Porcine round branch bivalent vaccine immunologic adjuvant containing three porcine interleukinins and manganese ions, composition and application

Abstract

The invention relates to the technical field of biology, in particular to a porcine round branch bivalent vaccine immunologic adjuvant containing three porcine interleukinins and manganese ions, a composition and application. The composition comprises porcine interleukin 2, porcine interleukin 4, porcine interleukin 6 and manganese ions and can be used for preparing a porcine round branch bivalent vaccine or an auxiliary preparation of the porcine round branch bivalent vaccine, and the porcine round branch bivalent vaccine immunologic adjuvant containing the three porcine interleukin comprises the compound. The immunologic adjuvant provided by the invention has the characteristics of safety and high efficiency, and can assist a vaccine to motivate animals earlier, faster and more lasting to obtain specific body fluid and cellular immune protection against porcine circovirus and mycoplasma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a porcine circovirus and mycoplasma hyopneumoniae bivalent vaccine immune adjuvant, a composition and an application containing three porcine interleukin and manganese ions. Background Art

[0002] Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are common animal infectious pathogens in large-scale pig farms in China. Infections with PCV2 and Mhp can both cause immunosuppression in the body, thereby secondary or mixed infections with other pathogens, greatly increasing the mortality rate of diseased pigs. More than 80% of pig farms in China are positive for PCV2 infection, with an incidence rate of 25% - 40% and a mortality and culling rate of about 10 - 15%. And more than 99% of pig farms have Mycoplasma hyopneumoniae infection. The average economic loss caused by Mhp infection to each pig in pig farms is more than 80 yuan, seriously affecting the breeding efficiency of pig farms.

[0003] Infections with PCV2 and Mhp have caused huge economic losses to the pig industry. Currently, the main countermeasures are mainly vaccination for prevention. Although the commercially available inactivated vaccine against porcine circovirus and mycoplasma (circovirus and mycoplasma bivalent vaccine) is undoubtedly more advantageous than single vaccines in terms of operational simplicity, the immune effect of the bivalent vaccine is inferior to that of single vaccines.

[0004] Therefore, it is urgent to develop a new animal vaccine immune adjuvant to enhance the immune effect of traditional circovirus and mycoplasma bivalent vaccines, relieve the epidemic prevention pressure of circovirus and mycoplasma infections on China's aquaculture industry, explore new technologies and new clues for regulating immune responses by animal immune molecules, and provide a new scientific basis and technical support for better preventing and controlling animal diseases. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a porcine circovirus and mycoplasma bivalent vaccine immune adjuvant, a composition and an application containing three porcine interleukin and manganese ions.

[0006] The technical solution of the present invention for solving the above technical problem is as follows:

[0007] The present invention provides a composition, including porcine interleukin - 2, porcine interleukin - 4, porcine interleukin - 6 and manganese ions.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Further, in the composition, the mass ratio of porcine interleukin - 2, porcine interleukin - 4, porcine interleukin - 6 and manganese ions is 0.05 - 1:0.05 - 1:0.05 - 1:50 - 200.

[0010] Furthermore, in the composition, the mass ratio of porcine interleukin-2, porcine interleukin-4, porcine interleukin-6 and manganese ions is 0.1 - 0.5:0.1 - 0.5:0.1 - 0.5:100 - 200.

[0011] The present invention also provides an application of the composition as described above. The composition can be used to prepare a porcine circovirus and mycoplasma pneumoniae bivalent vaccine, an immune adjuvant for the porcine circovirus and mycoplasma pneumoniae bivalent vaccine, or a vaccine potentiator for the porcine circovirus and mycoplasma pneumoniae bivalent vaccine.

[0012] Furthermore, the porcine circovirus and mycoplasma pneumoniae bivalent vaccine is a composite inactivated vaccine.

[0013] Furthermore, the auxiliary preparation includes a vaccine adjuvant and a vaccine potentiator.

[0014] The present invention also provides a porcine circovirus and mycoplasma pneumoniae bivalent vaccine immune adjuvant containing three porcine interleukins, including the composition as described above.

[0015] Furthermore, it further includes pharmaceutically or immunologically acceptable excipients.

