CN109200284B

CN109200284B - Mycoplasma hyopneumoniae live vaccine mucosal immune adjuvant, and preparation method and application thereof

Info

Publication number: CN109200284B
Application number: CN201811160766.0A
Authority: CN
Inventors: 熊祺琰; 张珍珍; 邵国青; 冯志新; 韦艳娜; 王佳; 白昀; 甘源; 张磊
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2022-01-11
Anticipated expiration: 2038-09-30
Also published as: CN109200284A

Abstract

The invention provides a mucosal immune adjuvant of a live vaccine of mycoplasma hyopneumoniae, a preparation method and application thereof, and relates to the field of veterinary vaccines. The mucosal adjuvant is a solution containing a recombinant protein CTB-P97R1 expressed by fusion of a cholera toxin B subunit and a mycoplasma hyopneumoniae P97R1 antigen. The invention also provides a preparation method of the mucosal adjuvant, which comprises the steps of preparing 10mg/ml recombinant protein CTB-P97R1 mother liquor and then diluting with a solvent. The invention also provides application of the mucosal immune adjuvant in preparation of a live vaccine of mycoplasma hyopneumoniae. The mucosa adjuvant is an aqueous compound adjuvant, the preparation method is simple and convenient, the adjuvant has stable property, has no damage to the vitality of the mycoplasma hyopneumoniae, can be compatible with an aerosol protective agent, and the mixture still has no damage to the vitality of the mycoplasma hyopneumoniae, is suitable for aerosol immunization of a live vaccine, and can be used for remarkably improving the mucosa immune protective efficacy of the vaccine and prolonging the immune duration.

Description

Mycoplasma hyopneumoniae live vaccine mucosal immune adjuvant, and preparation method and application thereof

Technical Field

The invention relates to the field of veterinary vaccines, in particular to a mucosal immune adjuvant of a live vaccine for mycoplasma hyopneumoniae, a preparation method and application thereof.

Background

Mycoplasma hyopneumoniae is a chronic respiratory infectious disease of swine caused by Mycoplasma hyopneumaniae (Mhp) and has the main symptoms of cough and asthma. Mainly causes the reduction of the feed conversion rate and the obstruction of the growth of the pigs, and has high morbidity and low mortality. The mycoplasma hyopneumoniae destroys cilia after infection, so that other respiratory bacteria and viruses are easy to generate secondary infection, thereby causing more serious epidemic situation. The disease is widely prevalent in the world, and causes great economic loss to the modern pig industry.

The mycoplasma hyopneumoniae is a refractory chronic infectious disease, the medicine treatment effect is not ideal enough, the mycoplasma hyopneumoniae is easy to relapse after stopping the medicine, and the vaccine prevention plays a key role in controlling the mycoplasma hyopneumoniae. At present, inactivated vaccines are mainly used for prevention at home and abroad, but because the mycoplasma hyopneumoniae has weak immunogenicity, the inactivated vaccines are often poor in protective efficacy, can only relieve symptoms and cannot block pathogen infection, and are high in cost, so that the actual requirements cannot be completely met. The genetic engineering vaccine is still in the research stage, and no product is on the market. The live vaccine can be proliferated in vivo transiently and has the advantages of small inoculation dose, good immunogenicity and lasting immune effect.

Researchers in China (China institute of veterinary medicine, Jiangsu province of agricultural science) do a lot of valuable work on the development of the swine mycoplasma pneumonia live vaccine, and the immunity efficacy of the developed live vaccine is superior to that of the inactivated vaccine, while the cost is relatively low and the market competitiveness is very strong. After being immunized by the respiratory mucosa, the live vaccine can be adhered and fixedly planted at the cilia of the bronchus to form an occupying effect, thereby effectively preventing the clinical wild virus infection and having unique advantages which are incomparable with the inactivated vaccine and the genetic engineering vaccine. However, the current live vaccine adopts an intrapulmonary injection immunization approach, which affects the wide popularization of the vaccine to a certain extent. Aerosol immunization, nasal drop immunization, etc., which are also immunizations via the mucosal route of the respiratory tract, are very promising alternatives. The mucosal immune system is the first line of defense of the body against the invasion of foreign pathogens, but the current vaccines for mucosal immunization are relatively few, and are largely related to the tendency of the physiological characteristics of mucosal parts to generate immune tolerance. In order to further enhance the mucosal immune stimulation capability of the live vaccine, adding an adjuvant suitable for mucosal immunity to the vaccine is an effective means.

In the prior art, no effective mucosal adjuvant aiming at the mycoplasma hyopneumoniae live vaccine exists. For this reason, the following difficulties mainly exist: firstly, the research on the mucosal immune adjuvant is still lacked at present, the mucosal immune adjuvant is influenced by unclear research on mucosal immune response to a great extent, the using effect and action principle of most adjuvant components on a mucosal part are not clear, and the adjuvant selection is difficult. Screening adjuvant components capable of effectively activating local mucosal immunity and cellular immunity by using an animal test according to the immunization requirement of the mycoplasma hyopneumoniae; secondly, for the live bacterial vaccine, the selection of the adjuvant also has specific requirements, the oil-in-water adjuvant is not applicable due to the destructiveness of the oil substance on the cell membrane, and even the oil-in-water and dual-phase adjuvants have high requirements on the stability and the preparation process, the water-based adjuvant can be preferably considered and screened by using an in vitro compatibility test; thirdly, when the live vaccine of mycoplasma hyopneumoniae is used for aerosol immunization, a spray protectant needs to be added to ensure the activity of the live vaccine in the atomization process, but the spray protectant is generally a special high-molecular component, and part of adjuvants cannot be matched with the spray protectant for use due to the chemical properties, the used solvent, the ion concentration, the pH value and the like (including unstable physicochemical properties after the two are mixed, damage to mycoplasma activity caused by the mixture, incapability of exerting the original spray protection effect of the mixture and incapability of exerting the original adjuvant activity of the mixture), so that the selected adjuvant can be matched with the spray protectant for use only through a series of experiments, and the use requirement of the live vaccine of mycoplasma hyopneumoniae can be met.

