CN113350495A - Streptococcus suis-haemophilus parasuis disease-porcine infectious pleuropneumonia triple subunit vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses a triple subunit vaccine for swine streptococcosis-haemophilus parasuis disease-swine contagious pleuropneumonia and a preparation method thereof. The triple subunit vaccine contains Streptococcus suis antigen proteins MRP, SLY and EF, Haemophilus parasuis antigen proteins AfuA, OppA2, CdtB and OppA, and Actinobacillus pleuropneumoniae antigen proteins ApxI, ApxII and OMP. Experiments prove that the vaccine can stimulate strong immune response of mice, has good cross protection effect on the mice attacked by different serotypes of streptococcus suis, haemophilus parasuis and actinobacillus pleuropneumoniae, and fully shows that the triple subunit vaccine prepared by the invention has good protection effect. Therefore, the invention lays a solid foundation for developing the high-efficiency, broad-spectrum and low-cost vaccine for the streptococcus suis disease-haemophilus parasuis disease-porcine infectious pleuropneumonia, and provides an effective technical means for preventing and treating the streptococcus suis disease, the haemophilus parasuis disease and the porcine infectious pleuropneumonia.

Description

Streptococcus suis-haemophilus parasuis disease-porcine infectious pleuropneumonia triple subunit vaccine and preparation method thereof

Technical Field

The invention relates to a triple subunit vaccine and a preparation method thereof, in particular to a triple subunit vaccine for swine streptococcosis-haemophilus parasuis disease-porcine infectious pleuropneumonia and a preparation method thereof, belonging to the technical field of veterinary medicines.

Background

Streptococcosis suis is a bacterial infectious disease caused by Streptococcus Suis (SS) and characterized by clinical symptoms of meningitis, septicemia, arthritis and the like of pigs, and is a major problem which troubles the pig industry all over the world for a long time. There are numerous serotypes of streptococcus suis, of which streptococcus suis serotype 2 (SS2) is also a zoonotic pathogen. People can be infected with the pathogen through a specific transmission path (such as wound exposure and the like) (Zhang diaza, Jinmeilin, Chen Chun, Streptococcus suis type 2 research progress (review). Breeding and feed, 2005,13-18.) and if the treatment is not timely, septicemia, meningitis and the like can be caused to cause poor prognosis, thus seriously threatening public health and safety.

The virulence factors of the streptococcus suis mainly comprise MRP, SLY, EF and the like. The several virulence factors are mainly found in the strains isolated in sick pigs, and the strains isolated in healthy pigs contain few virulence factors. Wisselink et al, which extracted MRP and EF from Streptococcus suis type 2, emulsified with adjuvant, immunized piglets found to have better immunoprotection against Streptococcus suis type 2 pathogenic strains (Wisselink HJ, N Stockhofe-Zurwieden, LAT Hilgers, et al, Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.Veterinary Microbiology,2002b:84,155-168), suggested that MRP and EF have the potential to develop into subunit vaccines. Hemolysin (sullysin) is secreted in the supernatant and is a member of the thiol-activated family of perforating toxins. Experiments have shown that the quantity of hemolysin produced by Streptococcus suis is related to the virulence of the strain (Norton PM, C Rolph, PN Ward, et al., episomal invasion and cell lysis by viral strains of Streptococcus suis infection by the presence of strain. Pathologens & diseases, 2010:26, 25-35.). However, studies have shown that it is not necessary in the pathogenic process of streptococci. SLY has hemolytic activity and is most hemolytic to human O-type erythrocytes.

Haemophilus parasuis disease is a bacterial infectious disease caused by Haemophilus Parasuis (HPS) and characterized by meningitis, pericarditis, arthritis and multiple serositis in pigs. This bacterium was first isolated and described in serous secretions of sick pigs by Glaser, a German scientist in 1910, so the disease is also known as Glaser's disease. Haemophilus parasuis is a serious hazard to the swine industry and is widely distributed worldwide. There are numerous serotypes of haemophilus parasuis and the current serotyping methods are not able to identify all haemophilus parasuis isolates. Serotype survey shows that serotypes popular at home and abroad are mainly serotypes with stronger toxicity such as 5 types, 4 types and 13 types (Liu Zheng Fei, Chua Xuanwang, Chenghun, and the like, research progress of haemophilus parasuis, animal medicine progress, 2003:24, 17-19). At present, the disease is in an obvious rising trend in intensive pig farms, mainly causes death of suckling piglets and weaned piglets, poor growth and development of the pigs, and low feed utilization rate, and has a common mixed infection phenomenon of haemophilus parasuis and other pathogens. The isolation rate of Haemophilus parasuis from clinically ill pigs is as high as 20%, which also causes great difficulty in the diagnosis and control of swine diseases (Kim J, HK Chung, T Jung, et al, Postweining multisystemic fashion wasting syndrome of pigs in Korea: evaluation, microscopical publications and surgery microorganisms. journal of Veterinary Medical Science,2002:64, 57).

Virulence factors of haemophilus parasuis mainly include AfuA, CDT, OppA2 and the like. The iron ABC transport substrate binding protein encoded by afuA gene mainly participates in transmembrane transport of Fe3+, has important role in the utilization of transferrin and lactoferrin, and in the process of bacterial infection of organism (Wei X, S Cao, L Zhang, et al, comprehensive protein analysis of the extracellular proteins of two Haemophilus paradoxus strains Nagasaki and SW114.Biochemical & biological Research Communications,2014:446, 997-1001). Charland studies have shown the presence of transferrin binding protein receptors in porcine serum (Charland N, CG D' Silva, RA Dumont, et al, Contact-dependent acquisition of transport-bound iron by two strands of microorganisms, 1995:41, 70). The afuA gene was found to be transcribed at a higher level in the virulent type 5 reference strain than in the avirulent type 3 reference strain. Cytolethal swelling toxin (CDT) is a protein toxin produced by many gram-negative bacteria, belongs to exotoxin which is unstable due to heat, and can cause swelling and death of eukaryotic cells. The holotoxin is composed of three subunits, namely CdtA, CdtB and CdtC. The research shows that the single CdtB has DNase activity, can enter cells and induce the breakage of DNA double chains in the nucleus, the CDTA and the CDTC can enhance the DNase I activity of the CDTB independently or together, the CDT holotoxin has the strongest toxicity to genes and can induce PK-15 cells to generate p 53-dependent apoptosis (Buboo, research on the cytotoxicity mechanism of the haemophilus parasuis lethal swolleninal toxin, the national academy of agricultural sciences, 2015). The oligopeptide permeases OppA and OppA2 belong to a member of the ABC binding cassette transporter family, which is involved in the transmembrane transport of proteins, polypeptides and various ions in the body. Researches find that ABC transporter is closely related to the virulence of bacteria, and participates in the uptake of external nutrition and metal ions by pathogenic bacteria and the adsorption of host cells. The family of proteins has good immunogenicity and can be used as candidate antigenic proteins of recombinant subunit vaccines (P L, B M, dW L, et al, Three differential phosphorus transport receptors encoded by the Mycobacterium tuberculosis gene and the bacterium present at the surface of Mycobacterium bovis BCG. Journal of Bacteriology,1997:179, 2900-.

Porcine infectious pleuropneumonia (Porcine infectious pleuropneumonia) is caused by Actinobacillus pleuropneumoniae (APP)Respiratory diseases of pigs. The pathological features are extensive cellulosic exudation, necrosis, hemorrhage, pleural adhesion of the lung, infiltration of neutrophils, lymphocytes and macrophages, vascular wall embolism, cellulose deposition, etc. If the bacterium is introduced into a clean pig herd which is not infected with actinobacillus pleuropneumoniae, acute outbreak can be caused, the mortality rate can reach 100 percent, if the pig herd is chronically infected, the mortality rate is greatly changed, but the morbidity is high, and once the pig is infected with the disease, the mortality rate is slow, the feed utilization rate is low, the daily gain is obviously reduced, and the like, so that the serious economic loss is brought to the pig raising industry (Janine T. Boss,

janson, Brian J.Sheehan, et al, Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of Infection, Microbes and Infection,2002,4: 225-. The division of APP into 15 serotypes based on capsular polysaccharide antigens has recently been reported for newly discovered types 16, 17 and 18 (JT Boss, Y Li, FC Robert, et al, comprehensive sequence analysis of the cellular polysaccharide of Actinobacillus pleuropneumoniae sera 1-18, and development of two multiplex PCRs for comprehensive capsule type microbiological analysis 2018,220: 83-89).

Pneumonia damage caused by APP is the result of multiple toxins acting together. Besides toxins, other virulence factors of APP also have some effect on pathogenesis. Including capsular polysaccharide, outer membrane protein, transferrin binding protein, adhesin, urease, protease, peroxide-reducing enzyme, Cu-Zn superoxide dismutase, etc. One of the most important virulence factors responsible for the pathogenesis of APP is the Apx toxin, an exotoxin that is secreted outside the cell. Including ApxI, ApxII, ApxIII and apxiv, apxiv is expressed only in vivo. ApxI is a strongly hemolytic, strongly cytotoxic protein with a molecular weight of 105 KDa; ApxII is a weakly hemolytic and weakly cytotoxic protein with a molecular weight of 103 KDa; ApxIII has no hemolytic activity but has strong cytotoxicity, previously called pleurotoxin, and has a molecular weight of 120 KDa. The pathogenic mechanism of Apx toxin is: the first line of defense to first invade the body, high concentrations of Apx toxin can form pores in phagocytes or other target cell membranes, causing cell swelling and death, and further causing tissue damage in terms of toxic effects on alveolar endothelial and epithelial cells, strong oxidation on macrophages and neutrophils, causing them to release oxygen residues, thus exhibiting toxic effects on host cells (Schaller, A., Kuhn, R., Kuhner, P., et al., Characterisation of apxIVA: a new RTX diagnostic of Actinobacillus pleuropneumoniae. microbiology,1999, 145: 2105-2116). Often, the major virulence factors of bacteria are also often the major protective antigens. The Apx toxin of APP, as well as an outer membrane protein Omp of around 40kDa, have proven to be highly immunogenic and are used in the development of subunit vaccines (F. Haesebrouck, K. Chiers, I.Van Overbeke, et al, Actinobacillus pleuropneumoniae infections in vaccines: the role of viral genes in pathogenesis and protection. vector Microbiology,1997,58: 239-.

