CN109200284A - A kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications - Google Patents

A kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications Download PDF

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CN109200284A
CN109200284A CN201811160766.0A CN201811160766A CN109200284A CN 109200284 A CN109200284 A CN 109200284A CN 201811160766 A CN201811160766 A CN 201811160766A CN 109200284 A CN109200284 A CN 109200284A
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adjuvant
live vaccine
mycoplasmal pneumonia
porcine mycoplasmal
vaccine
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CN109200284B (en
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熊祺琰
张珍珍
邵国青
冯志新
韦艳娜
王佳
白昀
甘源
张磊
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Jiangsu Academy of Agricultural Sciences
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Abstract

The present invention provides a kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications, is related to vaccine veterinary art.The mucosal adjuvant is the solution of the recombinant protein c TB-P97R1 expressed containing choleratoxin B subunit and mycoplasma hyopneumoniae P 97 R 1 Antigen Fusion.The present invention also provides the preparation methods of the mucosal adjuvant, prepare 10mg/ml recombinant protein c TB-P97R1 mother liquor, are then diluted using solvent.The present invention also provides application of the Mucosal Adjuvants in terms of preparing porcine mycoplasmal pneumonia live vaccine.Mucosal adjuvant of the present invention is aqueous compound adjuvant; preparation method is easy; adjuvant properties are stablized; it is not damaged to mycoplasma hyopneumoniae vigor; it can be compatible with aerosol protective agent; and mixture is still not damaged to mycoplasma hyopneumoniae vigor, is suitble to live vaccine aerosol immunity, is remarkably improved the mucosa-immune Vaccine effectiveness of vaccine using the adjuvant, extends immune duration.

Description

A kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications
Technical field
The present invention relates to vaccine veterinary arts, and in particular to a kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, system Preparation Method and its application.
Background technique
Porcine mycoplasmal pneumonia is one kind caused by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) The chronic respiratory communicable disease of pig, cardinal symptom are cough and asthma.Mainly cause the feed conversion rate decline and life of pig Length is obstructed, and disease incidence is high, the death rate is low.Cilium is destroyed after mycoplasma hyopneumoniae infection, leads to other respiratory tract bacteriums and virus It is easy to happen secondary infection, to cause more serious epidemic situation.The disease is worldwide widely current, and gives Pig Industry industry Cause heavy economic losses.
Porcine mycoplasmal pneumonia is a kind of intractable chronic infectious disease, and medication effect is not ideal enough, is easy after drug withdrawal multiple Hair, vaccine prevention play critical effect in control porcine mycoplasmal pneumonia.Currently, both at home and abroad mainly using inactivated vaccine into Row prevention, but since mycoplasma hyopneumoniae immunogenicity is weak, inactivated vaccine Vaccine effectiveness is often bad, and being only capable of mitigation symptom cannot Pathogen infection, and higher cost are blocked, actual needs can not be fully met.Recombinant vaccine is still in conceptual phase, It there is no launch.Live vaccine can of short duration proliferation in vivo, have that dosage of inoculation is small, immunogenicity is good, immune effect is lasting Advantage.
Studies in China person (China Veterinery Drug Inspection Office, Jiangsu Province Agriculture Science Institute) is in porcine mycoplasmal pneumonia live vaccine A large amount of and valuable work are done in exploitation, the live vaccine immune efficacy developed is better than inactivated vaccine, and cost is relatively It is low, there is the very strong market competitiveness.Live vaccine can stick field planting after being immunized by respiratory mucosa at bronchus cilium, Occupation time process is formed, the infection of clinical wild poison is effectively prevented, with the incomparable uniqueness of inactivated vaccine and recombinant vaccine Advantage.But live vaccine uses pulmonary injection immunization route at present, affects being widely popularized for vaccine to a certain extent.Aerosol Immune, Nasal immunization etc., it is immune again by respiratory mucosa approach, it is very potential alternative.Mucosa-immune System is the first line of defence that body resists foreign pathogen invasion, but relatively fewer by the vaccine of mucosa-immune at present, It is related to be largely intended to generation immune tolerance with the physilogical characteristics of mucosa.For the mucous membrane for further enhancing live vaccine Immunostimulatory potency adds into vaccine and the adjuvant of mucosa-immune is suitble to be effective means.