Disclosure of Invention

The invention mainly aims to disclose a mucosal immune adjuvant of a live vaccine of mycoplasma hyopneumoniae, which has no damage to the activity of the live vaccine of mycoplasma hyopneumoniae, can be used together with an aerosol spray protective agent, can enhance the immune stimulation capability of a mucosal route, and can strengthen the immune response of an important protective antigen P97R 1.

The invention also aims to provide a preparation method of the mucosal immunoadjuvant, which is simple and efficient.

The invention further aims to provide application of the mucosal immunoadjuvant.

A mucous membrane immune adjuvant of a live vaccine of mycoplasma hyopneumoniae is a solution containing a cholera toxin B subunit and a recombinant protein CTB-P97R1 expressed by fusion of mycoplasma hyopneumoniae P97R1 antigen, and the adjuvant contains 0.2-2mg/ml of recombinant protein CTB-P97R 1.

In the invention, the amino acid sequence of the recombinant protein CTB-P97R1 is shown as SEQ ID NO. 2.

In the invention, the recombinant protein CTB-P97R1 is prepared by the following method: fusing the R1 region gene of the mycoplasma hyopneumoniae adhesion factor P97 to the C end of a cholera toxin B subunit gene, and filling the fused gene into a pET-28a (+) plasmid to construct a recombinant plasmid pET28a-CTB-P97R 1; introducing the recombinant plasmid into escherichia coli to obtain a recombinant bacterium; and (3) inducing the recombinant bacteria to express CTB-P97R1 by IPTG, collecting the supernatant of the bacterial lysate, and purifying to obtain the recombinant protein CTB-P97R 1.

In the preferred technical scheme, the mucosal immunoadjuvant further contains 150-300 mu g/ml Poly (I: C) and 50-150 mu g/ml saponin.

In the invention, the saponin is quillaja saponin, ginsenoside or notoginsenoside.

The invention also provides a preparation method of the swine mycoplasma pneumonia live vaccine mucosal adjuvant, which comprises the steps of preparing 10mg/ml recombinant protein CTB-P97R1 mother liquor, and then diluting with a solvent, wherein the solvent is phosphate buffer solution.

In the preferred technical scheme, 1mg/ml of Poly (I: C) mother liquor and 1mg/ml of saponin mother liquor are prepared, and the solvents are phosphate buffer solutions; and mixing the recombinant protein CTB-P97R1 mother liquor, the Poly (I: C) mother liquor and the saponin mother liquor, and diluting by adopting a solvent.

The invention also provides application of the mucosal immune adjuvant of the live vaccine of the swine mycoplasma pneumonia in preparation of live vaccines of the swine mycoplasma pneumonia.

In the preferred technical scheme, the adjuvant and the spray protectant are mixed according to the volume ratio of 1:1, and then the mycoplasma hyopneumoniae vaccine strain culture or the freeze-dried powder is added and uniformly mixed to obtain the mycoplasma hyopneumoniae live vaccine solution.

In the application, the spray protective agent is an aqueous solution containing 600035-45 g/L polyethylene glycol, 3-5g/L cyclodextrin, 18-22g/L sodium thiosulfate and 18-22 g/L-histidine, and the pH value is 7.0-7.5.

The mucosal adjuvant has the following beneficial effects: firstly, the mucosal adjuvant is an aqueous compound adjuvant, the preparation method is simple and convenient, the adjuvant has stable property, the selected components have no damage to the activity of the mycoplasma hyopneumoniae, the mucosal adjuvant is suitable for being used as a live vaccine, and the mucosal adjuvant is safe to animals and has small side effect; secondly, the mucosal adjuvant can be compatible with an aerosol protective agent, and the mixed mixture still has no damage to the activity of mycoplasma hyopneumoniae and is suitable for aerosol immunization of live vaccines; thirdly, the mucosa adjuvant contains CTB-P97R1 recombinant protein (CTB part playing the role of the adjuvant), Poly (I: C) and saponin, the respective immune regulation advantages are comprehensively utilized, and the formed compound adjuvant can effectively activate local mucosa immune response and cell immune response through mucosa approach immunization, thereby meeting the requirements of mycoplasma hyopneumoniae immune protection; fourthly, the mucosal adjuvant contains CTB-P97R1, and fuses an important protective antigen P97R1 of mycoplasma hyopneumoniae to the C end of CTB, thereby being beneficial to further supplementing an antigen spectrum and strengthening the immune effect of the vaccine.

Through a series of in vivo and in vitro experiments, the special mucosal immune adjuvant for the live vaccine of the mycoplasma hyopneumoniae, which is nontoxic to the live vaccine of the mycoplasma hyopneumoniae, can be used together with an aerosol spray protective agent, can effectively stimulate an immune system in a mucosal immune way, and can specifically enhance the immune response of an important antigen P97R1, is developed, and can be used for remarkably improving the mucosal immune protective efficacy and prolonging the immune duration of the vaccine.