Streptococcus suis, Haemophilus parasuis and porcine contagious pleuropneumonia have similar clinical symptoms and are common respiratory diseases in pigs. The triple vaccine is used for immune prevention, so that the effect of one-injection multiple prevention can be achieved, the times of immunization can be greatly reduced, the consumption of manpower and material resources is reduced, and the triple vaccine is the research direction of vaccines for preventing and treating the three bacterial diseases. Streptococcus suis, Haemophilus parasuis and Actinobacillus pleuropneumoniae are all of numerous serotypes, and the cross-protection effect among different serotype strains is limited. Therefore, even with multivalent vaccines, the cross-protection effect of inactivated vaccines is still not ideal. The current vaccine research is more inclined to the development of subunit vaccines, and in recent years, the discovery of a plurality of pathogenic related protein molecules with good immunogenicity of HPS, SS and APP makes the research strategy of the subunit vaccines for the three bacterial diseases more attractive.

Because bacterial proteins, especially some toxin proteins, generally have large molecular weights, often above 100kDa, even up to 200kDa, artificial recombinant expression and purification of these proteins in vitro is often difficult. Either the yield is low or inclusion bodies are formed, which is not beneficial to large-scale production. Therefore, the analysis of the antigen structure of the predicted protein and the construction of the truncated expressed protein not only keep the immunogenicity of the whole protein, but also facilitate the expression and purification, which becomes the direction of efforts of various researchers. The inventor optimizes MRP, SLY and EF of streptococcus suis, ApxI, ApxII and OMP of actinobacillus pleuropneumoniae reported in the literature and AfuA, OppA2, CdtB and other antigen proteins of haemophilus parasuis identified by the inventor, so that the proteins are all expressed in a soluble form and are convenient to purify, and a good foundation is laid for developing streptococcus suis-haemophilus parasuis disease-porcine infectious pleuropneumonia triple subunit vaccines and large-scale production.

The streptococcus suis and haemophilus parasuis disease bigeminal genetic engineering subunit vaccine which is obtained in 2019 by a new veterinary certificate of the same kind in China is the first genetic engineering subunit vaccine aiming at the two bacterial diseases in the world, and mainly solves the problem of common pathogen multi-serotype coinfection in two clinical productions. But the practical application effect still needs further verification. At present, no triple subunit vaccine aiming at streptococcus suis, haemophilus parasuis and porcine infectious pleuropneumonia exists internationally, so that the invention has strong innovation and application value and wide application prospect.

Disclosure of Invention

The invention aims to provide a combined vaccine with good cross protection effect on swine streptococcosis, haemophilus parasuis and porcine contagious pleuropneumonia. In particular to a triple subunit vaccine which takes a plurality of main protective antigens of streptococcus suis, haemophilus parasuis and actinobacillus pleuropneumoniae as components.

The above purpose of the invention is realized by the following technical scheme:

the invention relates to a swine streptococcosis-haemophilus parasuis disease-swine contagious pleuropneumonia triple subunit vaccine, which contains MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis, and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae.

The invention utilizes bioinformatics software to analyze and predict the antigen structures of MRP, SLY and EF proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 proteins of haemophilus parasuis and ApxI, ApxII and OMP proteins of actinobacillus pleuropneumoniae, respectively constructs truncated expressed proteins with different antigen segments and different lengths, judges the reactogenicity and immunogenicity of the truncated expressed proteins through the reactivity with specific antiserum, and enables the target protein to be expressed in a soluble form and is beneficial to purification by optimizing different expression vectors, expression strains and culture and purification conditions. Preferably, MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB recombinant proteins of haemophilus parasuis, and ApxIIA recombinant proteins of actinobacillus pleuropneumoniae are proteins expressed after truncation, and are encoded by MRP, SLY and EF genes of streptococcus suis, CdtB gene of haemophilus parasuis, and ApxIIA genes of actinobacillus pleuropneumoniae, respectively, wherein one of the truncated MRP genes has a nucleotide sequence of SEQ ID NO: 1 is shown in the specification; a truncated sly gene having the nucleotide sequence of SEQ ID NO: 2 is shown in the specification; a truncated ef gene having the nucleotide sequence of SEQ ID NO: 3 is shown in the specification; a truncated cdtB gene having the nucleotide sequence of SEQ ID NO: 4 is shown in the specification; a truncated apxiia gene having the nucleotide sequence of SEQ ID NO: 8 is shown in the specification; a truncated apxIIA gene having the nucleotide sequence of SEQ ID NO: 9 is shown in the figure; the AfuA protein of haemophilus parasuis is encoded by the AfuA gene of haemophilus parasuis, and the nucleotide sequence thereof is SEQ ID NO: 5 is shown in the specification; the OppA protein of the haemophilus parasuis is coded by the oppA gene of the haemophilus parasuis, and the nucleotide sequence of the OppA protein is SEQ ID NO: 6 is shown in the specification; the OppA2 protein of Haemophilus parasuis is coded by the OPpA2 gene of Haemophilus parasuis, and the nucleotide sequence of the protein is SEQ ID NO: 7 is shown in the specification; the OMP protein of the actinobacillus pleuropneumoniae is coded by OMP gene of the actinobacillus pleuropneumoniae, and the nucleotide sequence is SEQ ID NO: shown at 10.

Of these, preferred concentrations of the MRP, SLY and EF recombinant proteins of Streptococcus suis, the CdtB, AfuA, OppA2 recombinant proteins of Haemophilus parasuis, and the ApxIA, ApxIIA, and OMP recombinant proteins of Actinobacillus pleuropneumoniae are 0.05-0.5mg/mL, more preferably 0.1-0.3mg/mL, and still more preferably 0.1mg/mL, respectively.

Wherein, preferably, the vaccine also contains an adjuvant, and the adjuvant comprises but is not limited to the following types: oil-in-water adjuvants, polymer and water adjuvants, water-in-oil adjuvants, aluminum hydroxide adjuvants, vitamin E adjuvants. More preferably, the adjuvant is ISA201 VG.

Among them, preferably, the triple subunit vaccine is in a dosage form of intramuscular, intradermal or subcutaneous administration, and more preferably in a dosage form of intramuscular administration.

Furthermore, the invention also provides a method for preparing the triple subunit vaccine, which comprises the following steps:

1) respectively obtaining gene sequences of MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae by a PCR (polymerase chain reaction) method; wherein, MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB recombinant proteins of haemophilus parasuis and ApxIA and ApxIIA recombinant proteins of actinobacillus pleuropneumoniae are expressed after truncation, and are respectively encoded by MRP, SLY and EF genes of streptococcus suis, cdtB genes of haemophilus parasuis and apxIA and apxIIA genes of actinobacillus pleuropneumoniae, wherein, the nucleotide sequence of one truncated MRP gene is SEQ ID NO: 1 is shown in the specification; a truncated sly gene having the nucleotide sequence of SEQ ID NO: 2 is shown in the specification; a truncated ef gene having the nucleotide sequence of SEQ ID NO: 3 is shown in the specification; a truncated cdtB gene having the nucleotide sequence of SEQ ID NO: 4 is shown in the specification; a truncated apxiia gene having the nucleotide sequence of SEQ ID NO: 8 is shown in the specification; a truncated apxIIA gene having the nucleotide sequence of SEQ ID NO: 9 is shown in the figure; the AfuA protein of haemophilus parasuis is encoded by the AfuA gene of haemophilus parasuis, and the nucleotide sequence thereof is SEQ ID NO: 5 is shown in the specification; the OppA protein of the haemophilus parasuis is coded by the oppA gene of the haemophilus parasuis, and the nucleotide sequence of the OppA protein is SEQ ID NO: 6 is shown in the specification; the OppA2 protein of Haemophilus parasuis is coded by the OPpA2 gene of Haemophilus parasuis, and the nucleotide sequence of the protein is SEQ ID NO: 7 is shown in the specification; the OMP protein of the actinobacillus pleuropneumoniae is coded by OMP gene of the actinobacillus pleuropneumoniae, and the nucleotide sequence is SEQ ID NO: 10 is shown in the figure;

2) cloning the truncated mrp gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-mrp; cloning the truncated sly gene to a pcold-sumo vector to construct a recombinant plasmid pcold-sly; cloning the truncated ef gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-ef; cloning the truncated cdtB gene to a pET-22b vector to construct a recombinant plasmid pET-22 b-cdtB; cloning the afuA gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-afuA; cloning oppA gene to pET-28a (+) carrier to construct recombinant plasmid pET-28 a-oppA; cloning oppA2 gene to pET-28a (+) vector to construct recombinant plasmid pET-28a-oppA 2; cloning the truncated apxIA gene to a pcold-sumo vector to construct a recombinant plasmid pcold-apxIA; cloning the truncated apxIIA gene to a pcold-sumo vector to construct a recombinant plasmid pcold-apxIIA; cloning the omp gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-omp;

3) respectively transforming the recombinant plasmids obtained in the step 2) into escherichia coli to obtain recombinant escherichia coli which respectively express MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae;

4) inducing and expressing the recombinant escherichia coli obtained in the step 3), centrifuging after carrying out ultrasonic treatment on the culture, purifying the obtained supernatant by using an affinity chromatography column to respectively obtain purified MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae;

5) mixing the purified recombinant proteins MRP, SLY, EF, CdtB, AfuA, OppA2, ApxI, ApxII and OMP, mixing the mixed protein solution with ISA201VG, and emulsifying to obtain the final product.