[0016] The present invention also provides a composite porcine circovirus and mycoplasma pneumoniae bivalent inactivated vaccine, including the porcine circovirus and mycoplasma pneumoniae bivalent vaccine immune adjuvant containing three porcine interleukins as described above and a porcine circovirus and mycoplasma pneumoniae bivalent vaccine antigen. The volume ratio of the porcine circovirus and mycoplasma pneumoniae bivalent vaccine immune adjuvant to the porcine circovirus and mycoplasma pneumoniae bivalent vaccine antigen is 1:0.5 - 1.5.

[0017] Furthermore, each milliliter of the porcine circovirus and mycoplasma pneumoniae bivalent vaccine antigen includes 10 7.0 TCID 50 of porcine circovirus type 2 strain 162 before inactivation and 10 8.0 CCU of Mycoplasma pneumoniae strain HP - G. And the mass of both is 0.1 - 1 μg. It also includes a solvent, and the solvent includes one or more of sterile water, sterile physiological saline, and sterile buffer solution.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) The composition of the present invention can synergistically enhance the immune response level of the porcine circovirus and mycoplasma pneumoniae bivalent inactivated vaccine, and improve and maintain a high level of immune protection and protection period of the porcine circovirus and mycoplasma pneumoniae bivalent inactivated vaccine;

[0020] (2) The water - soluble immune adjuvant for the porcine circovirus and mycoplasma pneumoniae bivalent vaccine containing three porcine interleukins of the present invention is safe and has no toxic side effects. It can be used as a safe and reliable new - type high - efficiency immune adjuvant for the porcine circovirus and mycoplasma pneumoniae bivalent inactivated vaccine, and is suitable for stimulating animals to obtain specific humoral and cellular immune protection against porcine circovirus and mycoplasma pneumoniae earlier, faster, and more persistently;

[0021] (3) The combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can stimulate a stronger immune response, manifested as a significant increase in the number of immune cells in peripheral blood;

[0022] (4) The combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly stimulate and improve animals to produce high levels of specific antibody titers earlier, protecting animals from porcine circovirus infection; and the high-level antibody titers are maintained for a longer period than in the control group;

[0023] (5) The combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly promote animals to produce more blood-specific TcEM, ThEM and ThCM cells, improve the specific cellular immunity and memory ability of animals, and resist the infection and invasion of porcine circovirus and Mycoplasma hyopneumoniae more persistently and at a high level;

[0024] (6) The combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly promote animals to produce more specific porcine circovirus antibodies and Mycoplasma hyopneumoniae antibodies, the number of lymphoid follicle B cells and activated B cells, significantly improve the specific humoral immunity level and memory response ability of animals, and better resist the infection and invasion of porcine circovirus and Mycoplasma hyopneumoniae. Description of the Drawings

[0025] Figure 1 It is a comparison chart of the changes in the body weights of mice in each experimental group of the immune adjuvant of the combined vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention;

[0026] Figure 2 It is a comparison of the relevant parameters of red blood cells in the peripheral blood of mice in each experimental group in Example 1 of the immune adjuvant of the combined vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention, Figure 2 In which a is a comparison chart of the change in the number of red blood cells, Figure 2 In which b is a comparison chart of the change in hemoglobin concentration, Figure 2 In which c is a comparison chart of the change in platelet count, Figure 2 In which d is a comparison chart of the change in mean corpuscular hemoglobin;

[0027] Figure 3 It is a comparison of the relevant parameters of white blood cells in the peripheral blood of mice in each experimental group in Example 1 of the immune adjuvant of the combined vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention, Figure 3 In which a is a comparison chart of the change in the number of white blood cells, Figure 3 In which b is a comparison chart of the change in the percentage of neutrophils, Figure 3 In which c is a comparison chart of the change in the percentage of lymphocytes, Figure 3 In which d is a comparison chart of the change in the percentage of monocytes;

[0028] Figure 4 It is a comparison chart of the percentage changes of various B lymphocyte subsets in the anticoagulated peripheral blood of mice in each experimental group on the 56th day in Example 2 of the immune adjuvant of the combined vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention;

[0029] Figure 5 For the immune adjuvant of the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine of the present invention, in Example 2, the comparison chart of the percentage changes of different T lymphocyte subsets in the anticoagulated peripheral blood of mice in each experimental group on the 56th day;

[0030] Figure 6 For the immune adjuvant of the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine of the present invention, in Example 3, the comparison chart of the detection results of the porcine circovirus antigen-specific antibody levels in each experimental group;

[0031] Figure 7 For the immune adjuvant of the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine of the present invention, in Example 3, the changes in the titer of Mycoplasma-specific antibodies in the plasma of the peripheral blood of mice in each experimental group. Detailed implementation mode

[0032] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0033] The composition of the present invention includes porcine interleukin-2 (IL-2), porcine interleukin-4 (IL-4), porcine interleukin-6 (IL-6), and manganese ions. Interleukin is short for interleukin (IL).

