary infection from other pathogens and death, causing huge economic losses to the pig industry (Nahar N, Turni C, Tram G, Blackall PJ, Atack JM. Actinobacillus pleuropneumoniae: The molecular determinants of virulence and pathogenesis. Adv Microb Physiol. 2021; 78: 179-216.).

[0003] Actinobacillus pleuropneumoniae belongs to the family Pasteurellaceae and the genus Actinobacillus. It is a Gram-negative cocci. Based on the differences in bacterial capsule and lipopolysaccharide antigens, APP is currently divided into 19 serotypes, each with different virulence and lack of cross-protection. The prevalent serotypes of APP vary between different regions in my country, and types 1, 3, 5, and 7 are the dominant serotypes. In most northern regions of my country, types 5 and 7 are prevalent, while in southern regions such as Fujian and Guangdong, types 1, 3, and 7 are prevalent (Zhu Xiugao, Li Yanqing. Infection and serotype distribution of Actinobacillus pleuropneumoniae in porcine[J]. Advances in Veterinary Medicine, 2017, 38(10):111-113.). Apx toxin is the main virulence factor of APP, including ApxⅠ, ApxⅡ, ApxⅢ, and ApxⅣ. Their toxicity varies, but they all play a vital role in the pathogenesis of APP. ApxⅣ exists in all APP serotypes. In addition, serotypes 1, 5, 9, and 11 also secrete ApxⅠ and ApxⅡ, serotypes 2, 3, 6, and 8 secrete ApxⅡ and ApxⅢ, type 10 secretes ApxⅠ, and types 7 and 12 secrete ApxⅡ (ITO H, SUEYOSHI M. The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacilluspleuropneumoniae serotype 15 [J]. Journal of Veterinary Medical Science, 2015, 77 (4): 483-486.). The subunit vaccines sold on the market have many antigen components, which give cross-protection to all serotypes to a certain extent (Sassu EL, Tobias TJ, Gottschalk M, Langford PR, Hennig-Pauka I. Update on Actinobacillus pleuropneumoniae-knowledge, gaps and challenges. Transboundary & Emerging Diseases, 2017.). The subunit vaccine (Apx) produced by Merck contains three exotoxins (ApxI, ApxII, ApxIII) and an outer membrane protein (OMPs) as antigens. High-purity OMPs will produce synergistic protection with toxins and have the function of improving cross-protection. Coglapix produced by Ceva can provide protection against all APP serotypes, and its antigen components include ApxI, ApxII, and ApxIII. The commercial subunit vaccine (Porcilis APP) produced by Novartis contains rApxII, rOmlA, rTbpB, and rCysL.

[0004] Pasteurella multocida (Pm) belongs to the family Pasteurellaceae, genus Pasteurella, and is the most important livestock and poultry pathogen among the more than 20 species of bacteria in the genus Pasteurella. The bacterium is a short rod or coccobacillus with blunt ends. It is a Gram-negative bacterium without flagella, non-motile, non-spore-forming, and facultatively anaerobic. According to the different components of capsular polysaccharide (K antigen) and lipopolysaccharide, Pasteurella multocida can be divided into five serogroups, namely A, B, D, E, F and 16 serotypes (Lu Chengping. Veterinary Microbiology [M]. 4th edition. Beijing: China Agriculture Press, 2007: 136-137.). Among them, type A mainly causes fowl cholera, types B and E mainly cause hemorrhagic septicemia, and type D mainly causes porcine atrophic rhinitis. Animals infected with Pm often show septicemia, hemorrhagic inflammation, or suppurative lesions in subcutaneous connective tissue, joints, and various organs. This bacterium can infect a variety of hosts, and different types of animals show different symptoms after infection. The diseases caused by pigs infected with Pm are swine plague and progressive atrophic rhinitis (PAR); humans can also be infected by animal scratches or bites (Al-Allaf AK, Harvey TC, Cunnington AR. Pericardial tamponade caused by Pasteurellamultocida infection after a cat bite. Postgrad Med J, 2001, 77: 199-200.). In addition, Pm also plays an important role in increasing the severity of primary lung damage caused by other pathogens.

