Porcine circovirus-streptococcus-haemophilus parasuis polyvalent vaccine and application

Abstract

The application belongs to the technical field of vaccines and provides a porcine circovirus-streptococcus-haemophilus parasuis multi-vaccine and application, the multi-vaccine comprising PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, inactivated SS2 bacteria liquid and inactivated HPS4 bacteria liquid.The application takes PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein and inactivated HPS4 and inactivated SS2 as antigens, produces according to the production process of a subunit vaccine, further adds an immune adjuvant and a vaccine excipient and the like, has good immunogenicity, the serum antibody produced has the characteristics of high titer, can be used for simultaneously preventing and treating PCV2, PCV3, PCV4, SS2 and HPS4 infections, and can achieve a better immune protection effect.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, and in particular relates to a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine and its application. Background Technology

[0002] Porcine circovirus (PCV) belongs to the genus Circovirus in the family Circoviridae. It is a non-enveloped, single-stranded circular DNA virus with a genome length of approximately 1.7-2.0 kb, making it one of the smallest known animal viruses. The virus particle has an icosahedral symmetry structure and a diameter of approximately 17-25 nm. Five genotypes, PCV1 to PCV5, have been identified. PCV2 is the key pathogen causing porcine circovirus-associated disease (PCVAD), while PCV3 and PCV4, as newly emerging members, have been continuously detected in pig herds in many countries around the world in recent years.

[0003] PCV2 is one of the most economically impactful viruses in the current swine industry. Infection can cause multisystemic wasting syndrome in weaned piglets, porcine dermatitis-nephropathy syndrome, and respiratory disease syndrome, clinically manifesting as growth retardation, emaciation, dyspnea, diarrhea, and lymphadenopathy. In terms of pathogenic mechanism, PCV2 does not rely solely on direct cell damage but works by interfering with the host's immune system. It can affect innate immune signaling, antigen presentation, and lymphoid tissue structure, leading to immunosuppression or imbalance, thereby promoting persistent viral replication and increasing the risk of secondary or co-infection. PCV3 is associated with reproductive disorders, myocarditis, and multisystemic inflammation, but is also commonly seen in subclinical infections without obvious symptoms. PCV4 can be detected in cases with respiratory and digestive symptoms; its pathogenicity is relatively mild, but its tissue distribution is stable. Epidemiological surveillance shows that the above three PCVs often co-infect in pig herds, frequently coexisting with bacterial pathogens, significantly increasing the incidence and severity of disease outbreaks.

[0004] Streptococcus suis (SS) and Haemophilus parasuis (HPS) are common bacterial pathogens in swine production. Streptococcus suis serotype 2 (SS2) is the most representative highly pathogenic serotype. It can be carried long-term as a resident bacterium in the upper respiratory tract and transforms into an invasive pathogen under stress, immunosuppression, or co-infection conditions, causing meningitis, septicemia, arthritis, and multi-organ damage, especially affecting the production performance of weaned piglets. SS2 is also an important zoonotic pathogen with clear public health significance. HPS is one of the main pathogens of Glasger's disease, characterized by polyfibrinous serositis as the core lesion. It often coexists with immunosuppressive pathogens or respiratory co-infections, increasing clinical complexity. Surveillance data from multiple regions show that HPS4 and HPS5 are both dominant serotypes, consistently detected in clinical cases.

[0005] In large-scale pig farming systems, mixed infections of PCV2, PCV3, and PCV4 with SS2 and HPS4 have become a common cause of respiratory disease syndromes. Immunosuppression induced by viral pathogens (especially PCV) creates conditions for bacterial colonization and invasion, while bacterial infection further amplifies the inflammatory response and exacerbates tissue damage. Studies have shown that PCV2 can promote SS2 transepithelial transport by weakening airway tight junctions and increasing barrier permeability; PCV2 infection can also upregulate fibronectin expression, enhancing HPS4 adhesion and colonization. Epidemiological surveys have also confirmed a high detection rate of triple co-infection of PCV2, SS2, and HPS4 in clinically healthy pig herds, suggesting that this combination not only exists in diseased groups but may also persist long-term in a latent carrier state, becoming the basis for disease amplification during periods of stress or immune fluctuations.