Drawings

FIG. 1 PCR identification of recombinant plasmid pET28a-CTB-P97R1, in lane 1: DNA molecular weight standard; lane 2: positive control (P97 gene fragment); lane 3: negative control; lane 4: and (3) taking the recombinant vector pET28a-CTB-P97R1 as a template and P97R1-up and P97R1-down as amplification products of primers.

FIG. 2 SDS-PAGE electrophoresis of purified fusion protein CTB-P97R1, wherein lane 1: protein molecular weight standards, lane 2: the purified fusion protein CTB-P97R 1.

FIG. 3 shows the results of ELISA assays for the ability of the fusion protein CTB-P97R1 to bind GM1 at different concentrations, with the addition of the fusion protein CTB-P97R1 in ng/well on the abscissa.

Figure 4 mycoplasma hyopneumoniae holoprotein antigen stimulates lymphocyte proliferation response, wherein "×" indicates a very significant difference compared to the non-adjuvanted control group, P < 0.01.

Fig. 5 mycoplasma hyopneumoniae P97R1 protein stimulated lymphocyte proliferation responses, where "×" indicates a very significant difference compared to the non-adjuvanted control group, P < 0.01.

Figure 6 levels of specific sIgA antibodies against whole mycoplasma hyopneumoniae, where "@" indicates a significant difference compared to the non-adjuvanted control group, P < 0.05; "x" indicates very significant difference compared to the non-adjuvanted control group, P < 0.01.

Figure 7 levels of specific sIgA antibodies against mycoplasma hyopneumoniae P97R1 protein, where "×" indicates a very significant difference compared to the non-adjuvanted control group, P < 0.01.

Figure 8 lung score indices after challenge for each group, percentage in the figure is the protection rate for each group, wherein "+" indicates that the lung score index was significantly different from the non-adjuvanted control group, P < 0.01.

Figure 9 comparison of protective efficacy of each aerosol vaccine, wherein "×" indicates a very significant difference compared to the challenge control group, P < 0.01.

Figure 10 shows lung scores and protection rates for various groups of animals after challenge, where ". x" indicates significant difference from the non-adjuvanted control group, P <0.01, where percentages are protection rates for each group.

Detailed Description

The invention will be further described and illustrated with reference to specific embodiments, but the invention is not limited thereto.

Example 1: construction of recombinant engineering bacterium BL21-pET28a-CTB-P97R1

In order to screen the mucosal immune adjuvant of the swine mycoplasma pneumonia live vaccine, a plurality of fusion proteins are designed. Through tests, only the fusion protein CTB-P97R1 is found to have better immune effect. The amino acid sequence of the fusion protein CTB-P97R1 is shown as SEQ ID NO. 2, and the gene sequence is shown as SEQ ID NO. 1.

This example describes the construction of a recombinant genetically engineered bacterium BL21-pET28a-CTB-P97R1 for the preparation of the fusion protein CTB-P97R 1.

According to the gene sequence (AY307389.1) of cholera toxin B subunit (CTB) recorded in GenBank, the CTB gene with a signal peptide part removed is obtained by adopting a gene synthesis mode (the specific sequence is shown in SEQ ID NO:3), and PagI and NheI enzyme cutting sites are respectively added at two ends. The synthetic sequence is purified after double digestion, and is enzymatically linked with pET-28a (+) carrier which is subjected to double digestion and purification by Nco I (Pag I and Nco I are isocaudarner enzymes) and Nhe I at 4 ℃ overnight by using T4-DNA ligase, a ligation product is transformed into BL21(DE3) competent cells, clones are picked, and positive clones are screened by the method of Nco I digestion and DNA sequencing. And (3) marking the positive clone, selecting the single clone and repeatedly testing once, and the result proves that the obtained single clone is successfully introduced into a recombinant vector pET28a-CTB carrying CTB genes (removing signal peptides), and the glycerol tube is prepared for storage.

According to the gene sequence (U50901) of Mycoplasma hyopneumoniae P97 in GenBank, a primer is designed, and the R1 region of the P97 gene is amplified by taking the total DNA of the Mycoplasma hyopneumoniae strain 168 as a template. Wherein, the sequence of the upstream primer P97R1-up (added with NheI enzyme cutting site) is 5'-AAAGCTAGCTTACCTCAGCCGCCAGCA-3'; the sequence of the downstream primer P97R1-down (with the addition of XhoI cleavage sites) is 5'-TTTCTCGAGAGCCATTGGGAAATAGTCTT-3'. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1 min, annealing at 54 ℃ for 1 min, extension at 72 ℃ for 30 sec, 30 cycles; extension at 72 ℃ for 10 min. The PCR product was digested with Nhe I and XhoI, purified, enzymatically ligated to pET28a-CTB recombinant vector digested in the same manner with T4-DNA ligase overnight at 4 ℃ to transform BL21(DE3) competent cells, clones were selected, and positive clones were selected by the above-mentioned PCR method (see FIG. 1) and DNA sequencing method. Marking out positive clones, picking up single clones, repeatedly verifying once by the same method, and proving a result that the obtained single clones are successfully introduced into a recombinant vector (named as pET28a-CTB-P97R1 recombinant vector) carrying CTB genes (removing signal peptides) and P97 gene R1 regions, and the single clones are named as BL21-pET28a-CTB-P97R1, and preparing and storing the glycerol tube.