[0005] The outer membrane protein of Pasteurella multocida plays a key role in the process of disease development caused by bacterial infection. PlpE (Pasteurella lipoprotein E) protein belongs to the lipoprotein in the outer membrane protein of Pasteurella multocida. It has been confirmed that PlpE protein can induce a high level of specific antibodies in mice and has certain immune protection (Wu JR, Shien JH, Shieh HK, Chen CF, Chang PC. Protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE). Vaccine, 2007, 25: 4140-4148.). Chinese scholars have studied the PlpE protein of porcine Pasteurella multocida and bovine Pasteurella multocida, and the results show that PlpE protein has good antigenicity and immune protection. VacJ is a highly conserved and widely distributed outer membrane lipoprotein in Pm strains. Many related studies have shown that lipoproteins can also induce the body to produce higher levels of antibodies (Sathish Bhadravati S, Abhinendra K, Revanaiah Y, Viswas KN. Immunogenicity of highly conserved recombinant VacJ outer membrane lipoprotein of Pasteurellamultocida. Vaccine, 2014, 32: 290-296.). In addition, outer membrane proteins OmpA, OmpH, Omp87, OmpW, etc. also have certain immune protection (Dabo SM, Taylor JD, Confer AW. Pasteurella multocida and bovine respiratory disease. Anim Health Res Rev, 2008, 8: 129-150.).

[0006] In order to effectively prevent and control the infection of APP and Pm and improve the efficiency of breeding, vaccination is one of the most effective means. At present, the research on related vaccines roughly includes three categories: inactivated vaccines, attenuated vaccines and genetic engineering vaccines. The commonly used porcine infectious pleuropneumonia vaccines in clinical practice are only inactivated vaccines and foreign subunit vaccines. However, inactivated vaccines cannot provide cross-protection against infection with different APP serotypes. The price of Merck's subunit vaccine is high, which increases the cost of breeding. In addition, only the outer membrane protein (OMP) of its vaccine components belongs to the subunit component, and the toxin antigen components (ApxⅠ, ApxⅡ, ApxⅢ) actually belong to toxoids. The vaccines used clinically to prevent multocida Pasteurella infection are mainly inactivated vaccines and attenuated vaccines. However, inactivated vaccines are limited by the limited protection between Pm serotypes and cannot be universally used. The immune effect of attenuated vaccines is easily affected by environmental factors and there is a risk of virulence reversal. In addition, whether it is inactivated vaccine or attenuated live vaccine, the protection period is short and multiple immunizations are required. With the implementation of a series of measures such as antibiotic restriction and ban, the difficulty of preventing and controlling the disease has increased, and the development of an effective vaccine is imminent. Subunit vaccines have the advantages of high cross-protection level, safety and low cost, and can effectively reduce lung damage and mortality, and have become an urgent need in the industry.

[0007] As the main pathogens of bacterial respiratory infections in pigs, Actinobacillus pleuropneumoniae and Pasteurella multocida both belong to the Pasteurellaceae family, and infection of pigs can cause similar respiratory symptoms. The use of a two-unit vaccine can achieve the effect of one shot for multiple defenses, which can not only reduce the number of immunizations and production costs, but also provide a strong level of cross-protection. Both Actinobacillus pleuropneumoniae and Pasteurella multocida have many serotypes, and the cross-protection between different serotypes is weak. Therefore, vaccine research at this stage is more inclined to two-unit vaccines. In recent years, with the discovery of multiple strong immunogenic antigenic proteins of Actinobacillus pleuropneumoniae and Pasteurella multocida, the creation of a two-unit vaccine for porcine infectious pleuropneumonia-porcine Pasteurella multocida has become possible.

[0008] Since many bacterial antigenic proteins have a large molecular weight, most of which are above 100KDa, it is difficult to recombinantly express and purify these proteins in vitro using genetic engineering methods, such as: insoluble protein expression increases the difficulty of purification and production costs; low protein yield and inability to produce on a large scale, etc. Therefore, by predicting and analyzing the antigenic structure of the protein through bioinformatics technology, and then truncating the protein on the basis of retaining most of its antigenic epitopes, the efficient and soluble expression of the protein is achieved, and the cost of downstream purification and large-scale production is reduced.