In the prior art, there are no the effective mucosal adjuvants for being directed to porcine mycoplasmal pneumonia live vaccine.To find out its cause, main There are following difficulties: first, the research of Mucosal Adjuvants is still extremely lacked at present, is largely answered by mucosa-immune The unsharp influence of research is answered, most adjuvant components are unclear in using effect, the action principle of mucosa, to assistant Agent selection brings comparable difficulty.Demand need to be immunized according to mycoplasma hyopneumoniae, using animal experiment to can effective activation office Portion's mucosa-immune and the adjuvant component of cellular immunity are screened；Second, for bacterium class live vaccine, in terms of adjuvant selection Also there is specific requirement, since oily substance is to the destructiveness of cell membrane, Water-In-Oil class adjuvant is often not suitable for, and even The requirement of oil-in-water class and two-phase adjuvant to its stability and preparation process is also very high, can pay the utmost attention to using aqueous adjuvants, benefit It is screened with external compatibility test；Third, porcine mycoplasmal pneumonia live vaccine carry out needing that spray is added when aerosol immunity Mist protective agent guarantees the activity of the live vaccine in atomization process, but spraying protective agent is generally special macromolecule component, and part is helped Agent cannot be with spraying protective agent with the use of (including two due to chemical property itself, solvent for use, ion concentration, pH etc. Physicochemical property is unstable, mixture has damage mycoplasma vigor, mixture cannot play original spraying protection after person's mixing Effect, mixture cannot play original adjuvanticity), therefore selected adjuvant must prove its energy by a series of experiments It is used cooperatively with spraying protective agent, is just able to satisfy the use demand of porcine mycoplasmal pneumonia aerosol live vaccine.
Summary of the invention
It is a primary object of the present invention to disclose a kind of pair of porcine mycoplasmal pneumonia live vaccine vigor it is undamaged, can be molten with gas Glue is sprayed that protective agent is used cooperatively, can enhancing oromucosal route immunostimulatory potency and can strengthen important protective antigens The Mucosal Adjuvants of the porcine mycoplasmal pneumonia live vaccine of P97R1 immune response.
It is a further object to provide the preparation method of above-mentioned Mucosal Adjuvants, this method is simple and efficient.
Another object of the present invention is to provide the application of above-mentioned Mucosal Adjuvants.
A kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, the mucosal adjuvant are containing choleratoxin B subunit With the solution of the recombinant protein c TB-P97R1 of mycoplasma hyopneumoniae P 97 R 1 Antigen Fusion expression, the adjuvant contains 0.2-2mg/ Ml recombinant protein c TB-P97R1.
In the present invention, the amino acid sequence of the recombinant protein c TB-P97R1 is as shown in SEQ ID NO:2.
In the present invention, the recombinant protein c TB-P97R1 is prepared with the following method: by mycoplasma hyopneumoniae stick because The area the R1 Gene Fusion of sub- P97 is fitted into building recombination matter in pET-28a (+) plasmid to the C-terminal of Cholera Toxin B Subunit Gene Grain pET28a-CTB-P97R1；The recombinant plasmid is imported into Escherichia coli, obtains recombinant bacterium；Using IPTG induction recombinant bacterium expression CTB-P97R1, collects cellular lysate liquid supernatant, and purifying obtains recombinant protein c TB-P97R1.
In preferred technical solution, the Mucosal Adjuvants also contain 150-300 μ g/ml Poly (I:C) and 50-150 μ g/ml saponin(e.
In the present invention, the saponin(e is quillaja saponin, ginsenoside or notoginsenoside.
The present invention also provides the preparation methods of the porcine mycoplasmal pneumonia live vaccine mucosal adjuvant, prepare 10mg/ml recombination Then PROTEIN C TB-P97R1 mother liquor is diluted using solvent, the solvent is phosphate buffer.
In preferred technical solution, Poly (I:C) mother liquor and 1mg/ml saponin(e mother liquor, solvent for preparing 1mg/ml are phosphorus Phthalate buffer；After the recombinant protein c TB-P97R1 mother liquor, Poly (I:C) mother liquor and saponin(e mother liquor are mixed, use is molten Dilution agent.