Example 2: expression and purification of fusion protein CTB-P97R1

The positive recombinant bacterium BL21-pET28a-CTB-P97R1 was shake-cultured overnight at 37 ℃ in LB medium (kanamycin-resistant). Inoculating the bacterial liquid into LB (kanamycin-resistant) liquid culture medium at a ratio of 2% the next day, and performing shaking culture at 37 ℃ until OD is reached₆₀₀Adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1.0mmol/L for induction expression, collecting bacterial liquid 4h after induction, centrifuging, collecting bacterial precipitation, and washing with PBS 1 time. Suspending the thallus in PBS buffer solution, performing ultrasonic lysis, centrifuging at 4 deg.C at 10000rpm for 30min, collecting lysis supernatant, purifying with His-Tag affinity chromatography column, dialyzing for 2 days to remove salt, and freeze drying. The purity of the fusion protein CTB-P97R1 was analyzed by 15% SDS-PAGE electrophoresis. As can be seen from FIG. 2, the purified fusion protein CTB-P97R1 was 90% pure and had a molecular weight of 21.5kDa, consistent with the expectation.

EXAMPLE 3 in vitro bioactivity assay of the fusion protein CTB-P97R1

The method comprises the following steps: the binding capacity of the fusion protein CTB-P97R1 to the ganglioside GM1 was examined by using the ganglioside GM 1-enzyme-linked immunosorbent (ELISA) assay. 96-well enzyme-linked plates were plated with carbonate buffer (containing 35mmol/L NaHCO) containing 5. mu.g/ml GM1₃、15mmol/L Na₂CO₃Aqueous solution of (d) coating overnight at 4 ℃ in 100. mu.l per well; PBST (0.1% Tween-20 in PBS buffer) 3 times, with 1% BSA PBS solution at 37 degrees C blocking for 2h, PBST after 3 times washing, each hole is added with 100 u l containing 500ng CTB-P97R1 (after purification) solution, 37 degrees C incubation for 2h, control with 100 degrees C boiling 10min treatment of CTB-P97R1 solution instead. After PBST washing, rabbit anti-CTB antibody (Bio-Rad, USA) diluted with PBS at a dilution of 1:4000 was added and incubated at 37 ℃ for 1 h. After PBST was washed 3 times, horseradish peroxidase-labeled goat anti-rabbit IgG (Roche Bodhisaku Bio Inc.) diluted with PBS at a dilution of 1:8000 was added and incubated at 37 ℃ for 1 hour. After washing, 100. mu.l of substrate TMB was added, the reaction was stopped with 50. mu.l of 2mol/L aqueous sulfuric acid, and the absorbance was measured at 450 nm. In addition, the ability of different concentrations of CTB-P97R1 to bind GM1 was examined using the same method.

As a result: as shown in FIG. 3, the fusion protein CTB-P97R1 can bind to GM1, and its binding to GM1 decreases with the decrease of the concentration of CTB-P97R1, therefore, the binding of the fusion protein CTB-P97R1 to GM1 can be dose-dependent. The above results demonstrate that the fusion protein CTB-P97R1 prepared in example 2 has similar in vitro activity to natural CTB.

Example 4 preparation of Mycoplasma hyopneumoniae live vaccine mucosal adjuvant

Respectively preparing fusion protein CTB-P97R1 mother liquor, Poly (I: C) mother liquor and saponin mother liquor, mixing the mother liquor, diluting with phosphate buffer solution to required concentration to obtain Mycoplasma hyopneumoniae live vaccine mucosal adjuvant

Preparing each mother solution: (1) preparation of fusion protein CTB-P97R1 mother liquor: preparing a purified fusion protein CTB-P97R1 by the method in example 2, and preparing 10mg/ml fusion protein CTB-P97R1 mother liquor by taking phosphate buffer solution (10mmol/L, pH7.2-7.4) as a solvent; (2) preparation of Poly (I: C) mother liquor: dissolving appropriate amount of Poly (I: C) (product name: VDN, Sedao Biotech Co., Ltd., Beijing) in phosphate buffer solution (10mmol/L, pH7.2-7.4) to prepare 1mg/ml Poly (I: C) mother liquor; (3) preparing a saponin mother solution: dissolving appropriate amount of Quillaja Saponaria Molina saponin (QS-21) in phosphate buffer solution (10mmol/L, pH7.2-7.4) to obtain 1mg/ml saponin mother liquor.

Preparation of a porcine mycoplasma pneumonia live vaccine mucosal adjuvant: when 100ml of adjuvant is taken as an example for preparation, a proper amount of 10mg/ml of recombinant protein CTB-P97R1 mother liquor is taken, meanwhile, 1mg/ml of Poly (I: C) mother liquor and 1mg/ml of saponin mother liquor are taken to be mixed, and certain dilution is carried out by phosphate buffer solution, thus obtaining the live vaccine mucosal adjuvant for swine mycoplasmal pneumonia, which contains the fusion protein CTB-P97R10.2-2 mg, the Poly (I: C)150-300 mu g and the saponin 50-150 mu g in each ml of solution.

Example 5 Activity Damage assay of adjuvants on live Mycoplasma hyopneumoniae vaccine

Vaccine: the freeze dried live vaccine (168 strain) for swine mycoplasma pneumonia is prepared with the culture of mycoplasma hyopneumoniae 168 strain obtained through conventional culture medium culture and through freeze drying. Each vial was filled with 2mL of M.hyopneumoniae 168 strain culture and lyophilized.