[0034] The composition of the present invention can be used to prepare a porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine or an auxiliary preparation for a porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine. Among them, the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine specifically refers to a vaccine that can prevent porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp).

[0035] The above composition of the present invention can synergistically enhance the immune response level of a porcine circovirus and Mycoplasma hyopneumoniae inactivated vaccine, improve and maintain a high level of immune protection and protection period of a porcine circovirus and Mycoplasma hyopneumoniae inactivated vaccine. At the same time, this composition has no safety toxic and side effects, and can be used as a new and highly efficient immune adjuvant for a porcine circovirus and Mycoplasma hyopneumoniae inactivated vaccine, which is suitable for stimulating animals to obtain specific humoral and cellular immune protection against porcine circovirus and Mycoplasma hyopneumoniae earlier, faster, and more persistently.

[0036] In addition, the above composition of the present invention provides the application of porcine interleukin in the adjuvant of a porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine, provides a new idea for the development and research of the adjuvant of a porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine, and also expands a new field for the application of porcine interleukin.

[0037] Preferably, in the composition of the present invention, the mass ratio of porcine interleukin-2, porcine interleukin-4, porcine interleukin-6, and manganese ions is 0.05-1:0.05-1:0.05-1:50-200.

[0038] Further preferably, in the composition, the mass ratio of porcine interleukin-2, porcine interleukin-4, porcine interleukin-6 and manganese ions is 0.1-0.5:0.1-0.5:0.1-0.5:100-200.

[0039] Further preferably, in the composition, the mass ratio of porcine interleukin-2, porcine interleukin-4, porcine interleukin-6 and manganese ions is 1:1:1:400.

[0040] When in use, calculated according to the average initial immunization body weight of mice at 18 g per mouse: porcine IL-2: 0.028 μg / g (w / w); porcine IL-4: 0.028 μg / g (w / w); porcine IL-6: 0.028 μg / g (w / w); Mn 2+ : 11.11 μg / g (w / w).

[0041] Preferably, the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine is a complex inactivated vaccine.

[0042] Preferably, the adjuvant preparation includes a vaccine adjuvant and a vaccine potentiator.

[0043] The porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine immunoadjuvant containing three porcine interleukins of the present invention includes the composition as described above, and also includes pharmaceutically and immunologically acceptable excipients.

[0044] Specifically, the pharmaceutically and immunologically acceptable excipients include solvents, and the solvents can be one or more of sterile water, sterile physiological saline, and sterile buffer solution.

[0045] The complex porcine circovirus and Mycoplasma hyopneumoniae bivalent inactivated vaccine of the present invention includes the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine immunoadjuvant containing three porcine interleukins as described above and the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine antigen, and the mass ratio of the two is 1:0.5-1.5. Further preferably, the ratio of the two is 1:1.

[0046] Preferably, it further includes the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine antigen and a solvent; each milliliter of the porcine circovirus and Mycoplasma hyopneumoniae bivalent vaccine antigen includes 107.0 TCID50 of inactivated porcine circovirus type 2 strain 162 and 108.0 CCU of Mycoplasma hyopneumoniae strain HP-G, and the mass of both is 0.1-1 μg; the solvent includes one or more of sterile water, sterile physiological saline, and sterile buffer solution.

[0047] Further preferably, in the vaccine, the concentration contents of the components of the adjuvant are as follows: porcine IL-2: 2.5 μg / ml; porcine IL-4: 2.5 μg / ml; porcine IL-6: 2.5 μg / ml; Mn 2+ : 1 mg / ml.

[0048] Preferably, the porcine circovirus and Mycoplasma hyopneumoniae bivalent inactivated vaccine containing the immunoadjuvant is administered by intramuscular or subcutaneous injection.