[0006] However, a combined immunization system for PCVs-SS2-HPS4 co-infection remains lacking. Existing vaccines mostly target single pathogens, failing to effectively address the complex infection ecosystem of multiple co-infections. Simply adding monovalent vaccines not only increases the number of immunizations and operational costs but also carries the risk of immune interference. Furthermore, the structural differences and immune coverage boundaries between PCV3 and PCV4-related antigens are not yet fully elucidated, making immunization strategies for multiple subtype co-infections even more challenging. Therefore, developing a combined immunization strategy for PCVs-SS2-HPS4 co-infection has significant theoretical and practical value. Summary of the Invention

[0007] The purpose of this invention is to provide a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine and its application, aiming to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On one hand, the present invention provides a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine, the multivalent vaccine comprising: PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, inactivated SS2 bacterial solution and inactivated HPS4 bacterial solution.

[0010] Furthermore, the PCV2 Cap protein, PCV3 Cap protein, and PCV4 Cap protein are mixed in equal proportions, and the content in the multivalent vaccine is 70 μg / 4mL, and the dosage is 70 μg / 4mL / head.

[0011] In the multivalent vaccine, the concentration of inactivated SS2 bacterial suspension is 1×10⁻⁶. 10 CFU / 4mL, dosage is 1×10 10 CFU / 4mL / head, the concentration of inactivated HPS4 bacterial solution is 6×10⁻⁶. 9 CFU / 4mL, dosage is 6×10 9 CFU / 4mL / head.

[0012] Furthermore, the multivalent vaccine also includes aluminum adjuvant and CpG.

[0013] Furthermore, in the multivalent vaccine, the aluminum adjuvant content is 0.8 mg / 4 mL, and the CpG content is 100 μg / 4 mL.

[0014] Furthermore, the method for preparing the inactivated SS2 bacterial solution includes the following steps:

[0015] Resuscitate SS2 on BHI plates, culture the bacteria, pick a single colony and inoculate it into BHI medium, shake and incubate, then add formaldehyde to inactivate it. After complete inactivation, centrifuge the bacterial solution, discard the supernatant, wash several times with sterile PBS, and then resuspend in sterile PBS to obtain the inactivated SS2 bacterial solution.

[0016] Furthermore, the method for preparing the inactivated HPS4 bacterial solution includes the following steps:

[0017] HPS4 was revived on BHI plates and cultured. A single colony was picked and inoculated into BHI medium containing 10% newborn calf serum and 20 μg / mL NAD. The culture was shaken and then inactivated with formaldehyde. After complete inactivation, the bacterial solution was centrifuged, the supernatant was discarded, and the solution was washed several times with sterile PBS. The solution was then resuspended in sterile PBS to obtain the inactivated HPS4 bacterial solution.

[0018] Furthermore, the bacterial culture specifically involves incubation at 37 ℃ for 10 h;

[0019] The specific shaking culture method is: shaking culture at 37 ℃ for 7-9 h;

[0020] The centrifugation process specifically involves centrifuging at 8000 rpm for 15 minutes.

[0021] On the other hand, the present invention provides a method for preparing a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine, comprising the following steps:

[0022] The PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, inactivated SS2 bacterial solution, and inactivated HPS4 bacterial solution were mixed and combined with aluminum adjuvant and CpG to prepare a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine.

[0023] Furthermore, the immunization schedule for the porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine is as follows: 4.0 mL of vaccine is injected intramuscularly into the neck of piglets. Two weeks after the first immunization, a second immunization is performed using the same dose and route.

[0024] On the other hand, the present invention provides the application of a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine in the preparation of a biological agent, the biological agent being used to prevent and treat PCV2, PCV3, PCV4, SS2 and HPS4 infections.

[0025] On the other hand, the present invention provides a method for detecting antibody titer in animals immunized with a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine, comprising the following steps:

[0026] S1. Antigen coating: The purified PCV2 Cap protein, PCV3 Cap protein, and PCV4 Cap protein were added to the coating solution as antigens and diluted to 2 μg / mL. 0.1 mL was added to each well of the ELISA plate and coated overnight at 4 ℃ for more than 12 h.

[0027] S2. Blocking treatment: Wash three times with PBST for 5 min each time, then add 0.1 mL of blocking solution (PBST containing 5% FBS) to each well and block at 37 ℃ for 2 h.