The following adjuvants were prepared separately: porcine mycoplasma pneumonia live vaccine mucosal adjuvant 1 (abbreviated as the mucosal adjuvant 1 of the present invention): was prepared by the method of example 4 using a solution containing 2mg/ml of the fusion protein CTB-P97R1, 300. mu.g/ml of Poly (I: C), and 150. mu.g/ml of saponin in PBS buffer (10mmol/L, pH 7.2-7.4). Control adjuvant 1: montanide IMS 251C (product of SEPPIC corporation), prepared according to the product specifications. Control adjuvant 2: 100mg/ml astragalus polysaccharide solution, and the solvent is PBS buffer solution. Control adjuvant 3: dissolving appropriate amount of carbomer 974P powder (product of Doubemon) in distilled water, and swelling to obtain 15mg/ml carbomer mother liquor; the pH of the carbomer mother liquor was adjusted to 7.2 with aqueous sodium hydroxide, and an appropriate amount of PBS buffer (10mmol/L, pH7.2-7.4) was added to give a final carbomer concentration of 2mg/ml, giving control adjuvant 3. Control adjuvant 4: montanide ISA 206 (product of SEPPIC corporation), prepared according to the product specification. Control adjuvant 5: montanide GEL (product of SEPPIC corporation), a 15% solution by mass was prepared according to the product specification. Control adjuvant 6: 2mg/ml hyaluronic acid solution, and the solvent is PBS buffer (10mmol/L, pH7.2-7.4).

And (3) toxicity detection: dissolving 1 bottle of Mycoplasma hyopneumoniae live vaccine (168 strain) lyophilized powder with each adjuvant, wherein the volume of adjuvant added into each bottle of lyophilized powder is 2ml, standing at 4 deg.C for 1h, sampling, determining CCU (color change unit), and operating three holes in parallel; meanwhile, PBS buffer solution is used for replacing adjuvant to dissolve 1 bottle of freeze-dried powder of the mycoplasma hyopneumoniae vaccine (168 strain) to be used as a control to detect CCU. Comparing the difference of CCU, and judging the activity damage condition of the adjuvant to the live vaccine.

The experimental results are as follows: as can be seen from Table 1, after incubation for 1h, compared with PBS buffer, the mucosal adjuvant 1 of the invention does not affect the activity of the live vaccine, and CCU has no obvious change. The control adjuvant 2, the control adjuvant 3, the control adjuvant 5 and the control adjuvant 6 also do not obviously influence the activity of the live vaccine, and the CCU has no obvious change; the control adjuvant 1 and the control adjuvant 4 groups have no color change in each well, and the CCU is 0, which indicates that the activity of the live vaccine is seriously damaged. The test results show that the mucosal adjuvant 1 has no damage to the activity of the live vaccine of the mycoplasma hyopneumoniae.

TABLE 1 Activity injury assay (CCU) for Mycoplasma hyopneumoniae live vaccines with adjuvants

Example 6: aerosol spray for preparing swine mycoplasma pneumonia live vaccine by matching adjuvant with spray protectant

Vaccine: the freeze dried live vaccine (168 strain) for swine mycoplasma pneumonia is prepared with the culture of mycoplasma hyopneumoniae 168 strain obtained through conventional culture medium culture and through freeze drying. Before lyophilization, each flask was filled with 2mL of culture.

Adjuvant: the mucosal adjuvant 1, the contrast adjuvant 2, the contrast adjuvant 3, the contrast adjuvant 5 and the contrast adjuvant 6 are the same as the embodiment 5.

Spray protectant: is aqueous solution (pH value 7.2) containing polyethylene glycol 600040 g/L, cyclodextrin 4g/L, sodium thiosulfate 20g/L, L-histidine 20 g/L.

Stability tests and results of adjuvant used with spray protectants: the adjuvants in the embodiment are mixed with the spray protectant according to the volume ratio of 1:1, and the mixture is uniformly shaken. Each mixed solution was left at 4 ℃ and 37 ℃ at the same time, and whether or not precipitation occurred was observed within 2 weeks. As a result: the mixture of control adjuvant 2 and spray protectant appeared cloudy and precipitated after being left at 37 ℃ for 2 weeks, and the mixture of the remaining adjuvant and spray protectant appeared normal.

The damage determination and the result of the active vaccine activity by the adjuvant and the spray protective agent are as follows: the adjuvants in the embodiment are mixed with the spray protectant according to the volume ratio of 1:1, and the mixture is uniformly shaken to obtain various diluents. Each diluent is dissolved with 1 bottle of swine mycoplasma pneumonia live vaccine (168 strain) freeze-dried powder to obtain a vaccine solution, and the dosage of each diluent is 2 mL. The vaccine solution prepared by each adjuvant is placed at 4 ℃ for 1h, then samples are taken for determining CCU, PBS buffer solution is used as a control to replace diluent, and the damage to the activity of the live vaccine is observed. As a result: as can be seen from table 2, the mixture of mucosal adjuvant 1 and spray protectant of the present invention did not affect live vaccine viability, the mixture of control adjuvant 2 and spray protectant reduced live vaccine viability by about 1 titer, the mixture of

control adjuvant

3 or 5 and spray protectant did not significantly affect live vaccine viability, and the mixture of control adjuvant 6 and spray protectant reduced live vaccine viability by about 4 titers.