The vaccines prepared by the present invention may be administered by a prime-boost regimen. For example, pigs may receive a second boost after a period of time (e.g., about 7, 14, 21, or 28 days) following the first vaccination. Typically, the dose for boosting is the same or lower than the dose administered for priming. In addition, a third booster immunization may be performed, for example 2-3 months, 6 months or a year after immunization.

The invention also evaluates the protection effect of the streptococcus suis-haemophilus parasuis disease-porcine infectious pleuropneumonia triple subunit vaccine on mice respectively attacking viruses of streptococcus suis, haemophilus parasuis and actinobacillus pleuropneumoniae, and simultaneously sets up a PBS and ISA201VG mixed emulsification control group. Each group of mice was challenged with M126 strain of Streptococcus suis serotype 2, GZ2 strain of serotype 9, HN10 strain of serotype 5 of Haemophilus parasuis, ZD12 strain of serotype 13, MD strain of serotype 5 of Actinobacillus pleuropneumoniae, and S-8 strain of serotype 7, respectively. It can be seen that the protective rates of the triple subunit vaccine on the streptococcus suis challenge mice are respectively 100% and 80%; the protection rate of the mouse with the haemophilus parasuis for attacking poison is 60 percent and 80 percent respectively; the protection rate of the mouse with the actinobacillus pleuropneumoniae attacking poison is 80 percent and 100 percent respectively. Is superior to the PBS and ISA201VG control group, has no protection on the virus attack of streptococcus suis, haemophilus parasuis and actinobacillus pleuropneumoniae, and all the virus attack mice die.

In the antibody level detection, the antibody titer of rMRP, rSLY, rEF, rCdtB, rAfuA, rOppA2, rApxI, rApxII and rOMP generated by the mice stimulated by the triple subunit vaccine is higher and is better than that of the PBS + ISA201VG control group.

Therefore, the invention further provides the application of the triple subunit vaccine in preparing medicaments for simultaneously preventing the streptococcus suis disease, the haemophilus parasuis disease and the porcine infectious pleuropneumonia.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a swine streptococcosis-haemophilus parasuis disease-swine contagious pleuropneumonia triple subunit vaccine, which can stimulate strong immune response of mice and has good cross protection effect on mice infected by different serotypes of streptococcus suis, haemophilus parasuis and actinobacillus pleuropneumoniae. Fully indicates that the triple subunit vaccine prepared by the invention has good protection effect. Therefore, the invention lays a solid foundation for developing the high-efficiency, broad-spectrum and low-cost vaccine for the streptococcus suis disease-haemophilus parasuis disease-porcine infectious pleuropneumonia, and provides an effective technical means for preventing and treating the streptococcus suis disease, the haemophilus parasuis disease and the porcine infectious pleuropneumonia.

Drawings

FIG. 1 shows the mrp, sly, ef, cdtB, afuA, oppA2, apxIA, apxIIA, omp gene clones;

wherein, M is DL 2000; 1: mrp,2: sly,3: ef,4: cdtB,5: afuA,6: oppA,7: oppA2,8: apxIA,9: apxIIA,10: omp;

FIG. 2 is a double restriction enzyme digestion verification of recombinant plasmid;

wherein, M is DL 15000; 1: pET-28a-mrp,2: pcold-sly,3: pET-28a-ef,4: pET-22b-cdtB, 5: pET-28a-afuA,6: pET-28a-oppA,7: pET-28a-oppA2,8: pcold-apxIA,9: pcold-apxIIA,10: pET-28 a-omp;

FIG. 3 is an SDS-PAGE detection of protein expression;

wherein M is molecular weight marker; 1: rMRP,2: rSLY,3: rEF,4: rCdtB,5: rAfuA,6: rOppA, 7: rOppA2,8: rApxI,9: rApxII,10: rOMP;

FIG. 4 shows Western-blot validation of the expression of each protein;

wherein M is molecular weight marker; 1: rMRP,2: rSLY,3: rEF,4: rCdtB,5: rAfuA,6: rOppA, 7: rOppA2,8: rApxI,9: rApxII,10: rOMP;

FIG. 5 shows ELISA antibody titer measurements for different proteins;

FIG. 6 shows the results of the challenge test.

Detailed Description

The invention will be better understood by the following further description of specific embodiments thereof, which are not to be construed as limiting the invention thereto.

EXAMPLE 1 expression purification of protein

1 extraction of bacterial genomes

The method comprises the steps of streaking Streptococcus suis 05ZYH33 glycerol bacteria stored in a laboratory in a THA culture medium (containing 10% horse serum), placing the bacteria in a constant-temperature incubator at 37 ℃ overnight, selecting a single colony, transferring the single colony to a THB culture medium (containing 10% horse serum), standing and culturing the colony in the incubator, carrying out amplification culture on the colony to 10mL of the THB culture medium (containing 10% horse serum), and extracting bacterial genomes by using a bacterial genome extraction kit according to instructions. And (4) placing the extracted genome in a-20 refrigerator for freezing and storing for later use.

The haemophilus parasuis HN10 glycerol strain stored in a laboratory is streaked in a TSA culture medium (containing 10 mug/mL NAD and 10% horse serum), then the culture medium is placed in a constant temperature incubator at 37 ℃ overnight, a single colony is picked and transferred to a TSB culture medium (containing 10 mug/mL NAD and 10% horse serum) to be subjected to standing culture in the incubator, the culture medium is expanded to 10mL of the TSB culture medium (containing 10 mug/mL NAD and 10% horse serum), and a bacterial genome is extracted by using a bacterial genome extraction kit according to the instruction. And (4) placing the extracted genome in a-20 refrigerator for freezing and storing for later use.

The actinobacillus pleuropneumoniae MD glycerol strain stored in a laboratory is streaked in a TSA culture medium (containing 10 mu g/mL NAD and 10% horse serum), the culture medium is placed in a constant temperature incubator at 37 ℃ overnight, a single colony is picked and transferred to a TSB culture medium (containing 10 mu g/mL NAD and 10% horse serum) to be subjected to static culture in the incubator, the culture medium is expanded to 10mL of the TSB culture medium (containing 10 mu g/mL NAD and 10% horse serum), and bacterial genome is extracted by using a bacterial genome extraction kit according to the instruction. And (4) placing the extracted genome in a-20 refrigerator for freezing and storing for later use.

2 construction and verification of recombinant plasmid

The invention utilizes bioinformatics software to analyze and predict the antigen structures of MRP, SLY and EF proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 proteins of haemophilus parasuis and ApxI, ApxII and OMP proteins of actinobacillus pleuropneumoniae, respectively constructs truncated expressed proteins with different antigen segments and different lengths, judges the reactogenicity and immunogenicity of the truncated expressed proteins through the reactivity with specific antiserum, and enables the target protein to be expressed in a soluble form and is beneficial to purification by optimizing different expression vectors, expression strains and culture and purification conditions.

Based on the above studies, the invention finally designs primers (table 1) according to the gene sequence of streptococcus suis 05ZYH33 strain (accession number CP000407.1), haemophilus parasuis SH0165 strain (accession number CP001321.1) and actinobacillus pleuropneumoniae L20 strain (accession number CP000569.1) in GenBank, and performs PCR according to the reaction program of table 2 with the bacterial genome as a template, to amplify the genes mrp, sley, slef, cdtB, afuA, oppA2, apxIA, apxiiia and omp genes of interest, wherein mrp, sley, ef, cdtB, apxIA and apxiiia are truncated expressed genes. Because PrimerSTAR Max DNA Polymerase has the characteristics of high specificity, high reaction sensitivity and high amplification efficiency, the annealing and extension time is greatly shortened due to the high annealing efficiency of the PrimerSTAR Max DNA Polymerase, and the PCR of the target gene can be denatured for 10s at 95 ℃ according to the specification; annealing at 55 ℃ for 10 s; extension of 72 ℃ for 30s, 30 cycles of the reaction sequence. After completion of PCR, nucleic acid electrophoresis was performed, and the results showed that the band sizes of the amplified mrp, sly, ef, cdtB, afuA, oppA2, apxIA, apxIIA and omp genes were located near the expected positions, respectively, as shown in FIG. 1. Wherein the nucleotide sequence of the truncated mrp gene is SEQ ID NO: 1 is shown in the specification; a truncated sly gene having the nucleotide sequence of SEQ ID NO: 2 is shown in the specification; a truncated ef gene having the nucleotide sequence of SEQ ID NO: 3 is shown in the specification; a truncated cdtB gene having the nucleotide sequence of SEQ ID NO: 4 is shown in the specification; an afuA gene, the nucleotide sequence of which is SEQ ID NO: 5 is shown in the specification; an oppA gene, the nucleotide sequence of which is SEQ ID NO: 6 is shown in the specification; an oppA2 gene, the nucleotide sequence of which is SEQ ID NO: 7 is shown in the specification; a truncated apxiia gene having the nucleotide sequence of SEQ ID NO: 8 is shown in the specification; a truncated apxIIA gene having the nucleotide sequence of SEQ ID NO: 9 is shown in the figure; an omp gene, the nucleotide sequence of which is SEQ ID NO: shown at 10.