[0049] Experimental verification shows that the combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly increase the numbers of white blood cells, neutrophils and lymphocytes in the peripheral blood of animals (p < 0.05), indicating that a stronger immune response is stimulated after vaccination, manifested as a significant increase in the number of immune cells in the peripheral blood.

[0050] The combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly stimulate animals to produce high levels of specific antibody titers earlier, protecting animals from porcine circovirus infection; and the high-level antibody titers are maintained for a longer period compared with the control group.

[0051] The combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly promote animals to produce more blood-specific TcEM (cytotoxic T effector memory cell), ThEM (Th effector memory) and ThCM (Th central memory) cells, improving the specific cellular immunity and its memory ability of animals, and resisting the infection and invasion of porcine circovirus and Mycoplasma hyopneumoniae more persistently and at a high level.

[0052] Meanwhile, the combined inactivated vaccine against porcine circovirus and Mycoplasma hyopneumoniae of the present invention can significantly promote animals to produce more specific porcine circovirus antibodies and Mycoplasma hyopneumoniae antibodies, follicular B cells (Follicular B cell, FOB) and activated B cell numbers, significantly improving the specific humoral immunity level and its memory response ability of animals, and better resisting the infection and invasion of porcine circovirus and Mycoplasma hyopneumoniae.

[0053] The present invention will be specifically described below through specific examples.

[0054] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, and the results are averaged. Among them, porcine interleukin-2 (IL-2, 652-P2-020 / CF, R&D Systems), 4 (IL-4, 654-P4-025 / CF, R&D Systems) and 6 (IL-6, 686-PI-025 / CF, R&D Systems) are all purchased from R&D Systems, and manganese adjuvant (Mn, product number MS0001) is purchased from Qimeng Biotechnology (Jiangsu) Co., Ltd.

[0055] The immunization procedures and groupings of the following examples are as follows:

[0056] Thirty 6-week-old female Kunming mice were randomly divided into 3 groups, with 10 mice in each group. They were respectively given intramuscular injections in the legs according to Table 1 and boosted immunization once in the third week.

[0057] Table 1 Immunization grouping for mouse experiments

[0058]

[0059] The body weights of the mice in each group were measured once a week, and anticoagulated blood from the tail vein (200 μL / mouse) was collected. The mice were euthanized on the 56th day after the first immunization, and the number of lymphocyte subsets was detected in the peripheral anticoagulated blood. The inactivated antigen of porcine circovirus type 2 and Mycoplasma hyopneumoniae (strain 162 + HP-G strain) was provided by Sichuan Huapai Biotechnology (Group) Co., Ltd. Each milliliter of this product contains 10 7.0 TCID 50 of porcine circovirus type 2 strain 162 before inactivation; and 10 8.0 CCU of Mycoplasma hyopneumoniae HP-G strain.

[0060] The results of the mouse body weights are shown by Figure 1 . During the entire immunization period, there were no significant differences in the body weights of the mice in the experimental group and the control group (p > 0.05), indicating that the new vaccine has good biosafety.

[0061] Example 1 Immunological changes in blood

[0062] For each mouse in each experimental group, 50 μL of EDTA anticoagulated blood samples were obtained, and the immunological changes in the whole blood of the mice were analyzed with reference to the operating procedures of the Tekang veterinary automatic five-part blood analyzer (TEK-VET5). The conventional results of the immunological changes in the peripheral anticoagulated blood of the mice in each group are shown in Figure 2 and 3 .

[0063] According to Figure 2 , it can be seen that there were no significant differences in 4 indexes such as red blood cells, hemoglobin concentration, platelets, and mean corpuscular hemoglobin in the peripheral blood of the mice in the treatment group and the control group at multiple time points (p > 0.05), indicating that the contents and physiological characteristics of red blood cells and platelets in the adjuvant treatment group and the control group of mice were stable after immunization, and no obvious side reactions occurred. White blood cells are the core of the immune system, including neutrophils, lymphocytes, and monocytes, etc., and are responsible for anti-infection, immune regulation, and tissue repair.