[0028] S3. Sample incubation: Dilute the serum of the experimental group and the serum of the control group with blocking buffer at a ratio of 1:800, and then add 0.1 mL to each well, with three replicates per group; incubate at 37 ℃ for 1.5 h;

[0029] S4. Secondary antibody incubation: Wash three times with PBST for 5 min each time, then add 0.1 mL of HPR-labeled goat anti-pig IgG (H+L) diluted 1:20000 with blocking buffer to each well, and incubate at 37 ℃ for 1 h;

[0030] S5. Color development and termination: Wash three times with PBST for 5 min each time, then add 0.1 mL to each well and react at 37 ℃ in the dark for 10 min. Then add 2M H2SO4 to each well to terminate the reaction.

[0031] S6. Result Detection and Judgment: Detect the OD of each well using a microplate reader. 450 Reading, when the OD of the positive serum well 450 A serum titer is obtained when the P / N ratio is greater than 2.1 compared to the negative serum titer (P / N≥2.1).

[0032] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0033] This invention uses PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, inactivated HPS4, and inactivated SS2 as antigens, and is produced according to the manufacturing process of subunit vaccines. It also adds immune adjuvants and vaccine excipients, etc., which have good immunogenicity and produce serum antibodies with high titers. It can be used to prevent and control and simultaneously prevent and treat PCV2, PCV3, PCV4, SS2 and HPS4 infection, and can achieve good immune protection effect. Attached Figure Description

[0034] Figure 1 The body temperature changes of piglets provided in the embodiments of the present invention;

[0035] Figure 2 The average relative daily weight gain of piglets provided in the embodiments of the present invention;

[0036] Figure 3 Antibody changes in piglets provided in embodiments of the present invention;

[0037] Figure 4 This is a comparison of the average relative daily weight gain of piglets infected with the corresponding pathogens, provided in the embodiments of the present invention. . Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0040] Example 1: Preparation of porcine circovirus-streptococcus-Haemophilus parasuis antigen:

[0041] 1) Prepare recombinant engineered strains Rosetta-PCV2, Rosetta-PCV3, and Rosetta-PCV4. The specific process is described in application number 202111457334.8, entitled "A PCV2, PCV3, and PCV4 Trivalent Subunit Vaccine and Its Preparation Method and Application".

[0042] 2) Isolation and acquisition of pathogenic bacteria: Streptococcus suis type 2 (SS2), Haemophilus parasuis type 4 (HPS4);

[0043] 3) Expression and purification of porcine circovirus Cap protein:

[0044] The recombinant engineered strains were cultured in a large-scale manner until the bacterial turbidity OD reached a certain level. 600 When the concentration of the induced bacterial culture reached 0.8, 1 mM IPTG (isopropyl galactothioglycoside) was added, and the culture was incubated overnight at 16 °C. The induced bacterial culture was centrifuged at 4 °C, 8000 rpm, and 3 min to collect the bacterial cell pellet. The pellet was washed twice with PBS (pH 8.0), resuspended in 30 mL of PBS, and lysed at 37 °C for 30 min with 1 mg / mL lysozyme. The pellet was then centrifuged and the supernatant was discarded. The pellet was resuspended in 30 mL of Binding Buffer (pH 8.0), and PMSF (phenylmethyl sulfonyl fluoride) was added to a final concentration of 1 mM. The bacterial cells were sonicated at 100 W for 3 s on ice, followed by a 3 s pause to lyse for 1 h. The supernatant was collected by centrifugation at 4 °C, 10000 rpm, and 30 min, which was the protein stock solution. After filtration through a 0.45 μm filter, the protein was purified using a pre-packed Ni-NTA purification resin column to obtain the Cap proteins of PCV2, PCV3, and PCV4.

[0045] 4) Inactivation of HPS4 and SS2:

[0046] HPS4 and SS2 were revived on brain heart broth (BHI) agar plates and incubated at 37 ℃ for 10 h. Single colonies of SS2 were picked and inoculated into BHI agar, and single colonies of HPS4 were picked and inoculated into BHI agar (containing 10% newborn calf serum + 20 μg / mL NAD (nicotinamide adenine dinucleotide)) and incubated at 37 ℃ with shaking for 7-9 h.