After electrophoresis, DNA recovery was performed according to the instructions of the gel recovery kit, and the plasmid pET-28a and pET-22b and the recovered mrp, sly, cdtB, afuA, oppA2 and omp were digested simultaneously with the corresponding endonucleases according to the system shown in Table 3. The digestion products were gel recovered and ligated according to the system in Table 4. The product was transformed into E.coli DH 5. alpha. competent cells, and the cells were plated on LB plates (containing 50. mu.g/. mu.L Kan) for resistance selection. ef. and recovering the apxIA and apxIIA amplification product gel, connecting the recovered apold-sumo plasmid with pcold-sumo plasmid by utilizing a clonExpress II recombinant connection kit, transforming the product into E.coli DH5 alpha competent cells, and coating the cells on a plate of LB (containing 50 mu g/mu L Kan) for resistance screening. Respectively inoculating the obtained monoclonals into LB (containing 50 mu g/mu L Kan) liquid culture medium, performing shake culture at 37 ℃ for 12h, and performing enzyme digestion and sequencing identification on the quality-improved particles. Specifically, the truncated mrp gene is cloned to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-mrp; cloning the truncated sly gene to a pcold-sumo (+) vector to construct a recombinant plasmid pcold-sly; cloning the truncated ef gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-ef; cloning the truncated cdtB gene to a pET-22b vector to construct a recombinant plasmid pET-22 b-cdtB; cloning the afuA gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-afuA; cloning oppA gene to pET-28a (+) carrier to construct recombinant plasmid pET-28 a-oppA; cloning oppA2 gene to pET-28a (+) vector to construct recombinant plasmid pET-28a-oppA 2; cloning the truncated apxIA gene to a pcold-sumo vector to construct a recombinant plasmid pcold-apxIA; cloning the truncated apxIIA gene to a pcold-sumo vector to construct a recombinant plasmid pcold-apxIIA; cloning the omp gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-omp.

The sequencing result shows that the sequence of the fragments of recombinant plasmids pET-28a-mrp, pcold-sly, pET-28a-ef, pET-22b-cdtB, pET-28a-afuA, pET-28a-oppA2, pcold-apxIA, pcold-apxIIA and pET-28a-omp, mrp, sly, ef, cdtB, afuA, oppA2, apxIA, apxIIA and omp has the coincidence rate of 100 percent with the gene sequence in GenBank, and has no base mutation, thereby proving that the gene fragments are successfully inserted into corresponding vectors. The recombinant plasmid is subjected to double enzyme digestion and then subjected to nucleic acid electrophoresis, the result shows that the size of the fragment separated after double enzyme digestion is consistent with that of the gene fragment, the ten gene fragments are further proved to be correctly cloned to corresponding vectors respectively, and the enzyme digestion result is shown in figure 2.

TABLE 1 primer sequences

TABLE 2 PCR reaction System

TABLE 3 digestion system

TABLE 4 connection System

3 expression and purification of recombinant target protein

Transferring a recombinant plasmid with correct sequencing verification into E.coli BL21(DE3) competent cells, coating thalli on LB (containing 50. mu.g/mL Kan or 100. mu.g/mL Amp) plates for resistance screening, picking recombinant strains pET-28a-mrp-BL21(DE3), pcold-sly-BL21(DE3), pET-28a-ef-BL21(DE3), pET-22b-cdtB-BL21(DE3), pET-28a-afuA-BL21(DE3), pET-28a-oppA-BL21(DE3), pET-28a-oppA2-BL21(DE3), pcold-apxIA-BL21(DE3), pcold-apxIIA-BL21(DE 6356), pET-28 a-82p 8653 (DE 21) and inoculating single LB containing 50. mu.g/mL LB (LB) or LB-21, then respectively transferring the recombinant bacteria into 10mL LB (containing 50 mu g/mL Kan or 100 mu g/mL Amp) liquid culture medium of 10 tubes, adding IPTG with the final concentration of 1mM when the OD600 of the bacterial liquid is approximately equal to 0.7, respectively carrying out protein induction on each recombinant bacteria at 37 ℃, 28 ℃ and 16 ℃, collecting after 6h, carrying out ultrasonic disruption on the bacteria, separating precipitates and supernatant through centrifugation, and detecting the expression condition of the target protein through SDS-PAGE to determine the optimal induction condition of each recombinant protein.

Inducing the constructed recombinant bacteria, collecting the bacteria, taking a proper amount of the bacteria after resuspension, carrying out SDS-PAGE, transferring the bacteria to a PVDF membrane, and carrying out Western-blot by using a commercial mouse His antibody as a primary antibody, wherein the result is shown in figure 4, and the fact that the protein can be expressed in the recombinant bacteria is proved.

After the induction expression conditions were determined, the newly streaked recombinant strains were inoculated and transferred to an expansion culture in 1L LB (containing 50. mu.g/. mu.L Kan or 100. mu.g/mL Amp) liquid medium, shaken at 200rpm at 37 ℃ to an OD600 of 0.7, and IPTG was added to a final concentration of 1mM, and each recombinant strain was induced under the optimum induction conditions. After the induction is finished, the thalli are collected, and after the ultrasonic crushing and the heavy suspension, the supernatant and the sediment are centrifugally separated. Then Ni resin is used for affinity chromatography purification of protein.

All recombinant proteins can be expressed in a soluble form by optimizing expression vectors and expression conditions. The recombinant protein was purified by Ni resin affinity chromatography and detected by SDS-PAGE. The results show that the purified rMRP, rSLY, rEF, rCdtB, rAfuA, rOppA2, rApxI, rApxII, rOMP are 82kDa, 55kDa, 30kDa, 38kDa, 61kDa, 56kDa, 66kDa, 62kDa, 40kDa in size, respectively, consistent with the expected results, as shown in FIG. 3.

Example 2 preparation and animal testing of Streptococcus suis-Haemophilus parasuis-porcine infectious pleuropneumonia triple subunit vaccine

Preparation of 1-triad subunit vaccine

The purified proteins rMRP, rSLY, rEF, rCdtB, rAfuA, rOppA2, rApxI, rApxII and rOMP in example 1 are mixed, and the mixed protein solution is mixed with ISA201VG and the like in mass ratio and then emulsified, so that the content of each protein in each dose of the emulsified vaccine is 20 mu g.

2 mouse immunization test

20 4-week-old SPF-grade C57 mice were randomly divided into 2 groups of 10 mice each, and 40 4-week-old SPF-grade BALB/C mice were randomly divided into 2 groups of 20 mice each. Mice were immunized according to the immunization schedule of table 5. The second immunization was performed 14d after the first immunization, and the dose and route of immunization were the same as those of the first immunization. And (4) carrying out tail-cutting blood collection on all mice 14d after the second immunization, separating serum, and subpackaging and freezing for later use.

TABLE 5 immunization protocol

3 detection of antibody levels in mouse serum

Recombinant proteins rEF, rSLY, rMRP, rOPPA2, rAfuA, rCdtB, rApxI, rApxII and rOMP were diluted to 2. mu.g/mL coated ELISA plates, 100. mu.L/well, overnight at 4 ℃ with coating buffer (0.1mol/L Na2CO3-NaHCO3, pH9.6), respectively. The serum (1:100) of 6 groups of mice, including C57, BALB/C mouse serum separated before immunization and experimental groups (C57 group and BALB/C group) and control groups (C57 group and BALB/C group) separated after 14d of a secondary immune, is taken as a primary antibody, goat anti-mouse IgG-HRP (1:10000) is taken as a secondary antibody, the color is developed by TMB color developing solution, and the value of OD450 is read by a microplate reader. The levels of antibodies to rMRP, rSLY, rEF, rCdtB, rAfuA, rOPPA2, rApxI, rApxII and rOMP, respectively, were detected in serum by indirect ELISA.

The results in FIG. 5 show that after the secondary immunization of the triple subunit vaccine, the antibody levels of 10 antigens in the serum of each group of mice are extremely obviously increased compared with those before the immunization (p is less than 0.0001); the antibody level in the serum of the mouse of the PBS + ISA201 adjuvant control group is not different from that before immunization; there was a very significant difference in the levels of each antibody after the second immunization (p <0.0001) for the control and the triple subunit vaccine groups. It was shown that the triple subunit vaccine can stimulate mice to produce high levels of antibodies to the above 10 antigens.

4 challenge test

After the second immunization 14d, 6 groups of mice were intraperitoneally challenged with freshly cultured SS 2464 strain, SS9 GZ2 strain, HPS5 HN10 strain, HPS13 ZD12 strain, APP5 MD strain and APP 7S-8 strain, respectively, at the minimum lethal dose MLD of the respective strains (SS 2: 5X 10S-8)⁷CFU/only, SS 9: 5X 10⁷CFU/only, HPS 5: 1.5×10⁸CFU/only, HPS 13: 5X 10⁷CFU/cartridge, APP 5: 1.5X 10⁸CFU/cartridge, APP 7: 7.5X 10⁸CFU/only). Mice were observed for mortality within a week and the vaccine protection rate was statistically determined.

The results of the experiment in figure 6 show that the PBS control mice all died during the observation period, and that the protection rates of the triple subunit vaccine were 100% (5/5), 80% (4/5), 60% (3/5), 80% (4/5), 80% (4/5) and 100% (5/5) for mice challenged with SS2, SS9, HPS5, HPS13, APP5 and APP7, respectively. The survival rate of the PBS + ISA201 adjuvant control group and the experimental group of mice is remarkably different (p <0.05) according to the statistic Log-Rank algorithm. The triple subunit vaccine has good protection effect on mice respectively infected with SS2, SS9, HPS5, HPS13, APP5 and APP 7.