The present invention also provides the porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants to prepare porcine mycoplasmal pneumonia epidemic disease living Application in terms of seedling.
In preferred technical solution, the adjuvant is mixed with spraying protective agent according to volume ratio for 1:1, pig is then added Mycoplasma pneumoniae vaccine strain culture or freeze-dried powder are uniformly mixed, obtain porcine mycoplasmal pneumonia live vaccine solution.
In this application, the spraying protective agent be 35-45g/L containing Macrogol 6000, it is cyclodextrin 3-5g/L, thio The aqueous solution of sodium sulphate 18-22g/L and L-Histidine 18-22g/L, pH 7.0-7.5.
Mucosal adjuvant of the present invention has the beneficial effect that: first, this mucosal adjuvant is aqueous compound adjuvant, preparation method letter Just, adjuvant properties are stablized, and selected ingredient is not damaged to mycoplasma hyopneumoniae vigor, are suitble to live vaccine to use, and to animal Safety, Small side effects；Second, mucosal adjuvant of the present invention can be compatible with aerosol protective agent, and mixed mixture is to pig Mycoplasma pneumoniae vigor is still not damaged, is suitble to live vaccine aerosol immunity；Third, mucosal adjuvant of the present invention contain CTB- P97R1 recombinant protein (playing adjuvant effect is the part CTB), Poly (I:C) and saponin(e fully utilize respective immune tune The compound adjuvant of section advantage, formation can effectively activate local mucosal immune response and cellular immunity to answer by the way that oromucosal route is immune It answers, meets the demand of mycoplasma hyopneumoniae immunoprotection；4th, mucosal adjuvant of the present invention contains CTB-P97R1, by pig pneumonia branch The important protective antigens P97R1 of substance is fused to the C-terminal of CTB, is conducive to further supplement spectrotype, and strengthening vaccine is immunized Effect.
The present invention by a series of internal and external test, have developed it is avirulent to porcine mycoplasmal pneumonia live vaccine, can be with It is that aerosol spray protective agent is used cooperatively, can under mucosal immune effective stimulus immune system, while can specificity The dedicated Mucosal Adjuvants of porcine mycoplasmal pneumonia live vaccine for enhancing important antigen P97R1 immune response, can be shown using the adjuvant It writes and improves the mucosa-immune Vaccine effectiveness of vaccine, extends immune duration.
Detailed description of the invention
The PCR of Fig. 1 recombinant plasmid pET28a-CTB-P97R1 identifies, wherein swimming lane 1:DNA molecular weight standard；Swimming lane 2: sun Property control (P97 genetic fragment)；Swimming lane 3: negative control；Swimming lane 4: using recombinant vector pET28a-CTB-P97R1 as template with P97R1-up and P97R1-down is the amplified production of primer.
The SDS-PAGE electrophoresis of the fusion protein CTB-P97R1 of Fig. 2 after purification, wherein swimming lane 1: protein molecular weight mark Standard, swimming lane 2: fusion protein CTB-P97R1 after purification.
Fig. 3 shows the ELISA testing result of various concentration fusion protein CTB-P97R1 combination GM1 ability, and abscissa is The additional amount of fusion protein CTB-P97R1 in every hole, unit are the holes ng/.
Fig. 4 mycoplasma hyopneumoniae whole bacterial protein antigenic stimulus lymphopoiesis response, wherein " * * " is indicated and no adjuvant Control group is extremely significant compared to difference, P < 0.01.
Fig. 5 mycoplasma hyopneumoniae P 97 R 1 albumen stimulates lymphopoiesis response, wherein " * * " is indicated and no vehicle control Group is extremely significant compared to difference, P < 0.01.
Fig. 6 is directed to the specific sIgA antibody level of the full bacterium of mycoplasma hyopneumoniae, wherein " * " is indicated and no adjuvant control group Compared to significant difference, P < 0.05；" * * " indicates that difference is extremely significant compared with no adjuvant control group, P < 0.01.
Fig. 7 is directed to the specific sIgA antibody level of mycoplasma hyopneumoniae P 97 R 1 albumen, wherein " * * " is indicated and no adjuvant Control group is extremely significant compared to difference, P < 0.01.
Fig. 8 each group attacks the Lung score index after poison, and the percentage in figure is the protective rate of each group, wherein " * * " indicates lung Dirty Scoring Index difference compared with no adjuvant control group is extremely significant, P < 0.01.