[0047] Then formaldehyde was added for inactivation. After complete inactivation, the bacterial solution was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the solution was washed twice with sterile PBS (pH 7.4) and resuspended with sterile PBS (pH 7.4) to obtain the inactivated bacterial solution.

[0048] Example 2: Preparation of a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine:

[0049] 1) Vaccine antigen dosage: Each antigen component is prepared according to the head dose, with 70 μg each of PCV2 Cap protein, PCV3 Cap protein, and PCV4 Cap protein, and the concentration of inactivated SS2 bacterial solution is 1×10⁻⁶. 10 The concentration of CFU-inactivated HPS4 bacterial solution was 6 × 10⁻⁶. 9 CFU, mix the antigens according to the above dosage.

[0050] 2) Adjuvant dosage: Aluminum adjuvant (Al) combined with CpG (cytosine-guanine oligodeoxynucleotide adjuvant) was used as an immune-enhancing adjuvant, with the dosage of aluminum adjuvant being 0.8 mg and the dosage of CpG being 100 μg;

[0051] 3) Mix the above components and prepare 4.0 mL of sterile phosphate-buffered saline (PBS) to obtain the porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine.

[0052] Example 3: Safety evaluation of the porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine:

[0053] 1. Animals: 6 Landrace piglets (4-6kg) aged 3-4 weeks;

[0054] Immunogen: The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine prepared in Example 2.

[0055] 2. Experimental procedure:

[0056] Three piglets were injected intramuscularly into their necks with 4.0 mL of vaccine per piglet. These three piglets served as a blank control group, and the piglets were observed for 14 consecutive days.

[0057] Observe the following indicators after vaccination: continuous temperature measurement for 7 days, weight change before and after vaccination, and observation of local and systemic adverse reactions after vaccination.

[0058] 3. Results:

[0059] 1) Body temperature monitoring results:

[0060] According to the vaccine safety testing requirements of the Chinese Veterinary Pharmacopoeia, piglets vaccinated with the above-mentioned vaccine were tested, and their body temperature was measured at different time points after vaccination. The results are as follows: Figure 1 As shown, there was no significant difference between the immunized group and the non-immunized control group (p>0.05).

[0061] 2) Weight monitoring results: The experimental piglets vaccinated with the above vaccines were weighed before immunization and 14 days after immunization, respectively, for both the immunized group and the non-immunized control group. The results are as follows: Figure 2 As shown, there was no significant difference in average daily weight gain between the immunized group and the control group (p>0.05), indicating that the vaccine had no significant effect on the weight gain of piglets.

[0062] 3) Clinical observation results after vaccination:

[0063] All the piglets in the above-mentioned vaccine trial group survived healthily during the 14-day observation period, showing good mental condition, no abnormalities in feeding and excretion, no obvious redness or induration at the injection site, and no visible granuloma formation.

[0064] The above monitoring results show that the porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine prepared in this embodiment of the invention did not cause any local or systemic adverse reactions after vaccination of piglets, and there were no significant differences in body temperature and average daily weight gain between the immunized group and the control group. The safety of the vaccine meets the requirements.

[0065] Experiment Example 4: Efficacy Evaluation of Porcine Circovirus-Streptococcus-Haemophilus parasuis Multivalent Vaccine

[0066] 1. Animals: Landrace piglets (4-6kg) aged 3-4 weeks;

[0067] Immunogen: The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine prepared in Example 2.

[0068] 2. Vaccination schedule:

[0069] The experimental group of piglets was injected with the vaccine via intramuscular injection in the neck, 4.0 mL / head. Two weeks later, the same dose and route were used for a second immunization. Meanwhile, the unimmunized piglets were used as the control group (challenge control group). All piglets in each group were isolated and raised under the same conditions.

[0070] 3. Antibody collection:

[0071] Whole blood and serum samples were collected from piglets in the experimental and control groups via the anterior vena cava at 0, 14, 21, and 28 days after immunization. Whole blood was stored at 4 ℃ and serum at -80 ℃.

[0072] 4. Antibody detection: PCVs antibodies in the serum of piglets in the experimental and control groups at different time points were detected by ELISA. The specific procedure is as follows:

[0073] ① Antigen coating: The PCVs Cap protein or HPS4 and SS2 whole bacterial protein purified in Example 1 were added to the coating solution and diluted to 2 μg / mL. 0.1 mL was used to coat each well of the ELISA plate and the plate was incubated overnight at 4 ℃ for more than 12 h.