[0009] The two-unit genetically engineered subunit vaccine for streptococcal disease and Haemophilus parasuis disease, which was approved for the national first-class new veterinary drug certificate in 2019, is the world's first genetically engineered subunit vaccine for these two bacterial diseases, proving that the development of combined subunit vaccines is currently a hot topic in vaccine research. At present, there are no two-unit subunit vaccines for porcine contagious pleuropneumonia and porcine Pasteurella multocida at home and abroad. Summary of the invention

[0010] The purpose of the present invention is to solve the problems existing in the prior art and to provide an antigen protein combination of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida and its application in the preparation of a porcine infectious pleuropneumoniae-porcine Pasteurella multocida bivalent subunit vaccine.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] A combination of antigenic proteins of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida, comprising proteins 1-6 whose amino acid sequences are respectively shown in SEQ ID NO.1-6. Among them, protein 1 whose amino acid sequences are shown in SEQ ID NO.1 is a truncated protein of porcine Actinobacillus pleuropneumoniae ApxⅠAN, protein 2 whose amino acid sequences are shown in SEQ ID NO.2 is a truncated protein of porcine Actinobacillus pleuropneumoniae ApxⅡAN, protein 3 whose amino acid sequences are shown in SEQ ID NO.3 is a truncated protein of porcine Actinobacillus pleuropneumoniae ApxⅢAN, protein 4 whose amino acid sequences are shown in SEQ ID NO.4 is a truncated protein of porcine Actinobacillus pleuropneumoniae OmpD, protein 5 whose amino acid sequences are shown in SEQ ID NO.5 is a truncated protein of porcine Pasteurella multocida PlpE, and protein 6 whose amino acid sequences are shown in SEQ ID NO.6 is a truncated protein of porcine Pasteurella multocida VacJ. Further, the nucleotide sequences encoding proteins 1-6 are respectively shown in SEQ ID NO.7-12.

[0013] The proteins 1-6 can be obtained by prokaryotic expression, the expression vector is preferably pET-sumo vector, and the expression host is preferably E. coli BL21 (DE3) or E. coli Transseta (DE3).

[0014] The invention discloses an application of the antigen protein combination of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida in the preparation of a bivalent subunit vaccine of porcine infectious pleuropneumoniae and porcine Pasteurella multocida.

[0015] A porcine contagious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine comprises the antigen protein combination of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida, and also comprises an adjuvant, wherein the adjuvant is preferably an aluminum hydroxide adjuvant.

[0016] The invention discloses an application of the porcine contagious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine in the preparation of medicines for preventing and treating porcine contagious pleuropneumonia and porcine pasteurellosis.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] (1) The truncated proteins selected in the antigen protein combination of the present invention are highly homologous in all serotypes of APP and Pm. By evaluating the immune protection of individual antigen proteins, antigen proteins with strong immune protection are combined to screen out the antigen combination with the best protection effect, and the subunit vaccine is prepared to provide cross-protection against different serotypes of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida. The protection rates against APP5 and APP7 strains are both 100%, and the protection rates against Pm A and Pm D strains are both 90%.

[0019] (2) The truncated proteins selected in the antigen protein combination of the present invention have been optimized to successfully achieve soluble expression, which is easy to express and purify in large quantities, thereby reducing production costs.

[0020] (3) The present invention lays a foundation for the development of an efficient, low-cost, broad-spectrum porcine contagious pleuropneumonia-porcine Pasteurellosis vaccine, and provides an effective technical means for the prevention and treatment of porcine contagious pleuropneumonia and porcine Pasteurellosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the detection of specific antibody levels in the sera of mice in different test groups.

[0022] Figure 2 It is the survival curve of mice in different experimental groups after virus attack. DETAILED DESCRIPTION

[0023] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0024] Example 1 Protein Expression and Purification

[0025] 1. Extraction of Bacterial Genomes

[0026] The APP1 and APP3 strains of Actinobacillus pleuropneumoniae stored in the laboratory were streaked on TSA plates containing 10% newborn calf serum and 0.01% NAD, and cultured overnight in a 37°C constant temperature incubator. Single colonies were picked and placed in 5 mL of TSB medium containing 10% newborn calf serum and 0.01% NAD, and cultured in a shaking incubator at 37°C and 180 rpm / min. The culture was then expanded to 10 mL of TSB medium containing 10% newborn calf serum and 0.01% NAD, and the bacterial genome was extracted using a bacterial genome extraction kit according to the instructions. The extracted genome was frozen in a -20°C refrigerator for later use.