Sequence listing

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)

<120> Streptococcus suis-haemophilus parasuis disease-porcine contagious pleuropneumonia triple subunit vaccine and preparation method thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2259

<212> DNA

<213> mrp

<400> 1

gttttcaagg aaagtgaaca gaacgagcaa ggtagcaaat atcgcgtcat tgctcaatgg 60

tcaggagatg aaaccactaa aggtatatat ggaaaaatct atatcgctac tcaggtttgg 120

acgactaaat tgggaacaaa cgagtgggga tggtttgact attctgatga ccaagctggt 180

ataaaattta ataacaaagg tttttggccg gcaggtgttc aaaatacact tcgaaatgct 240

actccagcta cagctgtaga gactacttat atctacaaag aaagttccaa gtatggtgat 300

gtcattgttg agtactacga tactgacgga aaacaaattg taaattcagt tgtagatact 360

cctaagtcag ctcttggcac agagtataat acagatgtgg accgtagacc agccagcttg 420

gttgctgctg atgggacagt ctacttctac aaagaagtta agtctgattc agctaagaca 480

accggtacag tagttgcagg tacgacaact gttaagtatg tttacgaaaa agctggtagc 540

gttaatgtta acttcgttga catcaatggt aaagtaatca aagctcctgt ttcagatgaa 600

aaagatgcga aacctggtta caattatgat accgacttgg atcagaaatt agcttccatc 660

acttttgaag gcaaggaata caaacttgtt cctgctggtg attatccggt tggtaaagtt 720

ggcaagggaa ataacttgat tgaagttggt aataatactg cgaaaggtat tgacccaaca 780

acaggcaaaa ttgaagccgg tgttaacaaa gaagttacct atgtctatag agcagtgaca 840

ggttctgtag ttgtaaatta caaagataca gaaggtaatg tgattaaaga tccagaaacg 900

gatgtgtctg atgcaccggt tggagatgct tatactacaa ctgacaagaa accaaacgaa 960

atcatcacaa aagatggatc acgctatgtt cttgttccat ctaagacaga tggtgaggaa 1020

aatggtaaag ttatcgaagg aacaatcaca gtaacttatg tttaccagaa agttgcaaac 1080

tggattccag agattccaaa tgtaccagaa acagaccgtc caaaagtacc ttacccattt 1140

gacccaacag agccagacga gccaatcgat ccaacgacac caggaacaaa tggcgaggtt 1200

ccaaatattc cttacgttcc aggatataca ccggttgatc ctaaggataa cacgccgttg 1260

aaaccaattg atccaaatga tccaggtaag ggttatgtac caccaacacc agaaaatcca 1320

ggtgttgata caccaattcc ttatgttcca gttaaaaaag tcgtaactaa ccacgttgat 1380

gaagagggta accctattgc accgcaagaa gagggaacaa aaccaaacaa atcaatccca 1440

ggttacgagt tcacaggtaa aactgttact gacgaagatg gcaacacaac tcacatctac 1500

aagaaaacac cagaagttaa gaatggtaca gttgttgtta actatgtaac agaagatggc 1560

acagttatca aggaacctgt aacagataca ccaacttctc cagaaggcac accatacgac 1620

actacagaca acaaacctaa gacaatcact ttcaaaggtg aagagtatga attggttcgt 1680

gttgacggta cagaaaacgg taaagttgta gaaggtgaaa cagttgtgac ttacgtttac 1740

cgtaaagtcg aaacacctgc taagaaagtt gtaactaacc acgttgatga agagggtaac 1800

cctgttgcgc cgcaagaaga gggaacaaaa ccaaacaaat caatcccagg ttacgaattt 1860

acaggtaaaa ctgttactga cgaagatggc aacacaactc acatctacaa gaaaacacct 1920

gctaagaaag ttgtgactaa ccacgttgat gaagaaggta accctattgc tccacaagag 1980

gatgggacaa caccaaaacg tcaaatttca ggttacgagt atgtgcgtac tgtagttgat 2040

gaagaaggta acacgacaca tatttatcgc aaactttcta ataaaccaac aacacctgag 2100

aaggaaactc ctgcaaaacc tcaagcaggt aaaaccgctt caggtaaagc tcaattgcca 2160

aatactggtg aggcttcatc tgtggcaggt gcgcttggta cagcaatgct tgtcgcaaca 2220

cttgcgtttg caagaaaacg tcgtcgtaac gaagattag 2259

<210> 2

<211> 1494

<212> DNA

<213> sly

<400> 2

atgagaaaaa gttcgcactt gattttaagc tcaatagtca gtttggcact cgtaggggtc 60

acaccattga gtgttcttgc agattccaaa caagatatta atcagtattt tcaaagcttg 120

acttacgagc cacaagagat tcttacaaat gagggagaat acattgataa tccgccagca 180

acaactggta tgttagaaaa cggacgtttt gtagtacttc gcagagaaaa gaagaatatt 240

acgaacaata gtgcagatat tgctgttatt gatgctaagg ctgcaaatat ttatccaggt 300

gctttattgc gtgctgacca aaatcttctg gataataatc caacgcttat cagtattgcg 360

cggggagatc tgacgcttag tttgaattta cctggtttgg ccaatgggga tagccacact 420

gttgtaaatt ctccaacaag aagtactgtt cgaacagggg tgaataacct tctgtctaaa 480

tggaataata cgtatgctgg agagtatggc aatacccaag cagagcttca atatgatgaa 540

acaatggcat acagtatgtc acaattgaaa acgaagttcg gaacctcttt tgaaaaaatt 600

gctgtaccat tagatatcaa ttttgatgcc gtgaattcgg gtgaaaaaca ggttcagatt 660

gttaacttta aacaaattta ttatacagtt agtgttgatg aaccagaatc tccaagcaag 720

ctttttgcag aagggacaac tgtagaagat ttgaaacgaa atgggataac agatgaggta 780

cctcctgttt atgtttccag cgtttcttat ggacgctcta tgttcatcaa gttagaaact 840

agcagtagga gtacccaagt tcaagccgca tttaaagcag ccatcaaagg cgttgatatt 900

agtggcaatg ctgagtatca agacattctg aaaaatactt cattctctgc ttatattttt 960

ggtggggatg caggtagcgc ggctactgtt gtgagcggaa atattgaaac actgaagaag 1020

attattgaag aaggtgcaag atacggaaaa ctcaatccag gtgttccgat ttcgtattca 1080

accaactttg tcaaagacaa tagacctgct cagattttga gcaattcaga gtacatagaa 1140

acaacttcaa cagtccataa tagcagtgca ttgacattgg atcattcagg tgcttatgtt 1200

gcgaaataca acattacttg ggaagaagta tcttacaatg aagctggaga agaagtttgg 1260

gaaccaaaag cttgggataa gaatggtgta aatctgacct cacactggag tgaaaccatt 1320

caaattccag gaaatgctcg caatcttcat gtcaatattc aagaatgtac aggattagca 1380

tgggagtggt ggagaacagt ttatgacaaa gatttaccac ttgttggtca acgtaaaata 1440

accatctggg gaacaacgtt atacccacag tatgcggatg aggtgataga gtaa 1494

<210> 3

<211> 870

<212> DNA

<213> ef

<400> 3

gatggagagt caggttctct tgtatcatca gacttaggta caaacactaa gattacttca 60

gaactggatc ctacgggagc aactgcaaac caaggagatg acggtcaatc ttcaactaag 120

tttaacgtta agattacagg taccggacct gctacagaag gtaccggcac ttataagctt 180

cgtgttggag aagataacta tccttttggt ccagagggga aacttgttga tggaaataaa 240

ccagaaaatg taggtttgac atctgtaaaa gttaccttcg taaaacatgc tacggtgtca 300

acaccagttt ctgttgaaaa tccagctaac ttaacgccag aagaaaaagc cgcagttatt 360

gctcaaatca agaaagacaa cgcagacaac gaaagattga agggcttgcc agattcagca 420

tttacagtta actcagatgg tactgtgtca gttgactaca gtgccggtgg tgtcaatgtt 480

gatggtgcga cagacattat taagaatgct gccacaaact tggcagatac acggaatcaa 540

gcaaaagcag aaatcgacac aaaattagct gaacataaaa aagctatcga agcaaaacgg 600

gatgaagcgt tttctaaaat tgatgatgac atttccttga gagcagaaca gagacaggct 660

gctaaggatg ccgttgctgc agctgctggg gatgctttga aagaattaga caacaaggcg 720

acagaagcaa aagaaaaaat tgataaagct acgacggcct cagaaatcaa tgatgctaag 780

actaatggtg agattaatct ggacagtgca gaagcagtag gcgaaaaagc tattaaccag 840

tcgaagcgca atcggcagag gacaaaggcg 870

<210> 4

<211> 834

<212> DNA

<213> cdtb

<400> 4

atgacgttat ttaatcgagc tatagcatta gtatgttatt tgcttcctca aattgtcttg 60

gcaaatttgg aaaactatac ggttgcaacg tggaacttgc aaggttcttc tgcgattaat 120

gaaagtaaat ggaacatcaa tgttcgtcag cttttgacgg ggcctcaagc ggcaggtatt 180

ttaatggttc aagaagcagg ctctttgcca tcaacagccg ttcatacacg ccgaatggtt 240

cagcctgagg gggttggctt tccgattgat gaatatgtgt ggaacttagg gacaagaagt 300

cgtccaaata atgtttatat ctattactct cgtcttgatg tgggggcaaa tcgagttaat 360

cttgcgatta ttgctcgtcg tatggcaagc gaagtgttcg tgattaattc cagttccagt 420

gtattaactt ctcgtccagc cataggtatt cgtattgatg acgatgcttt ctttagtatt 480

catgctcttt catctggagg agcagattca ctaagtttga ttcaaaatat tcatacgttc 540

tttaatactg agggaagacg acatattaat tggatggcgg taggggattt caatcgcgct 600

cctggtcgta tgcaagaggc tctagattct gaacctggac tgcgtaatgc tacactgatt 660

gttgcaccaa cggaacctac ccatcgttct ggtggagtat tggattatgc agtgttacac 720

aatgcgaatc aaaccacaca aaacacgacg gtcagtgcca gtattatgtt taatcagatg 780

agatcgcaga ttacctccga tcatttccca gtcagctttg taaaaaaacg ttaa 834

<210> 5

<211> 1041

<212> DNA

<213> afua

<400> 5

atgaaaaaat tgcaattaaa aaaatggttt tcaagccttt ttctcctttc accattagtc 60