[0074] ② Blocking treatment: Wash three times with PBST (phosphate buffer containing 0.1% Tween-20) for 5 min each time, then add 0.1 mL of blocking solution (PBST containing 5% fetal bovine serum) to each well and block at 37 ℃ for 2 h;

[0075] ③ Sample incubation: Dilute the serum of the experimental group and the serum of the control group with blocking buffer at a ratio of 1:800, and then add 0.1 mL to each well, with three replicates per group; incubate at 37 ℃ for 1.5 h;

[0076] ④ Secondary antibody incubation: Wash three times with PBST for 5 min each time, then add 0.1 mL of HPR (horseradish peroxidase) labeled goat anti-pig IgG (H+L) diluted 1:20000 with blocking buffer to each well, and incubate at 37 ℃ for 1 h;

[0077] ⑤ Color development and termination: Wash three times with PBST for 5 min each time, then add 0.1 mL to each well and react at 37 ℃ in the dark for 10 min. Then add 2M H2SO4 to each well to terminate the reaction.

[0078] ⑥ Result detection and interpretation: Detect the OD of each well using a microplate reader. 450 The serum titer is determined by reading the value of the positive serum well, which is greater than 2.1 compared to the value of the negative serum well.

[0079] ⑦ Results of humoral immune response testing:

[0080] like Figure 3 As shown, 14 days after the first immunization, some pigs in the immunized group had already begun to show positive ELISA antibodies against PCV3, PCV4, and HPS4 (P / N≥2.1); while 14 days after the second immunization, all pigs in the immunized group were positive for antibodies against all five antigens, while the control group remained negative. In addition, the immunized pigs had developed high antibody levels 14 days after the second immunization, with antibody titers for all antigens exceeding 1:51200.

[0081] 5. Piglet challenge protection experiment, the specific process is as follows:

[0082] ① Grouping: 13 days after the second vaccination, 3 pigs were randomly selected from the vaccine-immunized group and the challenge control group and transferred to separate animal pens for rearing. 14 days after the second vaccination, they were weighed and their weight was recorded.

[0083] ② Pathogen inoculation: 14 days after the second immunization, the immunized group and the challenge control group were inoculated with PCV2 virus solution (containing 10... 5 TCID 50 / mL), PCV3 virus solution (containing 10 5.5 TCID 50 / mL) and PCV4 virus solution (containing 10 5.5 TCID 50 / mL), 6.0 mL / head (2.0 mL nasal drops, 4.0 mL intramuscular injection); simultaneously, inject 1 mL of HPS4 (containing 6×10) intraperitoneally. 9 CFU) and SS2 1 mL (containing 8×10 9 CFU);

[0084] ③ Weight monitoring: In the virus challenge group, 3 animals from each of the vaccine immunization group and the challenge control group were weighed and their weights were recorded on day 28; in the bacterial infection group, 3 animals from each of the vaccine immunization group and the infection control group were weighed and their weights were recorded on day 14 after infection.

[0085] ④ Evaluation results of vaccine protective efficacy:

[0086] like Figure 4 As shown, in terms of growth indicators, the average relative daily weight gain of the immunized piglets was significantly higher than that of the non-immunized control group (P<0.05). The weight gain of the non-immunized control group was significantly suppressed after infection with the corresponding pathogen, while the immunized group maintained good growth and development after infection with the corresponding pathogen.

[0087] The above results confirm that immunizing animals with a mixture of the three recombinant proteins PCV2 Cap, PCV3 Cap, and PCV4 Cap provided in this embodiment of the invention, as well as inactivated SS2 and HPS4, exhibits good immunogenicity. The resulting serum antibodies have high titers and protective effects, and can be used as a multivalent vaccine to simultaneously prevent and treat PCV2, PCV3, PCV4, SS2, and HPS4 infections, achieving good immune protection for piglets.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine, characterized in that, The multivalent vaccine includes: PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, inactivated SS2 bacterial solution, and inactivated HPS4 bacterial solution.