[0027] The swine Pasteurella multocida type A strain stored in the laboratory was streaked on a TSA plate containing 10% newborn calf serum, placed in a 37°C constant temperature incubator for overnight culture, and a single colony was picked and placed in 5 mL of TSB medium containing 10% newborn calf serum, placed in a shaker at 37°C and 180 rpm / min for culture, and then expanded to 10 mL of TSB medium containing 10% newborn calf serum, and the bacterial genome was extracted using a bacterial genome extraction kit according to the instructions. The extracted genome was frozen in a -20°C refrigerator for later use.

[0028] 2. Construction and verification of recombinant plasmid

[0029] The invention uses bioinformatics software to analyze and predict the transmembrane region, signal peptide and antigenic epitope of ApxⅠAN, ApxⅡAN, ApxⅢAN, OmpD, LppB, OmlA and TbpB proteins of porcine Actinobacillus pleuropneumoniae and PlpE, VacJ, OmpA, OmpH and OmpW proteins of porcine Pasteurella multocida, respectively construct truncated expressed proteins containing different antigenic epitopes and different lengths, and finally achieve efficient and soluble expression of target proteins by optimizing different expression vectors and host strains as well as culture and purification conditions, and facilitate mass preparation and purification.

[0030] Based on the above research, primers for truncated expression proteins and pET-sumo plasmid were designed (Table 1). Using the bacterial genome as a template, PCR was performed according to the procedure in Table 2 to amplify the target gene fragments ApxⅠAN, ApxⅡAN, ApxⅢAN, OmpD, LppB, OmlA, TbpB and PlpE, VacJ, OmpA, OmpH, OmpW, and pET-sumo. Since PrimerSTAR Max DNA Polymerase has the characteristics of high specificity, high reaction sensitivity, and high amplification efficiency, and its high annealing efficiency greatly shortens the annealing and extension time, according to the instructions, the PCR of the target gene can use the reaction program of pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 10s; annealing at 58℃ for 10s; extension at 72℃ for 30s; and final extension at 72℃ for 2min, 30 cycles. After PCR was completed, nucleic acid electrophoresis was performed, and the results showed that the band sizes of the amplified ApxⅠAN, ApxⅡAN, ApxⅢAN, OmpD, LppB, OmlA, TbpB, PlpE, VacJ, OmpA, OmpH, OmpW and pET-sumo were located near the expected positions, respectively. Among them, a truncated gene ApxⅠAN, whose nucleotide sequence is shown in SEQ ID NO.7; a truncated gene ApxⅡAN, whose nucleotide sequence is shown in SEQ ID NO.8; a truncated gene ApxⅢAN, whose nucleotide sequence is shown in SEQ ID NO.9; a truncated gene OmpD, whose nucleotide sequence is shown in SEQ ID NO.10; a truncated gene LppB, whose nucleotide sequence is shown in SEQ ID NO.13; a truncated gene OmlA, whose nucleotide sequence is shown in SEQ ID NO.14; a truncated gene TbpB, whose nucleotide sequence is shown in SEQ ID NO.15; a truncated gene PlpE, whose nucleotide sequence is shown in SEQ ID NO.11; a truncated gene VacJ, whose nucleotide sequence is shown in SEQ ID NO.12; a truncated gene OmpA, whose nucleotide sequence is shown in SEQ ID NO.16; a truncated gene OmpH, whose nucleotide sequence is shown in SEQ ID NO.17; a truncated gene OmpW, whose nucleotide sequence is shown in SEQ ID NO.18.

[0031] Table 1 Primer sequences

[0032]

[0033]

[0034] Note: Underline indicates homology arms.