tttgcagctt ctaatgctca ggctgaagga aagctgaccg tttattgtgg cgtgcaaaac 120

aaggtgtgtg aagatctgac gaagcgtttt tcacaaaaat ataatgtgga aactcagttt 180

attcacggtg gaacagggac aattttaggt aagctgaaag cggaaaaaga taatccgcaa 240

gcggatattt ggtatggcgg cactattgag ccacatttcc aagcagggca gatggggcta 300

ttagaagcct atcgttcacc tttacaagcg gaggttttac ctcaatttaa agcattgcaa 360

gagagcgaag caggtaaata tacctcgatt gcgtatttga tggtgcttgg ttttggtatt 420

aacaccgaaa aattaaagca gttagggatt gatgctccga aaaaatgggc agatttgctt 480

gatcctcgct taaaaggcga agtgcaatta gctgatcctc gcacttcagg aacaatgtac 540

acgacgatga ttacgcttat tcagttaatg ggcgaggaaa aagcctttga gtacttgaaa 600

aaattagatg gcaacatttc tcaatatgtg aaaagtacct tagtgacctc taacctttct 660

cgtggagaga gtgctgtgac agttggcttt gctcacggct atgcttcaga aaaagagaaa 720

ggtgcaccgg ttgattatgt gttacctaaa gatggggtag gttatgcttt aggggctgca 780

agtattatta aaggtgcgag aaaccttgat aatgcgaaat tgtttatgga ttgggttctg 840

tctaaagagg ttcaagaaat tccttggcgt gaccacggac tttatcaaac accaaccaat 900

gtgaaagcag aagttgcccc acaatcacct aaaatagatg gggtgaaatt ggttgatgtg 960

gattatgccc gttttggttc aagcgaggaa gggaagcgtt tggtagacaa atggctcttt 1020

aatatcaaat tatcgaattg a 1041

<210> 6

<211> 1638

<212> DNA

<213> oppa

<400> 6

atgcaaacaa cctttacccg ttctttatta gccagtgcga ttgcgcttgg tctttctgtt 60

tcggcttttg cagctaaagt gcctgaaggt acggtgcttg cagagaagca agagatcatt 120

attaacaata gctcagaacc atcaagtttt gacccacata aaacagaagg tgtgccagaa 180

gctcaagttt cttatcagtt acttgaaggt ttagtcacca aagattctgc gggcgaaatc 240

attcctggtg tggctgaaac ctggaaaagt tctgatgatt tcaaaacctg gacttttaat 300

ttacgcaaaa atgcaaaatg gtctaatggc gaacctgtta ccgcacacga ttttgagttc 360

tcgcttaaac gtttaggcga tccgaaaacg gcttcacctt atgcaagtta cttaaactat 420

cttcaagttg aaaatgctca agacatcatt gatggtaaaa aagcaccgag tgagttagga 480

gttaaagccg ttgatgatta cactttagaa atcaaattaa gcaatcctgt gccttactta 540

gtcggtatga tgacgcacca aacgatgttg cctgtgccaa aagcggtcgt tgagaaatta 600

ggtgatgcgt gggtgaaaaa agagaactat gtaggtaacg gtgcttataa attagttgag 660

catgtgatta atgaaaaaat cgtgtttgaa cgtaacccat tgtattggaa tgataaagaa 720

accgtgatta ataaagcgac attcttagct attcaaaatg caagtacaga cgttcaacgt 780

tatcgtgcgg gtgacttaca tatcaccagc tacggattgc ctccagagca gttcccgaca 840

ttgaaaaaag aaattccaaa tgaagtgttt gtaacccgta cgctttcaac ttactactac 900

gagccaaaca acgaaaaagc accatttaac gatgtgcgtg tgcgtaaggc gttaaacttg 960

gcattagatc gtagtgtgat tacggataaa gtattagggc aagggcaaac gccaacctat 1020

gtgtttacac cgccgtacat tacagaaggt cacttaattc aacaacctga gtactcaaaa 1080

caggatatgg catctcgtaa agcagaggcg attaagttgt tagaagaagc tggctttagc 1140

aaagcgaacc ctcttaaatt tacgctttta tataacacta acgagaacca taagaaaatt 1200

gcgattgcag cacaatcgat cttaaaacaa aacacgggcg gtttagtgga tattaaactt 1260

gaaaaccaag agtggaaaac tttcttagat acacgccgtg cgggtaacta tgatgttgca 1320

cgtgcaggtt gggcggctga ctataaccaa gcgtcgactt tcggtaaata cttcttgtct 1380

aactcaagta acaataccgc tcgttataag agtgcggctt atgatgctga aatcaatgcg 1440

gcatataaag cgggtaacgc agaagaacgt gcggcagctt atgcaaaagc ggaagctcag 1500

ttagcgaaag attatgcgat cattcctatc tataactatg taaacccacg tttggttaag 1560

ccattcgtga aaggttatga gggtaaagat ccgcaggatg atattttatt aagaaacctt 1620

tatatcatta agcagtaa 1638

<210> 7

<211> 1542

<212> DNA

<213> oppa2

<400> 7

atgaaattag ttgcaggtgt tggtgctggt ttagcattct caggttcaat cggtactttt 60

gcttctcagg cttatgctgc accagcaaaa ggttcaacta tcgaaacagg tattgcttat 120

ccgatttcaa cgggttttga cccaatgagt tcaactggtg catcttcaat ggcggctaat 180

atccacattt ttgaaggttt agttgattta cacccagcaa ctcgccagcc atatctagca 240

cttgcagcga aagaaccaga aaaagttgac gatgtaactt atcgcatcac tttacgtgac 300

ggtgcggtat tccataacgg ttcagcagta accagtgctg atgttgtgtt ttcatttgag 360

cgtgtattag atccaaatac aaaatcactc tttgcacaat tcatcccatt cattaaatca 420

gtcactgcag tggatcaaaa aacagttgaa tttaaattga aatatccatt tgcattattc 480

aaagaacgtt taaccattat caaaatagta ccaaaagcat taattgaagc tcaaggtcaa 540

tcagtctttg atgcaaaccc tgctggtact ggtccatata aatttgtttc agcagtcaaa 600

gatgaccgta tcgtatttga agcaaaccct gcttacacag gtccatatcc tgcaactgtt 660

gaaaaaatga catggttctt actctttgat gacgcagcac gtgttgcagc acaagagtca 720

ggtcgtgtac aagcgattga aaacgtacct tacttagatg cggatcgctt aaaacgtaaa 780

gcagctgtag aatcagtgca atcattcggc ttaattttct taatgtttaa ctgtgaaaaa 840

gcaccgttta acaacaagaa agtacgtcaa gcattacaat atgcaattga tacacaaaaa 900

ttagttgatg tggtgttctt aggcaacgca aaacctgcga catcttatgt tcaagactct 960

cacccagact atgtgaaagc ctcaacagtc tatgatttcg atccgaaaaa agcggctgca 1020

ttgttgaaag aagcgggtgt agataaactt gagtttacaa cacgttcaac cgcacataaa 1080

tgggtagtgg actctgttca aatgatcctt gaagactgga acaagatccc tggtgtaaaa 1140

gtgacaaaca tcgcttcaca atcaccatac aatgacggtg ttgatgcagg taactttgaa 1200

gtattaatcg caccaggtga cccatcagta ttcggtaacg acttagactt attattaagc 1260

tggtggtacc gtggtgatgt atggccgaaa aaacgtttcc gctggtcaaa tacacctgaa 1320

tatgcggaag ttcaaaaact tcttgacgct gcggtggctg cgaaaactcc ggcagaagca 1380

cgtgaaattt ggggtaaagc gattaacatc attgctgaag aagcagcact ttatccaatt 1440

attcaccgta aacttccaac agcttggagt aacaaagcat tagatggctt taaaccatta 1500

tcaaccacag gtatgtcatt cattggtgta agccgtaaat aa 1542

<210> 8

<211> 3069

<212> DNA

<213> apxia

<400> 8

atggctaact ctcagctcga tagagtcaaa ggattgattg attcacttaa tcaacataca 60

aaaagtgcag ctaaatcagg tgccggcgca ttaaaaaatg gtttgggaca ggtgaagcaa 120

gcagggcaga aattaatttt atatattccg aaagattatc aagctagtac cggctcaagt 180

cttaatgatt tagtgaaagc ggcggaggct ttagggatcg aagtacatcg ctcggaaaaa 240

aacggtaccg cactagcgaa agaattattc ggtacaacgg aaaaactatt aggtttctcg 300

gaacgaggca tcgcattatt tgcacctcag tttgataagt tactgaataa gaaccaaaaa 360

ttaagtaaat cgctcggcgg ttcatcggaa gcattaggac aacgtttaaa taaaacgcaa 420

acggcacttt cagccttaca aagtttctta ggtacggcta ttgcgggtat ggatcttgat 480

agcctgcttc gtcgccgtag aaacggtgag gacgtcagtg gttcggaatt agctaaagcg 540

ggtgtggatc tagccgctca gttagtggat aacattgcaa gtgcaacggg tacggtggat 600

gcgtttgccg aacaattagg taaattgggc aatgccttat ctaacactcg cttaagcggt 660

ttagcaagta agttaaataa ccttccagat ttaagccttg caggacctgg gtttgatgcc 720

gtatcaggta tcttatctgt tgtttcggct tcattcattt taagtaataa agatgccgat 780

gcaggtacaa aagcggcggc aggtattgaa atctcaacta aaatcttagg caatatcggt 840

aaagcggttt ctcaatatat tattgcgcaa cgtgtggcgg caggcttatc cacaactgcg 900

gcaaccggtg gtttaatcgg ttcggtcgta gcattagcga ttagcccgct ttcgttctta 960

aatgttgcgg ataagtttga acgtgcgaaa cagcttgaac aatattcgga gcgctttaaa 1020

aagttcggtt atgaaggtga tagtttatta gcttcattct accgtgaaac cggtgcgatt 1080

gaagcggcat taaccacgat taacagtgtg ttaagtgcgg cttccgcagg tgttggggct 1140

gctgcaaccg gctcattagt cggtgcgccg gtagcagctt tagttagtgc aatcaccggt 1200

attatttcag gtattttaga tgcttctaaa caggcaatct tcgaacgagt tgcaacgaaa 1260

ttagcgaata agattgacga atgggagaaa aaacacggta aaaactattt tgaaaacggt 1320

tatgacgccc gccattccgc attcttagaa gatacctttg aattgttatc acaatacaat 1380

aaagagtatt cggtagagcg tgtcgttgct attacgcaac agcgttggga tgtcaatatc 1440

ggtgaacttg ccggcattac tcgcaaaggt tctgatacga aaagcggtaa agcttacgtt 1500

gatttctttg aagaaggaaa acttttagag aaagaaccgg atcgttttga taaaaaagtg 1560

tttgatccgc ttgaaggtaa aatcgacctt tcttcaatta acaaaaccac tttattgaaa 1620

tttgttacgc cggtctttac cgcaggtgaa gagattcgtg agcgtaagca aaccggtaaa 1680