The Vaccine effectiveness of each aerosol vaccine of Fig. 9 compares, wherein " * * " indicates that difference is extremely significant compared with attacking malicious control group, P<0.01。
Figure 10 is shown attack poison after groups of animals Lung score situation and protective rate, wherein " * * " indicate and no adjuvant pair According to group than significant difference, P < 0.01, wherein percentage is the protective rate of each group.
Specific embodiment
The present invention is described further in conjunction with specific embodiments and demonstration, but content included by the present invention is not It is confined to this.
Embodiment 1: the building of recombination engineering BL21-pET28a-CTB-P97R1
In order to screen the Mucosal Adjuvants of porcine mycoplasmal pneumonia live vaccine, a variety of fusion proteins are devised.Through overtesting, Only discovery fusion protein CTB-P97R1 has preferable immune effect.The amino acid sequence of fusion protein CTB-P97R1 such as SEQ Shown in ID NO:2, gene order such as SEQ ID NO:1.
The present embodiment describes the recombination engineering bacteria BL21-pET28a-CTB- for being used to prepare fusion protein CTB-P97R1 The building process of P97R1.
According to the gene order (AY307389.1) for the choleratoxin B subunit (CTB) included in GenBank, using base Because the mode of synthesis obtains the CTB gene (particular sequence is shown in SEQ ID NO:3) of removal signal peptide moiety, and distinguish at both ends Add Pag I and I restriction enzyme site of Nhe.It will be purified after above-mentioned composition sequence double digestion, with Nco I (Pag I be with Nco I isocaudarner) PET-28a (+) carrier with I double digestion of Nhe and after purification is connected with 4 DEG C of T4-DNA ligase overnight enzymes, connection product conversion BL21 (DE3) competent cell, picked clones, with I digestion of Nco and DNA sequencing method screening positive clone.Positive colony is drawn Line, simultaneously repeated authentication is primary for picking monoclonal, as a result proves that the monoclonal obtained has successfully imported carrying CTB gene (removal letter Number peptide) recombinant vector pET28a-CTB, preparation glycerol tube saves.
According to mycoplasma hyopneumoniae P97 gene order (U50901) in GenBank, design primer is with mycoplasma hyopneumoniae 168 plants of total DNAs are the area R1 of template amplification P97 gene.Wherein, upstream primer P97R1-up (being added to NheI restriction enzyme site) Sequence is as follows: 5 '-AAAGCTAGCTTACCTCAGCCGCCAGCA-3 '；Downstream primer P97R1-down (is added to XhoI digestion Site) sequence it is as follows: 5 '-TTTCTCGAGAGCCATTGGGAAATAGTCTT-3 '.PCR reaction condition are as follows: 94 DEG C of initial denaturations 10 minutes；94 DEG C are denaturalized 1 minute, and 54 DEG C are annealed 1 minute, and 72 DEG C extend 30 seconds, 30 circulations；72 DEG C extend 10 minutes.PCR is produced Object Nhe I and XhoI double digestion are connect with using the pET28a-CTB recombinant vector of same method digestion with T4-DNA after purification 4 DEG C of enzyme overnight enzymes connect, and connection product converts BL21 (DE3) competent cell, picked clones, with above-mentioned PCR method (see Fig. 1) and DNA sequencing method screening positive clone.Positive colony is crossed, picking monoclonal is primary using same procedure repeated authentication, knot Fruit proves that the monoclonal obtained has successfully imported the recombinant vector (life of carrying CTB gene (removal signal peptide) and the area P97 gene R1 Entitled pET28a-CTB-P97R1 recombinant vector), which is named as BL21-pET28a-CTB-P97R1, prepares glycerol Pipe saves.