[0035] Table 2 PCR reaction system

[0036] Reaction components volume 2×Phanta Max Buffer 25μL dNTP Mix 1μL Upstream primer 2μL Downstream primer 2μL Phanta Max super-Fidelity DNA polymerase 1μL template 1.5μL <![CDATA[ddH 2 The]]> 17.5μL total 50μL

[0037] The linearized pET-sumo plasmid was efficiently recombined with the recovered ApxⅠAN, ApxⅡAN, ApxⅢAN, OmpD, LppB, OmlA, rTbpB and PlpE, VacJ, OmpA, OmpH, OmpW using the clonExpress II recombination ligation kit according to the system shown in Table 3. The product was propagated into E.coli DH5α competent cells, and the bacterial solution was then spread on LA plates containing 75μg / μL Amp for resistance screening. The obtained monoclones were inoculated into LB medium containing 75μg / μL Amp, and after shaking and culturing at 37℃ for 12h, the plasmid was extracted for PCR or double enzyme digestion identification, and sent to Qingke Biotechnology Company for sequencing identification. Specifically, the present invention clones the truncated ApxⅠAN gene into the pET-sumo vector to construct a recombinant plasmid pET-sumo-ApxⅠAN; clones the truncated ApxⅡAN gene into the pET-sumo vector to construct a recombinant plasmid pET-sumo-ApxⅡAN; clones the truncated ApxⅢAN gene into the pET-sumo vector to construct a recombinant plasmid pET-sumo-ApxⅢAN; clones the truncated OmpD gene into the pET-sumo vector to construct a recombinant plasmid pET-sumo-OmpD; clones the truncated LppB gene into the pET-sumo vector to construct a recombinant plasmid pET-sumo-LppB; clones the truncated OmlA gene into the pET-sumo vector to construct a recombinant plasmid pET- sumo-OmlA; the truncated TbpB gene was cloned into the pET-sumo vector to construct the recombinant plasmid pET-sumo-TbpB; the truncated PlpE gene was cloned into the pET-sumo vector to construct the recombinant plasmid pET-sumo-PlpE; the truncated VacJ gene was cloned into the pET-sumo vector to construct the recombinant plasmid pET-sumo-VacJ; the truncated OmpA gene was cloned into the pET-sumo vector to construct the recombinant plasmid pET-sumo-OmpA; the truncated OmpH gene was cloned into the pET-sumo vector to construct the recombinant plasmid pET-sumo-OmpH; the truncated OmpW gene was cloned into the pET-sumo vector to construct the recombinant plasmid pET-sumo-OmpW.

[0038] These recombinant expression plasmids were identified by PCR and sequencing, and all were consistent with expectations, with no sequence mutations, proving that the genes were successfully inserted into the corresponding vectors.

[0039] Table 3 Homologous recombination reaction system

[0040] Reaction components volume Linearized vector 2μL Purpose fragment 2μL 5×CE MultiS Buffer 4μL Exnase MultiS 2μL <![CDATA[ddH 2 The]]> 10μL total 20μL

[0041] 3. Expression and purification of recombinant target protein

[0042] The recombinant plasmid verified by sequencing was transferred into E. coli BL21 (DE3) or E. coli Transseta (DE3) competent cells, and then the cells were spread on LA (containing 75 μg / mL The resistance screening was carried out on the Amp) plate, and the single clones of the recombinant strains pET-sumo-ApxⅠAN-Transseta(DE3), pET-sumo-ApxⅡAN-Transseta(DE3), pET-sumo-ApxⅢAN-Transseta(DE3), pET-sumo-OmpD-BL21(DE3), pET-sumo-LppB-BL21(DE3), pET-sumo-OmlA-BL21(DE3), pET-sumo-TbpB-BL21(DE3), pET-sumo-PlpE-BL21(DE3), pET-sumo-VacJ-BL21(DE3), pET-sumo-OmpA-BL21(DE3), pET-sumo-OmpH-BL21(DE3), and pET-sumo-OmpW-BL21(DE3) were picked and inoculated into LB (containing 75 μg / mL Amp) liquid culture medium, and then transferred to 200mL LB (containing 75μg / mL Amp) liquid culture medium, and waited for the bacterial liquid OD 600 When the pH reaches 0.6-0.8, take an appropriate amount of bacterial solution as a blank control (without IPTG), add IPTG with a final concentration of 1mM to the remaining bacterial solution, and induce protein in each recombinant bacterium at 37℃ for 4h, 28℃ for 8h, and 16℃ for 16h. After the induction, collect the cells by centrifugation, resuspend and crush, collect the supernatant and precipitate, and perform SDS-PAGE electrophoresis. Collect the cells and detect the expression of the target protein by SDS-PAGE.