[0064] According to Figure 3It can be seen that on the 7th day and the 28th day after the first immunization (7 days after the booster immunization), the contents of white blood cells, neutrophils, lymphocytes and monocytes in the peripheral blood of the mice in the adjuvant treatment group were significantly higher than those in the control group C3 (p < 0.05); and on the 28th day, the contents of white blood cells, neutrophils and lymphocytes in the peripheral blood of the mice in the treatment group were significantly higher than those in the control groups C1 and C3 (p < 0.05). This indicates that the mice in the adjuvant treatment group had a stronger immune response after vaccination with the vaccine combined with the novel adjuvant, manifested as a significant increase in the number of immune cells in the peripheral blood.

[0065] Example 2 Effect of the Immune Adjuvant on the Immune Efficiency of the Vaccine and the Establishment of Long-Term Immune Memory

[0066] In this example, after vaccination, the effect of the immune adjuvant on the immune efficiency of the vaccine and the establishment of long-term immune memory was investigated. Specifically, flow cytometry was used to analyze the lymphocyte subsets in the peripheral anticoagulated blood of mice. The experimental steps were as follows:

[0067] 1) Add 1 μl of each of the flow antibodies against mouse CD45 (CD45 Monoclonal Antibody (30-F11), Super Bright TM 600, eBioscience TM , 63-0451-82), CD3 (BD Pharmingen TM FITC Hamster Anti-Mouse CD3e, 553061), CD4 (CD4 MonoclonalAntibody (GK1.5), eFluor TM 450, eBioscience TM , 48-0041-82), CD8 (CD8a MonoclonalAntibody (53-6.7), PerCP-Cyanine5.5, eBioscience TM , 45-0081-82), CD44 (CD44Monoclonal Antibody (IM7), APC, eBioscience TM , 17-0441-82) and CD62L (CD62L (L-Selectin) Monoclonal Antibody (MEL-14), PE, eBioscience TM , 12-0621-82) to a 1.5 ml EP tube 1 containing 100 μl of mouse peripheral anticoagulated blood (EDTA·2K anticoagulant), incubate in the dark at 4 °C for 30 min.

[0068] 2) Add 1 ml of 1× RBC lysis buffer (prepared with deionized water) to the above reaction system. After lysing at room temperature for 5 min, centrifuge at 1500 rpm and 4 °C for 5 min to remove the supernatant.

[0069] 3) Washing: Add 200 μl of PBS (containing 0.5% BSA) to the cell pellet in step 2, pipette to mix well, centrifuge at 1500 rpm and 4 °C for 5 min to remove the supernatant, and wash twice.

[0070] 4) Resuspend the cells with 500 μl of PBS (containing 0.5% BSA), then add 2% paraformaldehyde to a final volume and fix in the dark at 4 °C, and load onto the machine.

[0071] The following staining protocol was processed using the same steps as above:

[0072] Add 1 μl of each of the flow antibodies anti-mouse CD19 (BD, CD19 BUV395, 563557), IgM (ThermoFisher, lgM APC, 17-5790-82), and IgD (ThermoFisher, lgD FITC, 11-5993-85) to tube 2.

[0073] T cells are defined as the CD3 + / CD45 + population. According to the different expressions of CD4 and CD8, T cells can be further divided into four major subsets. These subsets are CD4 + TH cells, CD8 + TC cells, CD4 + / CD8 + double-positive (DP) cells, and CD4 - / CD8 - double-negative (DN) cells. The latter are mainly composed of γδ+ cells under normal circumstances. When analyzing CD44 and CD62L in TH and TC lymphocytes, three different subsets can be distinguished: CD62LhiCD44neg / lo cells, CD62L hi CD44 hi central memory (CM) cells, and CD62L neg / lo CD44 hi effector memory (EM) cells. B cells are defined as the CD19 + population. When analyzing IgM and IgD in B cells, four different subsets can be distinguished: Follicular (FO) B cells (B FO , IgM - , IgD +)、Marginal zone(MZ)B cells(IgM + ,IgD + )、Transitional Bcells(IgM + ,IgD - ) and Class-switched or activated B cells(IgM - ,IgD - ). The gating logic of flow cytometry in this example is shown in Table 2.