Embodiment 2: the expression and purification of fusion protein CTB-P97R1
Positive restructuring bacterium BL21-pET28a-CTB-P97R1 is vibrated using LB culture medium (kalamycin resistance) at 37 DEG C Overnight incubation.Bacterium solution is inoculated in LB (kalamycin resistance) fluid nutrient medium by next day in 2% ratio, and 37 DEG C of shaken cultivations are extremely OD600=0.4-0.6, the isopropyl-β-D-thiogalactoside (IPTG) that final concentration of 1.0mmol/L is added carry out induction table It reaches, 4h collects bacterium solution after induction, and centrifuging and taking bacterial sediment is washed 1 time with PBS.Thallus is suspended in PBS buffer solution, ultrasound Wave cracking, 10000rpm is centrifuged 30min at 4 DEG C, takes cracking supernatant His-Tag affinity chromatography column separating purification fusion egg It is white, 2 days desalinations of dialysing, freeze-drying.With the purity of 15%SDS-PAGE electrophoretic analysis fusion protein CTB-P97R1.It can from Fig. 2 To see, fusion protein CTB-P97R1 purity after purification is consistent up to 90%, molecular weight 21.5kDa with expection.
The Bioactivity of 3 fusion protein CTB-P97R1 of embodiment detects
Method: using Ganglioside GM1-Enzyme-linked Immunosorbent Assay (ELISA) testing inspection fusion protein CTB-P97R1 with The binding ability of Ganglioside GM1.It (is containing 35mmol/L that 96 hole elisa plates, which use the carbonate buffer solution containing 5 μ g/ml GM1, NaHCO3、15mmol/L Na2CO3Aqueous solution) 4 DEG C of coatings overnight, every 100 μ l of hole；PBST (the PBS containing 0.1%Tween-20 Buffer) washing 3 times, with the PBS solution containing 1%BSA in 37 DEG C of closing 2h, after PBST is washed 3 times, every hole is added 100 μ l and contains There is the solution of 500ng CTB-P97R1 (after purification), 37 DEG C of incubation 2h boil the CTB- of 10min processing in control with 100 DEG C P97R1 solution substitution.After PBST is washed, addition is the diluted rabbit-anti CTB antibody (U.S. 1:4000 according to dilution with PBS Bio-Rad company), 37 DEG C of incubation 1h.After PBST is washed 3 times, addition is the diluted horseradish mistake of 1:8000 according to dilution with PBS The goat anti-rabbit igg (Wuhan doctor's moral biotech firm) of oxide enzyme label, 37 DEG C of incubation 1h.100 μ l substrates are added after washing TMB terminates reaction with the aqueous sulfuric acid of 50 μ l 2mol/L, detects absorbance value under 450nm.In addition, using phase Tongfang Method investigates the ability of various concentration CTB-P97R1 combination GM1.
As a result: as shown in figure 3, fusion protein CTB-P97R1 can in conjunction with GM1, with the reduction of CTB-P97R1 concentration, It also declines in conjunction with GM1, therefore, fusion protein CTB-P97R1 can and GM1 combination have dose dependent.The above results The fusion protein CTB-P97R1 for proving prepared by embodiment 2 has the similar external activity of natural CTB.
The preparation of 4 porcine mycoplasmal pneumonia live vaccine mucosal adjuvant of embodiment
Fusion protein CTB-P97R1 mother liquor, Poly (I:C) mother liquor, saponin(e mother liquor are prepared respectively, then above-mentioned mother liquor is mixed It closes, is diluted to required concentration with phosphate buffer up to porcine mycoplasmal pneumonia live vaccine mucosal adjuvant
The preparation of each mother liquor: (1) preparation of fusion protein CTB-P97R1 mother liquor: using method preparation purifying in embodiment 2 Fusion protein CTB-P97R1 afterwards prepares 10mg/ml's with phosphate buffer (10mmol/L, pH7.2-7.4) for solvent Fusion protein CTB-P97R1 mother liquor；(2) preparation of Poly (I:C) mother liquor: suitable Poly (I:C) (Beijing Saden biology difficult to understand is taken Science and Technology Ltd., ProductName: VDN) it is dissolved in phosphate buffer (10mmol/L, pH7.2-7.4), prepare 1mg/ml's Poly (I:C) mother liquor；(3) preparation of saponin(e mother liquor: suitable quillaja saponin (QS-21) is taken to be dissolved in phosphate buffer In (10mmol/L, pH7.2-7.4), the saponin(e mother liquor of 1mg/ml is prepared.