[0043] After the induction expression conditions were determined, the newly streaked recombinant strain was inoculated and transferred to 1L LB (containing 75μg / mL Amp) liquid medium for expansion culture, and shaken at 37°C and 200rpm until OD 600 When the pH is 0.6-0.8, IPTG with a final concentration of 1mM is added to induce each recombinant bacterium under its optimal induction conditions. After the induction is completed, the bacteria are collected, crushed under high pressure, centrifuged at low temperature, and the supernatant and precipitate are separated. The supernatant is then purified by affinity chromatography using Ni resin.

[0044] After optimization of expression vector and expression conditions, all recombinant proteins can be expressed in soluble form. The recombinant proteins were purified by Ni resin affinity chromatography and detected by SDS-PAGE. The results showed that the sizes of purified ApxIAN, ApxIIAN, ApxIIIAN, OmpD, LppB, OmlA, TbpB and PlpE, VacJ, OmpA, OmpH, OmpW were 52kDa, 26kDa, 51kDa, 55kDa, 54kDa, 50kDa, 70kDa and 52kDa, 38kDa, 48kDa, 35kDa, 32kDa, respectively, which were consistent with expectations. Further Western Blot analysis showed that the recombinant proteins were successfully expressed and had good antigenicity.

[0045] Example 2 Screening of antigenic proteins of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida and evaluation of the optimal antigen combination

[0046] 1. Screening of antigenic proteins of Actinobacillus pleuropneumoniae

[0047] Take the purified porcine pleuropneumoniae Actinobacillus protein OmpD, LppB, OmlA, TbpB of Example 1, dilute appropriately according to its concentration and mix with aluminum hydroxide adjuvant 1:1 equal volume, prepare a subunit vaccine of a single protein after full combination, and finally make the content of each protein in each dose of vaccine 50μg. Take 48 SPF-grade 5-week-old female BALB / c mice and randomly divide them into 6 groups, each with 8 mice. According to the mouse test scheme in Table 4, immunization and attack are carried out. After the first immunization, the second immunization is carried out 14d after the first immunization. The immunization method is multi-point injection of the neck and back skin. The immunization dose and route are the same as the first immunization. Finally, the APP5 type strain is used for attacking, and the survival of each group of mice is observed for one week. The results show that all mice in the control group died, the survival rate of mice in the Merck commercial vaccine group was 67.5%, and the survival rate of mice in the OmpD group, OmlA group, and LppB group was 37.5%, which is higher than the TbpB group with a survival rate of 12.5% ​​(the results are shown in Table 5).

[0048] Table 4 Mouse experimental scheme

[0049]

[0050] Table 5 Immune protection rate of each group after challenge with APP5 strain

[0051]

[0052] 2. Evaluation of the best antigen combination for Actinobacillus pleuropneumoniae

[0053] Take the purified porcine pleuropneumoniae Actinobacillus protein ApxIAN, ApxIIAN, ApxIIIAN, OmpD, LppB of Example 1, dilute appropriately according to their concentrations and mix them, mix the mixed protein solution with aluminum hydroxide adjuvant in equal volumes at a ratio of 1:1, and prepare a subunit vaccine containing multiple proteins after full combination, and finally make the content of each protein in each dose of vaccine 20 μg. Take 120 SPF-grade 5-week-old female BALB / c mice and randomly divide them into 6 groups, each with 20 mice. Perform immunization according to the mouse test scheme in Table 6. The second immunization was carried out 14 days after the first immunization, and the immunization method was subcutaneous multi-point injection at the back of the neck. The immunization dose and route were the same as those of the first immunization. Finally, APP5 and APP7 strains were used for toxicity attack, and continuous observation for one week was performed to count the survival of mice in each group. The results show that the second vaccine group (three toxins + OmpD) has the highest protection rate against APP5 and APP7 strains, both of which are 90% (the results are shown in Tables 7 and 8). Therefore, the combination of ApxIAN, ApxIIAN, ApxIIIAN and OmpD was selected as the antigen combination of porcine contagious pleuropneumonia subunit vaccine.