[0074] Table 2 Gating logic of flow cytometry

[0075]

[0076]

[0077] Figure 4 are the proportions of each B lymphocyte subset in the anticoagulated peripheral blood of mice on the 56th day. On the 56th day, the proportions of Activated and FO B cells in the treatment group were significantly higher than those in the control group (p < 0.05). While the proportions of MZ and Transitional B cells were significantly lower than those in the control group (p < 0.05). It shows that transitional B cells in the experimental group were mainly transformed into FO and Class-switched B cells, preparing for the next step of transformation into memory B lymphocytes. Transitional cells represent the last stage before differentiating into a more mature pre-immune B cell population, indicating that the addition of adjuvant promoted the activation and differentiation of B cells, and provided a basis for the immune system to produce more antibodies with high affinity for antigens, while promoting the formation of memory B cells, further proving that adding adjuvant is more conducive to improving vaccine immune efficiency and establishing long-term immune memory.

[0078] Figure 5 are the proportions of each T lymphocyte subset in the anticoagulated peripheral blood of mice on the 56th day. On the 56th day, the CD4 + T EM , CD8 + T CM and T EM in the peripheral blood of the treatment group mice were all significantly higher than those in the control group (p < 0.05), while CD4 + and CD8 + T cells were all significantly lower than those in the control group (p < 0.05), which may be related to the increase in the transformation and maturation of various immunologically active T cell subsets (such as CD4 + T EM , CD8 + T CM and CD8 + TEM ) It is related to CD8 T cells. CD8 T cells can directly participate in cytotoxicity to eliminate virus-infected cells. Central memory T cells can exist in the body for a long time and can respond rapidly when encountering the same antigen again, providing long-term immunity, while effector memory T cells provide rapid immune protection after repeated infections. The results show that this adjuvant can enhance the immune response of mice after repeated infections and provide stronger long-term immune memory and immune protection effects.

[0079] Example 3 Neutralization / antigen-specific antibody assay

[0080] (1) Porcine circovirus antigen-specific antibody

[0081] Referring to the improved method of the porcine circovirus type 2 ELISA antibody detection kit of Guangdong Yueyang Biotechnology, the secondary antibody was replaced with goat anti-mouse IgG labeled with horseradish peroxidase from Solarbio (product number: SE131) for the determination of mouse-derived antigen-specific antibodies.

[0082] Figure 6 It is a dynamic graph of the content of porcine circovirus antigen-specific antibodies in the peripheral blood plasma of mice. As can be seen from the figure, at day 0 and day 7, there were no significant differences in the content of porcine circovirus antigen-specific antibodies among all mice (p > 0.05). From day 14 to day 56, the levels of this virus antibody in the plasma of the experimental group mice were significantly higher than those of the control group (p < 0.05), indicating that the vaccine antigen combined with the novel immune adjuvant can stimulate mice to present a stronger specific humoral immune response, the level of virus-specific antibodies was significantly increased, and its antiviral immune protection ability was significantly enhanced.

[0083] (2) Determination of Mycoplasma hyopneumoniae specific antibody - indirect hemagglutination method

[0084] The experimental materials required for the determination of Mycoplasma hyopneumoniae specific antibody are as follows:

[0085] 1) Prepare freeze-dried Mycoplasma antigen-sensitized red blood cell products. Among them, the mass percentage of Mycoplasma antigen-sensitized red blood cells is 10%, and it is diluted to 2% with 1 / 15 mol / L PBS (pH value 7.2) when in use for IHA test.

[0086] 2) Prepare positive and negative control sera and store them at 2 - 8°C or frozen.

[0087] 3) Microreaction plate: 96-well V-shaped organic glass plate or plastic plate.

[0088] 4) Diluent: 1 / 15 mol / L PBS (pH value 7.2) containing 1% healthy rabbit serum (inactivated at 56°C for 30 minutes).

[0089] The specific determination steps are as follows:

[0090] 1) Treatment of the serum to be tested: The serum to be tested is inactivated at 56°C for 30 minutes.

[0091] 2) Adding PBS diluent: Add 25 μl of diluent to each well of the reaction plate.

[0092] 3) Diluting the serum: Add 25 μl of the serum to be tested, positive serum, and negative serum to the wells in the first column respectively, then perform serial dilution to the 12th well, and finally discard 25 μl.

[0093] 4) Adding mycoplasma antigen-sensitized erythrocytes: Add 25 μl of 2% mycoplasma antigen-sensitized erythrocytes to each well; for the antigen control, add 25 μl of diluent + 25 μl of antigen-sensitized erythrocytes.