tacgaatata tgaccgaatt attcgttaaa ggtaaagaaa aatgggtggt aaccggtgtg 1740

cagtcacata atgcgattta tgactatacg aatcttatcc aattagcgat agataaaaaa 1800

ggtgaaaaac gtcaagtgac cattgaatct catttgggtg agaaaaatga tcgtatatat 1860

ctttcatccg gttcatctat cgtatatgcg ggtaacggac atgatgtagc atattacgat 1920

aaaaccgata caggttactt aacatttgac ggacaaagtg cacagaaagc cggtgaatat 1980

attgtcacta aagaacttaa agctgatgta aaagttttaa aagaagtggt taaaactcag 2040

gatatttcag ttggaaaacg cagtgaaaaa ttagaatatc gtgattatga gttaagccca 2100

ttcgaacttg ggaacggtat cagagctaaa gatgaattac attctgttga agaaattatc 2160

ggtagtaatc gtaaagacaa attctttggt agtcgcttta ccgatatttt ccatggtgcg 2220

aaaggcgatg atgaaatcta cggtaatgac ggccacgata tcttatacgg agacgacggt 2280

aatgatgtaa tccatggcgg tgacggtaac gaccatcttg ttggtggtaa cggaaacgac 2340

cgattaatcg gcggaaaagg taataatttc cttaatggcg gtgatggtga cgatgagttg 2400

caggtctttg agggtcaata caacgtatta ttaggtggtg cgggtaatga cattctgtat 2460

ggcagcgatg gtactaactt atttgacggt ggtgtaggca atgacaaaat ctacggtggt 2520

ttaggtaagg atatttatcg ctacagtaag gagtacggtc gtcatatcat tattgagaaa 2580

ggcggtgatg atgatacgtt attgttatcg gatcttagtt ttaaagatgt aggatttatc 2640

agaatcggtg atgatcttct tgtgaataaa agaatcggag gaacactgta ttaccatgaa 2700

gattacaatg ggaatgcgct cacgattaaa gattggttca aggaaggtaa agaaggacaa 2760

aataataaaa ttgaaaaaat cgttgataaa gatggagctt atgttttaag ccaatatctg 2820

actgaactga cagctcctgg aagaggtatc aattacttta atgggttaga agaaaaattg 2880

tattatggag aaggatataa tgcacttcct caactcagaa aagatattga acaaatcatt 2940

tcatctacgg gtgcatttac cggtgatcac ggaaaagtat ctgtaggctc aggcggaccg 3000

ttagtctata ataactcagc taacaatgta gcaaattctt tgagttattc tttagcacaa 3060

gcagcttaa 3069

<210> 9

<211> 2871

<212> DNA

<213> apxiia

<400> 9

atgtcaaaaa tcactttgtc atcattaaaa tcgtccttac aacaaggatt gaaaaatggg 60

aaaaacaagt taaatcaagc aggtacaaca ctgaagaatg gtttaactca aactggtcat 120

tctctacaga atggggctaa aaaattaatc ttatatattc ctcaaggcta tgattcgggt 180

caaggaaatg gagttcaaga tttagttaaa gctgctaatg atttaggtat tgaagtatgg 240

cgagaagaac gcagcaattt ggacattgca aaaactagct ttgatacaac tcagaaaatt 300

ctaggtttta ctgatagagg aattgtatta tttgcacctc agctagataa tttattaaag 360

aagaatccta aaattggcaa tacattagga agtgcttcta gcatctcaca aaatataggt 420

aaagccaata ctgtattagg tggtattcaa tctattttag gatctgtttt atctggagta 480

aatctgaatg aattacttca aaataaagat cctaatcaat tagaacttgc aaaagcaggg 540

ctagaactga ctaatgaatt agttggtaat attgctagct cggtgcaaac tgtagatgca 600

tttgcagaac aaatatctaa actaggttca catttacaga atgtgaaagg attaggagga 660

ttgagtaata aattacaaaa tctaccagat ctaggaaaag caagtttagg tttggacatt 720

atctctggtt tactttctgg agcatctgca ggtctcattt tagcagataa agaggcttca 780

acagaaaaga aagctgccgc aggtgtagaa tttgctaacc aaattatagg taatgtaaca 840

aaagcggtct catcttacat tcttgcccaa cgagtcgctt caggtttgtc ttcaactggt 900

cctgtcgctg cattaatcgc atctacagtt gcactagctg ttagccctct ttcattctta 960

aatgtagctg ataagtttaa acaagctgat ttaatcaaat catattctga acgcttccaa 1020

aaattaggat atgatggaga tcgtttatta gctgattttc accgtgagac aggaactatt 1080

gatgcttctg taacaacaat taacactgct ttagcagcta tctccggtgg agttggagct 1140

gcaagcgcgg gttctctagt cggagctcca gttgcgttac tcgttgctgg tgttacggga 1200

cttattacaa ctattctaga atattctaaa caagccatgt ttgaacatgt tgcaaataag 1260

gttcatgaca gaatagttga atgggagaaa aaacataata aaaactattt tgagcaaggt 1320

tatgattctc gtcatttagc tgatttacaa gacaatatga agtttcttat caatttaaat 1380

aaagaacttc aggctgaacg cgtagtagct attacccaac aaagatggga taaccaaatt 1440

ggagacctag cggcaattag ccgtagaacg gataaaattt ccagtggaaa agcttatgtg 1500

gatgcttttg aggaggggca acaccagtcc tacgattcat ccgtacagct agataacaaa 1560

aacggtatta ttaatattag taatacaaat agaaagacac aaagtgtttt attcagaact 1620

ccattactaa ctccaggtga agagaatcgg gaacgtattc aggaaggtaa aaattcttat 1680

attacaaaat tacatataca aagagttgac agttggactg taacagatgg tgatgctagc 1740

tcaagcgtag atttcactaa tgtagtacaa cgaatcgctg tgaaatttga tgatgcaggt 1800

aacattatag aatctaaaga tactaaaatt atcgcaaatt taggtgctgg taacgataat 1860

gtatttgttg ggtcaagtac taccgttatt gatggcgggg acggacatga tcgagttcac 1920

tacagtagag gagaatatgg cgcattagtt attgatgcta cagccgagac agaaaaaggc 1980

tcatattcag taaaacgcta tgtcggagac agtaaagcat tacatgaaac aattgccacc 2040

cacccaacaa atgttggtaa tcgtgaagaa aaaattgaat atcgtcgtga agatgatcgt 2100

tttcatactg gttatactgt gacggactca ctcaaatcag ttgaagagat cattggttca 2160

caatttaatg atattttcaa aggaagccaa tttgatgatg tgttccatgg tggtaatggt 2220

gtagacacta ttgatggtaa cgatggtgac gatcatttat ttggtggcgc aggcgatgat 2280

gttatcgatg gaggaaacgg taacaatttc cttgttggag gaaccggtaa tgatattatc 2340

tcgggaggta aagataatga tatttatgtc cataaaacag gcgatggaaa tgattctatt 2400

acagactctg gcggacaaga taaactggca ttttcggatg taaatcttaa agacctcacc 2460

tttaagaaag tagattcttc tctcgaaatc attaatcaaa aaggagaaaa agttcgtatt 2520

gggaattggt tcttaaaaaa tgatttggct agcacagttg ctaactataa agctacaaat 2580

gaccgaaaaa ttgaggaaat tattggtaaa ggaggagaac gtattacatc aaaacaagtt 2640

gataaactga ttaaggaggg taacaatcaa atctctgcaa aagcattatc caaagttggg 2700

aatgattaca atacgagtaa agatagacag aacgtatcta atagcttagc aaaattgatt 2760

tcttcagtcg aaagctttac gtcttcctca aactttagga ataatttagg agcatatgtt 2820

ccttcatcaa taaatgtctc gaataatatt caattagcta gagccgctta a 2871

<210> 10

<211> 1104

<212> DNA

<213> omp

<400> 10

atgaatattg caacaaaatt aatagccggt ttagtcgcag gtttagtgct taccgcatgt 60

agtggcggcg gctcatcggg ttcatcgcct aaaccaaatt cggaatctac gcctaaggtt 120

gatatgtccg caccaaaagc ggagcagcca aaaaaagagg aagctccgca agcggatagc 180

ccgaaagcag aaaaaccaaa aagtattgct ccactgatga tggaaaaccc aaaagtagag 240

aaacagaaag aaaataacct acaagagaaa agtccaaagg cagacgaacc gcaagtaatg 300

gatccaaaat taggtgctcc acaaaaagat gatcagaagt tagaagaacc taagaataaa 360

agtaatgcgg aaattcttaa ggaattaggg attaaggata ttacttcagg gacaattagt 420

atttccgata ttgaattgaa tctacaatta gatagcaatg ataatgtgaa aatatctttg 480

ttaaatgaga atttaatgcg tgataattta acgattaata ataagattgc aggttcggat 540

attagaacgt taaaagattc ttcaggtaga ttgttaggtt attatggtta tgtgcaattg 600

aatcaagtta cacaagactc tcgtgaccca gataattata agcatcagtt tgaaaatcat 660

tatttactgt ctatgaatga tgctgagaaa atattaccag aaaagtcgtt agaatataaa 720

ggtagtatga tttacggata taatacttct ggaaatgaaa agcttactgc agaagtgaat 780

gctaaatatg atagttcaac taaaaaatta tcaatgaaag tatatgataa tgatcgttat 840

tggaaattag gcgaagtaat gagtaacaat gttagattac cagaagaaaa agttgatggt 900

gtgaaagttg attctgacgg aacaattaat gctcgtttat atttaagcac tgaagaacca 960

ttaaaattaa cccctgacgc caatttctcc ggtggtattt ttgggaaaaa cggtgaagta 1020

ctggcaggaa aagcggaaag cattaaggga gaatggcaag gcgtaatcgg tgctacggca 1080

acaacaaaag aagataaaaa ataa 1104

Claims (10)

1. The porcine streptococcosis-haemophilus parasuis disease-porcine contagious pleuropneumonia triple subunit vaccine is characterized in that the vaccine contains MRP, SLY and EF recombinant proteins of the porcine streptococcosis, CdtB, AfuA, OppA and OppA2 recombinant proteins of the haemophilus parasuis, and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae.