[0054] Table 6 Mouse test protocol

[0055]

[0056] Table 7 Immune protection rate of each group after challenge with APP5 strain

[0057]

[0058] Table 8 Immune protection rate of each group after challenge with APP7 strain

[0059]

[0060] 3. Screening of porcine Pasteurella multocida antigen protein

[0061] Take the purified porcine Pasteurella multocida proteins OmpD, PlpE, VacJ, OmpA, OmpH, and OmpW of Example 1, dilute them appropriately according to their concentrations, and mix them in equal volumes of 1:1 with aluminum hydroxide adjuvant, and prepare a subunit vaccine of a single protein after full combination, and finally make the content of each protein in each dose of vaccine 50 μg. Take 128 SPF-grade 5-week-old female BALB / c mice and randomly divide them into 8 groups, each with 16 mice. Perform immune attack according to the mouse test scheme in Table 9. Perform the second immunization 14d after the first immunization, and the immunization method is multi-point injection of the neck and back skin. The immunization dose and route are the same as the first immunization. Finally, use PmA type and PmD type to attack the virus, observe continuously for one week, and count the survival of each group of mice. The results showed that the cross-protection of the VacJ group against PmA and PmD was good, 50% and 62.5% respectively, while the protection rate of the PlpE group against PmA was extremely high, 100%, and against PmD was low, only 25%, and the protection rates of other protein groups were all low (results are shown in Tables 10 and 11). Therefore, VacJ and PlpE were selected as the antigen combination of the subunit vaccine of porcine Pasteurella multocida.

[0062] Table 9 Mouse test plan

[0063]

[0064]

[0065] Table 10 Immune protection rate of each group after challenge with PmA strain

[0066]

[0067] Table 11 Immune protection rate of each group after challenge with PmD strain

[0068]

[0069]

[0070] Example 3 Preparation of a combined subunit vaccine against porcine contagious pleuropneumonia and Pasteurella multocida and evaluation of its immune effect

[0071] 1. Preparation of a combined subunit vaccine for porcine contagious pleuropneumonia and porcine Pasteurella multocida

[0072] The purified proteins ApxIAN, ApxIIAN, ApxIIIAN, OmpD, PlpE and VacJ in Example 1 were taken and appropriately diluted according to their concentrations and then mixed. The mixed protein solution was mixed with aluminum hydroxide adjuvant in an equal volume ratio of 1:1, and after sufficient combination, the content of each protein in each dose of vaccine was 25 μg.

[0073] 2. Evaluation of the immune effect of bivalent subunit vaccines

[0074] Take 80 SPF-grade 5-week-old female BALB / c mice and randomly divide them into a control group and a two-unit subunit vaccine group, with 40 mice in each group. According to the mouse test scheme in Table 12, immunization and challenge were performed. The second immunization was performed 14 days after the first immunization, and the immunization method was multi-point injection of the neck and back skin. The immunization dose and route were the same as the first immunization. Before the first immunization, the second immunization and the challenge, the orbital vein blood of each group of mice was collected and the serum was packaged and frozen for standby use. Before the challenge, the control group and the two-unit subunit vaccine group were divided into 4 groups, each with 10 mice, and APP5, APP7, PmA, and PmD strains were used for challenge, respectively.