[0094] 5) Reaction: After adding the samples, place on a micro oscillator and shake for about 15 seconds, then let stand at room temperature for 1 - 2 hours to determine the result.

[0095] 6) Judgment: When the titer of the positive serum ≥ 1:64, the titer of the negative serum ≤ 1:4, and there is no autoagglutination in the antigen-sensitized erythrocyte control well, the test is valid. The highest dilution of the serum showing (++) hemagglutination reaction is taken as the end point of the serum titer. When the titer of the serum to be tested ≥ 1:16, it is judged as positive; when the serum titer ≤ 1:8, it is judged as negative.

[0096] 7) Judgment criteria: The highest dilution of the serum showing (++) hemagglutination reaction is taken as the end point of the serum titer. Fixed pathogen dilution serum method; then calculate the mycoplasma specific antibody titer of the serum.

[0097] The results are shown in Table 3 and Figure 7 as follows. On day 0, the specific antibodies against Mycoplasma hyopneumoniae in the plasma of all mice were less than 2 1 . From day 7 to day 42, the rising trends of antibodies in the experimental group and control group C1 were similar, but on day 56, the mycoplasma neutralizing antibody titers of the mice in the experimental group (> 2 12 ) were significantly better than those of control group C1. It shows that the specific antibody titers in the plasma of the mice in the treatment group can be maintained at a high level for a longer time and can play a better immune protection role.

[0098] Table 3 Mycoplasma hyopneumoniae neutralizing antibody

[0099] Group / Valence Day 0 Day 7 Day 14 Day 28 Day 42 Day 56 A <![CDATA[< 2 1 > <![CDATA[2 2 > <![CDATA[2 6 > <![CDATA[> 2 12 > <![CDATA[> 2 12 > <![CDATA[> 2 12 > C1 <![CDATA[< 2 1 > <![CDATA[2 2 > <![CDATA[2 6 > <![CDATA[> 2 12 > <![CDATA[> 2 12 > <![CDATA[2 12 > C3 <![CDATA[<2 1 > <![CDATA[< 2 1 > <![CDATA[<2 1 > <![CDATA[< 2 1 > <![CDATA[<2 1 > <![CDATA[< 2 1 >

[0100] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that Including porcine interleukin 2, porcine interleukin 4, porcine interleukin 6 and manganese ions.

2. A composition according to claim 1, characterized in that In the composition, the mass ratio of the porcine interleukin 2, the porcine interleukin 4, the porcine interleukin 6 and the manganese ion is 0.05-1:0.05-1:0.05-1:50-200.

3. A composition according to claim 2, characterized in that In the composition, the mass ratio of the porcine interleukin 2, the porcine interleukin 4, the porcine interleukin 6 and the manganese ion is 0.1-0.5:0.1-0.5:0.1-0.5:100-200.

4. Use of the composition according to any one of claims 1 to 3, characterized in that: The composition can be used to prepare a porcine round bacillus bivalent vaccine, an immune adjuvant for a porcine round bacillus bivalent vaccine, or a vaccine enhancer for a porcine round bacillus bivalent vaccine.

5. The use of a composition according to claim 4, characterized in that: The porcine round bacillus bivalent vaccine is a compound inactivated vaccine.

6. An immune adjuvant for a porcine round bacillus bivalent vaccine containing three porcine interleukins and manganese ions, characterized in that: The invention comprises the composition according to any one of claims 1 to 3.

7. The porcine round bacillus bivalent vaccine immune adjuvant containing three porcine interleukins and manganese ions according to claim 6, characterized in that: Pharmaceutically acceptable excipients are also included.

8. A composite porcine roundworm bivalent inactivated vaccine, characterized in that: It comprises the porcine round rod bivalent vaccine immune adjuvant and porcine round rod bivalent vaccine antigen as described in claim 6 or 7, and the volume ratio of the porcine round rod bivalent vaccine immune adjuvant and the porcine round rod bivalent vaccine antigen is 1:0.5-1.

5.

9. A composite porcine round bacillus bivalent inactivated vaccine according to claim 8, characterized in that: Each milliliter of the porcine circovirus bivalent vaccine antigen includes 10 7.0 TCID 50 and Mycoplasma hyopneumoniae HP-G strain 10 8.0 CCU, and the masses of both are 0.1-1 μg, and also include a solvent, and the solvent includes one or more of sterile saline and sterile buffer.

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