TABLE 2 viability impairment of live vaccines against Mycoplasma hyopneumoniae by mixtures of adjuvants with spray protectants

Atomization effect test and results when the adjuvant is used together with the spray protectant: mixing the adjuvants with still-acceptable compatibility (mucosa adjuvant 1,

contrast adjuvants

2, 3, and 5) with spray protectant at volume ratio of 1:1, and shaking for mixing to obtain each diluent. In the control, PBS buffer was mixed as a diluent with the spray protectant instead of adjuvant. Each diluent is dissolved with 1 bottle of swine mycoplasma pneumonia live vaccine (168 strain) freeze-dried powder to obtain a vaccine solution, and the dosage of each diluent is 2 mL. And (3) carrying out atomization test on the vaccine solution prepared by each adjuvant, adopting an ultrasonic atomizer, carrying out atomization oscillation frequency of 1.7MHz, and after spraying for 5 minutes, collecting aerosol and carrying out particle size determination. The results in table 3 show that when the mucosal adjuvant 1 and the control adjuvant 2 of the present invention are used in combination with a spray protectant, the atomization is normal, and the particle size is not significantly changed; when the control adjuvant 3 is matched with a spray protectant for use, the fog output is reduced, the atomization is slow, the time required by atomization is prolonged to 2-3 times, and the particle size is reduced to a certain extent; when the control adjuvant 5 was used in combination with the spray protectant, atomization was not possible.

Table 3 aerosol spray test of adjuvant-complexed spray protectant for live vaccine of mycoplasma hyopneumoniae

In conclusion, the diluent obtained by mixing the mucosal adjuvant 1 and the spray protectant has good stability, and the diluent and the live vaccine are incubated for 1h together, so that the activity of the vaccine is not influenced, and the good atomization performance is maintained.

In addition, the experiment in this example was also performed on the mucosal adjuvant 2 of the live vaccine of mycoplasma hyopneumoniae (abbreviated as the mucosal adjuvant 2 of the present invention). The mucosa adjuvant 2 of the invention is a solution containing fusion protein CTB-P97R 1300 mu g/ml, Ploy (I: C)200 mu g/ml and saponin 62.5 mu g/ml, and the solvent is PBS buffer solution (10mmol/L, pH7.2-7.4). Experimental results show that the mucosal adjuvant 2 has similar effect with the mucosal adjuvant 1. The diluent obtained by mixing the mucosa adjuvant 2 and the spray protectant has good stability, and the diluent and the live vaccine are incubated for 1h together, so that the activity of the vaccine is not influenced, and the good atomization performance is maintained.

Example 7: detection of mucosa adjuvant for enhancing mucosa immune protection efficacy of swine mycoplasma pneumonia live vaccine

Vaccine: the freeze dried live vaccine (168 strain) for swine mycoplasma pneumonia is prepared with the culture of mycoplasma hyopneumoniae 168 strain obtained through conventional culture medium culture and through freeze drying. Before lyophilization, each flask was filled with 2mL of M.hyopneumoniae 168 strain culture.

Adjuvant: the mucosal adjuvant 2 of the invention is the same as in example 6. Control adjuvant 7: is a solution containing 200 mug/ml of Ploy (I: C) and 62.5 mug/ml of saponin, the solvent is PBS buffer solution (10mmol/L, pH7.2-7.4), and only the fusion protein CTB-P97R1 is lacked, similar to the preparation method of the mucosal adjuvant 1. Control adjuvant 2 and control adjuvant 3 were the same as in example 5.

Animals: and (3) detecting the mycoplasma hyopneumoniae antigen antibody at 7-15 days old as a negative healthy ternary pig.

Preparing a vaccine: each adjuvant in this example was mixed with a spray protectant (same as example 6) at a volume ratio of 1:1 to prepare a diluted solution. Dissolving live vaccine lyophilized powder with each diluent to obtain 2.5 × 10⁷Vaccine solution CCU/ml. In addition, 2.5X 10 was prepared by replacing the diluent with PBS buffer (10mmol/L, pH7.2-7.4)⁷Vaccine solution CCU/ml.

Immunization and toxin counteracting: animals were randomized into 7 groups of 12 animals each. G1 group as negative control groupNo immunity and no toxic attack; g2 group is a non-adjuvant control group, and is a vaccine solution prepared by immunizing PBS buffer solution; the G3-6 groups are respectively a mucosal adjuvant 2 group, a contrast adjuvant 7 group, a contrast adjuvant 2 group and a contrast adjuvant 3 group, and vaccine solutions prepared by immunizing the mucosal adjuvant 2, the contrast adjuvant 7, the contrast adjuvant 2 and the contrast adjuvant 3 respectively. The immunization method is aerosol immunization, and comprises the following specific steps: placing a group of piglets in a piglet atomization box, pouring the vaccine solution into an ultrasonic atomizer for spraying, wherein the atomization oscillation frequency is 1.7MHz, and after atomization is finished, allowing the piglets to continuously stay in the atomization box for 30 minutes. The vaccine immunization amount of each pig is 5 multiplied by 10⁷The CCU. G7 group is control group for counteracting toxic pathogen, and only for counteracting toxic pathogen without immunization. 2 months after immunization, the G2-G7 groups were detoxified by mycoplasma hyopneumoniae JS strain, and the piglets of the G1-G7 groups were subjected to a necropsy 28 days later.