[0075] Table 12 Mouse test plan

[0076]

[0077] 3. Detection of mouse serum specific antibody levels

[0078] The recombinant proteins ApxIAN, ApxIIAN, ApxIIIAN, OmpD, PlpE, and VacJ were diluted to 4 μg / mL with coating buffer (25 mmol / L carbonate buffer, pH = 9.6) and coated on ELISA plates, 100 μL / well, overnight at 4°C. The serum of mice with the two-unit vaccine before the first immunization, before the booster immunization, and before the challenge and the serum of mice in the control group (PBS + aluminum hydroxide adjuvant group) (1:500) were used as the primary antibody, and goat anti-mouse IgG-HRP (1:5000) was used as the secondary antibody. After sufficient incubation, TMB color development solution was used for 30 minutes, and the color development was stopped with color development stop solution, and then the OD was read with an enzyme reader. 450 The values ​​were used to detect and compare the differences in the specific antibody levels of ApxIAN, ApxIIAN, ApxIIIAN, OmpD, PlpE and VacJ in the serum of each group of mice.

[0079] The results showed that after booster immunization with the bivalent subunit vaccine, the specific antibody levels of ApxIAN, ApxIIAN, ApxIIIAN, OmpD, PlpE and VacJ in the serum of each group of mice were significantly higher than those before the initial immunization (p<0.01); there was no significant difference in the antibody level in the serum of mice in the PBS+aluminum hydroxide adjuvant control group before and after immunization; after booster immunization, there were significant differences in the antibody levels between the bivalent subunit vaccine group and the control group (p<0.05, p<0.01) (see Figure 1 ). It showed that the bivalent subunit vaccine could stimulate mice to produce high levels of specific antibodies against the above six antigens.

[0080] 4. Virus challenge test

[0081] Fourteen days after booster immunization, mice in each group were challenged with freshly cultured APP5, APP7, PmA and PmD strains at their respective minimum lethal doses (MLD) by intraperitoneal injection (APP5: 2×10 8 CFU / pc, APP7: 3×10 8 CFU / piece, PmA: 30CFU / piece, PmD: 2×10 6 The clinical symptoms and mortality of the mice were observed within one week, and the immune protection rate of the two-unit subunit vaccine was statistically analyzed.

[0082] The test results showed that all mice in the PBS control group died during the observation period, and the protection rates of the two-unit subunit vaccine against mice challenged with APP5, APP7, PmA and PmD strains were 100% (10 / 10), 100% (10 / 10), 90% (9 / 10), and 90% (9 / 10), respectively (see Table 13 for the results). According to the statistical Log-Rank algorithm, the survival rate of mice in the PBS+aluminum hydroxide adjuvant control group and the two-unit subunit vaccine group was significantly different (p<0.01) (see Table 13 for the results). Figure 2 ). The results showed that the bivalent subunit vaccine had a good immune protection effect on mice infected with APP5, APP7, PmA and PmD strains respectively.

[0083] Table 13 Immune protection rate of dual subunit vaccine against different strains after infection

[0084]

[0085]

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A combination of antigenic proteins of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida, Features: It comprises proteins 1-6 whose amino acid sequences are shown as SEQ ID NO.1-6 respectively.

2. The antigen protein combination according to claim 1, Features: The nucleotide sequences encoding proteins 1-6 are shown in SEQ ID NOs. 7-12 respectively.

3. The antigen protein combination according to claim 1, Features: Proteins 1-6 were obtained by prokaryotic expression.

4. The antigen protein combination of porcine Actinobacillus pleuropneumoniae and porcine Pasteurella multocida according to claim 3, Features: The prokaryotic expression vector is pET-SUMO vector and the host is Escherichia coli.

5. Use of the antigen protein combination according to any one of claims 1 to 4 in the preparation of a combined subunit vaccine for porcine contagious pleuropneumonia and porcine Pasteurella multocida.

6. A porcine contagious pleuropneumonia-swine pasteurella multocida bivalent subunit vaccine, Features: Comprising the antigen protein combination according to any one of claims 1 to 4.

7. The porcine contagious pleuropneumonia-porcine Pasteurella multocida bivalent subunit vaccine according to claim 6, Features: An adjuvant is also included.

8. The porcine contagious pleuropneumonia-porcine Pasteurella multocida bivalent subunit vaccine according to claim 7, Features: The adjuvant is aluminum hydroxide adjuvant.

9. Use of the bivalent subunit vaccine of porcine contagious pleuropneumonia and porcine Pasteurellosis multocida according to any one of claims 6 to 8 in the preparation of a medicament for preventing and treating porcine contagious pleuropneumonia and porcine Pasteurellosis multocida.

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