2. The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine according to claim 1, characterized in that, The PCV2 Cap protein, PCV3 Cap protein, and PCV4 Cap protein are mixed in equal proportions, and the content of each protein in the multivalent vaccine is 70 μg / 4 mL, with a dosage of 70 μg / 4 mL / head. In the multivalent vaccine, the concentration of inactivated SS2 bacterial suspension is 1×10⁻⁶. 10 CFU / 4mL, dosage is 1×10 10 CFU / 4mL / head, the concentration of inactivated HPS4 bacterial solution is 6×10⁻⁶. 9 CFU / 4mL, dosage is 6×10 9 CFU / 4mL / head.

3. The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine according to claim 1, characterized in that, The multivalent vaccine also includes aluminum adjuvant and CpG.

4. The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine according to claim 3, characterized in that, In the multivalent vaccine, the aluminum adjuvant content is 0.8 mg / 4 mL, and the CpG content is 100 μg / 4 mL.

5. The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine according to claim 1, characterized in that, The method for preparing the inactivated SS2 bacterial solution includes the following steps: Resuscitate SS2 on BHI plates, culture the bacteria, pick a single colony and inoculate it into BHI medium, shake and incubate, then add formaldehyde to inactivate it. After complete inactivation, centrifuge the bacterial solution, discard the supernatant, wash several times with sterile PBS, and then resuspend in sterile PBS to obtain the inactivated SS2 bacterial solution.

6. The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine according to claim 1, characterized in that, The method for preparing the inactivated HPS4 bacterial culture includes the following steps: HPS4 was revived on BHI plates and cultured. A single colony was picked and inoculated into BHI medium containing 10% newborn calf serum and 20 μg / mL NAD. The culture was shaken and then inactivated with formaldehyde. After complete inactivation, the bacterial solution was centrifuged, the supernatant was discarded, and the solution was washed several times with sterile PBS. The solution was then resuspended in sterile PBS to obtain the inactivated HPS4 bacterial solution.

7. The porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine according to claim 5 or 6, characterized in that, The bacterial culture was specifically carried out by incubating at 37 ℃ for 10 h. The specific shaking culture method is: shaking culture at 37 ℃ for 7-9 h; The centrifugation process specifically involves centrifuging at 8000 rpm for 15 minutes.

8. A method for preparing a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine as described in any one of claims 1-7, characterized in that, Includes the following steps: The PCV2 Cap protein, PCV3 Cap protein, PCV4 Cap protein, inactivated SS2 bacterial solution, and inactivated HPS4 bacterial solution were mixed and combined with aluminum adjuvant and CpG to prepare a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine.

9. The use of a porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine as described in any one of claims 1-7 in the preparation of biological agents, characterized in that, The biological agent is used to prevent and treat PCV2, PCV3, PCV4, SS2 and HPS4 infections.

10. A method for detecting antibody titer in animals immunized with the porcine circovirus-streptococcus-Haemophilus parasuis multivalent vaccine as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Antigen coating: The purified PCV2 Cap protein, PCV3 Cap protein, and PCV4 Cap protein were added to the coating solution as antigens and diluted to 2 μg / mL. 0.1 mL was added to each well of the ELISA plate and coated overnight at 4 ℃ for more than 12 h. S2. Blocking treatment: Wash three times with PBST for 5 min each time, then add 0.1 mL of blocking solution to each well and block at 37 ℃ for 2 h. S3. Sample incubation: Dilute the serum of the experimental group and the serum of the control group with blocking buffer at a ratio of 1:800, and then add 0.1 mL to each well, with three replicates per group; incubate at 37 ℃ for 1.5 h; S4. Secondary antibody incubation: Wash three times with PBST for 5 min each time, then add 0.1 mL of HPR-labeled goat anti-pig IgG (H+L) diluted 1:20000 with blocking buffer to each well, and incubate at 37 ℃ for 1 h; S5. Color development and termination: Wash three times with PBST for 5 min each time, then add 0.1 mL to each well and react at 37 ℃ in the dark for 10 min. Then add 2M H2SO4 to each well to terminate the reaction. S6. Result Detection and Judgment: Detect the OD of each well using a microplate reader. 450 Reading, when the OD of the positive serum well 450 A serum titer is obtained when the serum titer is greater than 2.1 times that of a negative serum titer.

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