Evaluation of immune indexes: 2 weeks after immunization, 3 animals from each group were collected for anticoagulation and bronchoalveolar lavage fluid (BALF). Separating Peripheral Blood Mononuclear Cells (PBMC) from anticoagulated blood, and detecting the proliferation level of lymphocytes by using mycoplasma hyopneumoniae holomycoprotein and P97R1 protein for stimulation, wherein the specific method refers to Vet J,2014,199: 268-; taking bronchoalveolar lavage fluid (BALF), and detecting the level of a specific sIgA antibody aiming at mycoplasma hyopneumoniae holobacteria and the level of a specific sIgA antibody aiming at P97R1 protein, wherein the specific method is disclosed in Vaccine,2013,31:1305 and 1311.

And (3) toxin attacking protection evaluation: the remaining 9 animals were sacrificed in each group 28 days after challenge, and the lesions in the lungs of the experimental pigs were scored by reference to the method reported by MADEC and KOBISCH (1982) (journal Rech. Portine en France,1982,14:405,412) and the mean value was calculated. The whole lung is divided into 7 lobes, namely, the left heart lobe (LCL), the left tip lobe (LAL), the Left Diaphragm Lobe (LDL), the right heart lobe (RCL), the right tip lobe (RAL), the Right Diaphragm Lobe (RDL) and the accessory lobe (IL). The lesion score per lung was the sum of the dorsal and ventral lesion scores of the 7 lobes described above, with a total score of 28. And (3) dividing each lung lobe into 0-4 minutes according to the area of the injury, wherein the injury is 0 minute, 1-25% of the area injury is 1 minute, 26-50% of the area injury is 2 minutes, 51-75% of the area injury is 3 minutes, and 76-100% of the area injury is 4 minutes. The method for calculating the toxicity attack protection rate (see Vet J,2014,199: 268-: (mean value of challenge control group score-mean value of immunization group score)/(mean value of challenge control group score-mean value of negative control group score) × 100%.

And (3) test results:

as shown in figures 4 and 6, the mucosal adjuvant 2 of the invention can significantly enhance the proliferation response of mycoplasma hyopneumoniae sIgA antibody in BALF and lymphocyte in blood, and has statistical difference compared with a non-adjuvant control group, and the immunostimulation enhancing capability is better than that of 3 control adjuvants. Meanwhile, fig. 5 and 7 show that mucosal adjuvant 2 of the present invention can also enhance sIgA antibody level against P97R1 antigen in BALF and lymphocyte proliferation response in blood, and the effect is also superior to that of 3 control adjuvants. The results show that the mucosal adjuvant 2 can specifically enhance local mucosal immune response and cellular immune response, has good immune stimulation capability, and can further strengthen the immune response to an important protective antigen P97R 1.

As shown in fig. 8, after challenge, lung lesions of animals of mucosal adjuvant 2 groups of the invention were scored as 2.44, and the protection rate against challenge was 91.34%; the lung lesion score of the animal of the adjuvant-free control group is 9.67, and the toxic attack protection rate is only 55.95%, which shows that the mucosal adjuvant 2 can obviously improve the protection effect of the live vaccine. And the other 3 control adjuvant groups have no statistical difference compared with the non-adjuvant control group, and the aerosol immune effect of the swine mycoplasma pneumonia live vaccine cannot be obviously enhanced due to the fact that the differences exist between the adjuvant components, compatibility or atomization performance and the mucosal adjuvant 2 in the invention.

Example 8: comparison of protective efficacy of the live vaccine containing the mucosal adjuvant 2 for the mycoplasma hyopneumoniae with that of the existing live vaccine for the mycoplasma hyopneumoniae

Immunization and toxin counteracting: animals were randomized into 4 groups of 5 animals each. The G1 group is a negative control group, and is not immunized and not attacked; group G2 (immunization group using the technique of the invention) the live vaccine prepared by the mucosal adjuvant 2 of the invention was used for aerosol immunization, the specific method is as follows. Group G3 (panel using prior art immunization) aerosol immunization of live vaccines was performed with prior art. G4 group is control group for counteracting toxic pathogen, and only for counteracting toxic pathogen without immunization. And (3) performing virus challenge with mycoplasma hyopneumoniae JS strain 2 months after immunization, and performing autopsy 28 days after virus challenge. And (3) toxin attacking protection evaluation: the same as in example 7.

The live vaccine and aerosol immunization method prepared by the mucosal adjuvant 2 provided by the invention in the embodiment comprises the following steps of mixing the mucosal adjuvant 2 (same as the embodiment 6) and the spray protective agent (same as the embodiment 6) according to the volume ratio of 1:1 to obtain a diluent. Dissolving the live vaccine (168 strain) of mycoplasma hyopneumoniae in diluent to obtain 2.5 × 10 lyophilized powder⁷Vaccine solution CCU/ml. Spraying with ultrasonic atomizer, wherein the atomization oscillation frequency is 1.7MHz, and the immunization dose per pig is 5 × 10⁷CCU。

The existing mycoplasma hyopneumoniae live vaccine aerosol immunization method (patent 'a mycoplasma hyopneumoniae atomized live vaccine and its preparation and inspection method ZL 201210275639.1'): dissolving lyophilized powder of Mycoplasma hyopneumoniae vaccine (168 strain) in deionized water solution (pH 7.5) containing 50mg/ml glycerol and 1mg/ml polyvinylpyrrolidone to obtain 2.5 × 10⁷Vaccine solution CCU/ml. Spraying with medical lower respiratory tract deposition type sprayer, air compressor pump pressure of 0.2M Pa, and immunization dose of 5 × 10 for each pig⁷CCU。

And (3) test results: as shown in figure 9, the live vaccine of Mycoplasma hyopneumoniae prepared by using the mucosal adjuvant 2 of the invention is used for aerosol immunization according to the method, the lung score is 3, and the protection rate is 87.13%. And the existing aerosol immunization technology of the swine mycoplasma pneumonia live vaccine is used for immunization, the lung score is 10.4, and the protection rate is 50.50%. The embodiment proves that the effect of the live vaccine prepared by the mucosal adjuvant 2 is obviously better than that of the prior art.