2. The triple subunit vaccine of claim 1 wherein the MRP, SLY and EF recombinant proteins of streptococcus suis, the CdtB recombinant proteins of haemophilus parasuis, and the ApxIIA and ApxIIA recombinant proteins of actinobacillus pleuropneumoniae are truncated expressed proteins encoded by the truncated MRP, SLY and EF genes of streptococcus suis, the CdtB gene of haemophilus parasuis, and the ApxIIA and ApxIIA genes of actinobacillus pleuropneumoniae, respectively, wherein one of the truncated MRP genes has the nucleotide sequence of SEQ ID NO: 1 is shown in the specification; a truncated sly gene having the nucleotide sequence of SEQ ID NO: 2 is shown in the specification; a truncated ef gene having the nucleotide sequence of SEQ ID NO: 3 is shown in the specification; a truncated cdtB gene having the nucleotide sequence of SEQ ID NO: 4 is shown in the specification; a truncated apxiia gene having the nucleotide sequence of SEQ ID NO: 8 is shown in the specification; a truncated apxIIA gene having the nucleotide sequence of SEQ ID NO: 9 is shown in the figure; the AfuA protein of haemophilus parasuis is encoded by the AfuA gene of haemophilus parasuis, and the nucleotide sequence thereof is SEQ ID NO: 5 is shown in the specification; the OppA protein of the haemophilus parasuis is coded by the oppA gene of the haemophilus parasuis, and the nucleotide sequence of the OppA protein is SEQ ID NO: 6 is shown in the specification; the OppA2 protein of Haemophilus parasuis is coded by the OPpA2 gene of Haemophilus parasuis, and the nucleotide sequence of the protein is SEQ ID NO: 7 is shown in the specification; the OMP protein of the actinobacillus pleuropneumoniae is coded by OMP gene of the actinobacillus pleuropneumoniae, and the nucleotide sequence is SEQ ID NO: shown at 10.

3. The triple subunit vaccine of claim 1, wherein the concentration of the MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA2 recombinant proteins of haemophilus parasuis, and ApxIA, apxiiia, OMP recombinant proteins of actinobacillus pleuropneumoniae are 0.05-0.5mg/mL, respectively.

4. The triple subunit vaccine of claim 1, wherein the concentration of the MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA2 recombinant proteins of haemophilus parasuis, and ApxIA, apxiiia, OMP recombinant proteins of actinobacillus pleuropneumoniae are 0.1-0.3mg/mL, respectively.

5. The triple subunit vaccine of claim 1, wherein the concentration of the MRP, SLY and EF recombinant proteins of streptococcus suis, the CdtB, AfuA, OppA2 recombinant proteins of haemophilus parasuis, and the ApxIIA, OMP recombinant proteins of actinobacillus pleuropneumoniae are 0.1mg/mL, respectively.

6. The triple subunit vaccine of claim 1, further comprising an adjuvant, wherein said adjuvant includes, but is not limited to, the following: oil-in-water adjuvants, polymer and water adjuvants, water-in-oil adjuvants, aluminum hydroxide adjuvants, vitamin E adjuvants.

7. The triple subunit vaccine of claim 6 wherein the adjuvant is ISA201 VG.

8. The triple subunit vaccine of claim 1, wherein the triple subunit vaccine is administered intramuscularly, intradermally or subcutaneously, preferably intramuscularly.

9. A method of making the triple subunit vaccine of any one of claims 1 to 8, comprising the steps of:

1) respectively obtaining gene sequences of MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae by a PCR (polymerase chain reaction) method; wherein, MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB recombinant proteins of haemophilus parasuis and ApxIA and ApxIIA recombinant proteins of actinobacillus pleuropneumoniae are expressed after truncation, and are respectively encoded by MRP, SLY and EF genes of streptococcus suis, cdtB genes of haemophilus parasuis and apxIA and apxIIA genes of actinobacillus pleuropneumoniae, wherein, the nucleotide sequence of one truncated MRP gene is SEQ ID NO: 1 is shown in the specification; a truncated sly gene having the nucleotide sequence of SEQ ID NO: 2 is shown in the specification; a truncated ef gene having the nucleotide sequence of SEQ ID NO: 3 is shown in the specification; a truncated cdtB gene having the nucleotide sequence of SEQ ID NO: 4 is shown in the specification; a truncated apxiia gene having the nucleotide sequence of SEQ ID NO: 8 is shown in the specification; a truncated apxIIA gene having the nucleotide sequence of SEQ ID NO: 9 is shown in the figure; the AfuA protein of haemophilus parasuis is encoded by the AfuA gene of haemophilus parasuis, and the nucleotide sequence thereof is SEQ ID NO: 5 is shown in the specification; the OppA protein of the haemophilus parasuis is coded by the oppA gene of the haemophilus parasuis, and the nucleotide sequence of the OppA protein is SEQ ID NO: 6 is shown in the specification; the OppA2 protein of Haemophilus parasuis is coded by the OPpA2 gene of Haemophilus parasuis, and the nucleotide sequence of the protein is SEQ ID NO: 7 is shown in the specification; the OMP protein of the actinobacillus pleuropneumoniae is coded by OMP gene of the actinobacillus pleuropneumoniae, and the nucleotide sequence is SEQ ID NO: 10 is shown in the figure;

2) cloning the truncated mrp gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-mrp; cloning the truncated sly gene to a pcold-sumo vector to construct a recombinant plasmid pcold-sly; cloning the truncated ef gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-ef; cloning the truncated cdtB gene to a pET-22b vector to construct a recombinant plasmid pET-22 b-cdtB; cloning the afuA gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-afuA; cloning oppA gene to pET-28a (+) carrier to construct recombinant plasmid pET-28 a-oppA; cloning oppA2 gene to pET-28a (+) vector to construct recombinant plasmid pET-28a-oppA 2; cloning the truncated apxIA gene to a pcold-sumo vector to construct a recombinant plasmid pcold-apxIA; cloning the truncated apxIIA gene to a pcold-sumo vector to construct a recombinant plasmid pcold-apxIIA; cloning the omp gene to a pET-28a (+) vector to construct a recombinant plasmid pET-28 a-omp;

3) respectively transforming the recombinant plasmids obtained in the step 2) into escherichia coli to obtain recombinant escherichia coli which respectively express MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae;

4) inducing and expressing the recombinant escherichia coli obtained in the step 3), centrifuging after carrying out ultrasonic treatment on the culture, purifying the obtained supernatant by using an affinity chromatography column to respectively obtain purified MRP, SLY and EF recombinant proteins of streptococcus suis, CdtB, AfuA, OppA and OppA2 recombinant proteins of haemophilus parasuis and ApxIA, ApxIIA and OMP recombinant proteins of actinobacillus pleuropneumoniae;

5) mixing the purified recombinant proteins MRP, SLY, EF, CdtB, AfuA, OppA2, ApxI, ApxII and OMP, mixing the mixed protein solution with ISA201VG, and emulsifying to obtain the final product.

10. Use of a triple subunit vaccine according to any one of claims 1 to 8 in the manufacture of a medicament for the simultaneous prevention of streptococcosis suis, haemophilus parasuis and contagious pleuropneumonia suis.

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