Example 9: evaluation of swine mycoplasma pneumonia live vaccine mucous membrane immune protective phase containing mucous membrane adjuvant 2

Vaccine: porcine mycoplasma pneumonia live vaccine (168 strain) freeze-dried powder.

The mucosal adjuvant 2 of the invention is the same as in example 6.

Vaccine formulation: mixing the mucosa adjuvant 2 and the spray protectant (same as the embodiment 6) according to the volume ratio of 1:1 to obtain a diluent; the diluent is adopted to dissolve the freeze-dried powder of the mycoplasma hyopneumoniae vaccine (168 strain) to obtain 2.5 multiplied by 10⁷Vaccine solution CCU/ml. The PBS buffer solution is used for replacing the mucosa adjuvant 2 of the invention to be mixed with the spray protective agent to dissolve the live vaccine to prepare 2.5 multiplied by 10⁷CCU/ml of unadjuvanted control vaccine solution.

Immunization and toxin counteracting: animals were randomly divided into 4 groups of 12 animals each. The G1 group is a negative control group, and is not immunized and not attacked; g2 group is the mucosal adjuvant group of the invention, the vaccine solution prepared by immunizing the mucosal adjuvant 2 of the invention has the same immunization method as the example 7, and the vaccine immunization amount of each pig is 5 multiplied by 10⁷The CCU. The G3 group is a non-adjuvant control group, and the immunization method is the same as the G2 group by immunizing a non-adjuvant control vaccine solution. G4 group is control group for counteracting toxic pathogen, and only for counteracting toxic pathogen without immunization. 9 months after immunization, and 28 days after challenge. And (3) toxin attacking protection evaluation: the same as in example 7.

And (3) test results: as shown in fig. 10, the lung score of the non-adjuvanted control group was 12.67, and the protection rate was only 39.15%. The lung lesion score of the animals with the mucosal adjuvant group is 3.5, the virus attack protection rate reaches 85%, and the live vaccine with the mucosal adjuvant 2 can still provide good virus attack protection effect after 9 months of immunization, which indicates that the protection period of the live vaccine with the mucosal adjuvant 2 can reach 9 months after aerosol mucosal immunization.

SEQUENCE LISTING

<110> agricultural science and academy of Jiangsu province

<120> porcine mycoplasma pneumonia live vaccine mucosal immune adjuvant, preparation method and application thereof

<130> 20180930

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<170> PatentIn version 3.3

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Claims

1. A mixture of immunologic adjuvant and protective agent of mycoplasma hyopneumoniae live vaccine is characterized in that the mixture comprises a mucous membrane immunologic adjuvant and a spray protective agent; the mucosal adjuvant is a solution of a fusion protein CTB-P97R1 containing a cholera toxin B subunit and a mycoplasma hyopneumoniae P97R1 antigen, the adjuvant contains 0.2-2mg/ml of the fusion protein CTB-P97R1, 150-300 mu g/ml of Poly (I: C) and 50-150 mu g/ml of saponin, and the amino acid sequence of the fusion protein CTB-P97R1 is shown as SEQ ID NO: 2; the spray protective agent is an aqueous solution containing 600035-45 g/L of polyethylene glycol, 3-5g/L of cyclodextrin, 18-22g/L of sodium thiosulfate and 18-22g/L of L-histidine, and the pH value is 7.0-7.5.

2. The mixture according to claim 1, wherein the fusion protein CTB-P97R1 is prepared by the following method: fusing the R1 region gene of the mycoplasma hyopneumoniae adhesion factor P97 to the C end of a cholera toxin B subunit gene, and filling the fused gene into a pET-28a (+) plasmid to construct a recombinant plasmid pET28a-CTB-P97R 1; introducing the recombinant plasmid into escherichia coli to obtain a recombinant bacterium; and (3) inducing the recombinant bacteria to express CTB-P97R1 by IPTG, collecting the supernatant of the bacterial lysate, and purifying to obtain the fusion protein CTB-P97R 1.

3. The mixture according to claim 2, wherein the saponin is quillaja saponin, ginsenoside or notoginsenoside.

4. The method for preparing the mixture according to claim 1, wherein a phosphate buffer solution is used to prepare 10mg/ml of a mother solution of the fusion protein CTB-P97R 1; preparing 1mg/ml Poly (I: C) mother liquor and 1mg/ml saponin mother liquor, wherein the solvents are phosphate buffer solution; and mixing the fusion protein CTB-P97R1 mother liquor, the Poly (I: C) mother liquor and the saponin mother liquor, and diluting by adopting a solvent to obtain the porcine mycoplasma pneumonia live vaccine mucosal adjuvant.

5. Use of a mixture according to claim 1 for the preparation of a live vaccine against mycoplasma hyopneumoniae.

6. The application of claim 5, wherein the mucosal adjuvant is mixed with the spray protectant according to the volume ratio of 1:1, and then the mycoplasma hyopneumoniae vaccine strain culture or the freeze-dried powder is added and mixed uniformly to obtain the live vaccine solution for mycoplasma hyopneumoniae.