The preparation of porcine mycoplasmal pneumonia live vaccine mucosal adjuvant: when being prepared by taking 100ml adjuvant as an example, 10mg/ml is taken Recombinant protein c TB-P97R1 mother liquor it is appropriate, while take 1mg/ml Poly (I:C) mother liquor and 1mg/ml saponin(e mother liquor it is mixed Close, with phosphate buffer carries out centainly dilute to get into every ml solution containing fusion protein CTB-P97R1 0.2-2mg, The porcine mycoplasmal pneumonia live vaccine mucosal adjuvant of Poly (I:C) 150-300 μ g, saponin(e 50-150 μ g.
5 adjuvant of embodiment measures the Active lesions of porcine mycoplasmal pneumonia live vaccine
Vaccine: porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder is 168 plants of mycoplasma hyopneumoniae using routine culture The culture that base culture obtains is lyophilized.Per it is bottled enter 168 plants of cultures of 2mL mycoplasma hyopneumoniae be lyophilized.
Prepare following adjuvant respectively: porcine mycoplasmal pneumonia live vaccine mucosal adjuvant 1 (is abbreviated as mucosal adjuvant 1 of the present invention): It is the solution containing 2mg/ml fusion protein CTB-P97R1,300 μ g/ml Poly (I:C) and 150 μ g/ml saponin(es, solvent is PBS buffer solution (10mmol/L, pH7.2-7.4) is prepared according to method in embodiment 4.Compare adjuvant 1:Montanide IMS 251C (SEPPIC Products) is prepared referring to product description.Compare the astragalus polyose solution of adjuvant 2:100mg/ml, solvent For PBS buffer solution.Control adjuvant 3: it takes appropriate CARBOPOL 974P powder (Lubrizol Corp.'s product) to be dissolved in distilled water, fills it Swelling is divided to obtain the carbomer mother liquor of 15mg/ml；The pH value for adjusting carbomer mother liquor using sodium hydrate aqueous solution adds to 7.2 Appropriate PBS buffer solution (10mmol/L, pH7.2-7.4) makes the final concentration of 2mg/ml of carbomer, obtains control adjuvant 3.Control assistant Agent 4:Montanide ISA 206 (SEPPIC Products) is prepared referring to product description.Compare adjuvant 5:Montanide GEL (SEPPIC Products) is configured to the solution that mass percentage is 15% referring to product description.Compare adjuvant 6: The hyaluronic acid solution of 2mg/ml, solvent are PBS buffer solution (10mmol/L, pH7.2-7.4).
Toxicity detection: dissolving 1 bottle of porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder with above-mentioned each adjuvant respectively, and every bottle The adjuvant volume being added in freeze-dried powder is 2ml, is sampled after 4 DEG C of placement 1h, and (color change unit, color change measurement CCU Become unit), three holes of operation repetitive；1 bottle of porcine mycoplasmal pneumonia live vaccine (168 is dissolved with PBS buffer solution substitution adjuvant simultaneously Strain) freeze-dried powder, as control, detection CCU.The difference for comparing CCU judges adjuvant to the Active lesions situation of live vaccine.
Experimental result: as seen from Table 1, after being incubated for 1h, compared with PBS buffer solution, mucosal adjuvant 1 of the present invention does not influence epidemic disease living Seedling vigor, CCU is without significant change.Control adjuvant 2, control adjuvant 3, control adjuvant 5 and control adjuvant 6 also not appreciably affect epidemic disease living Seedling vigor, CCU is without significant change；And compare adjuvant 1 and control 4 groups of each holes of adjuvant it is non-discolouring, CCU 0 illustrates to live vaccine Vigor has damage very serious.Above-mentioned test result explanation, work of the mucosal adjuvant 1 of the present invention to porcine mycoplasmal pneumonia live vaccine Power is not damaged.
Each adjuvant of table 1 damages measurement (CCU) to porcine mycoplasmal pneumonia live vaccine vigor
Embodiment 6: the spraying protective agent of adjuvant cooperation is used to prepare the aerosol spray of porcine mycoplasmal pneumonia live vaccine
Vaccine: porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder is 168 plants of mycoplasma hyopneumoniae using routine culture The culture that base culture obtains is lyophilized.Before freeze-drying, 2mL culture is housed in every bottle.
Adjuvant: mucosal adjuvant 1 of the present invention, control adjuvant 2, control adjuvant 3, control adjuvant 5, control adjuvant 6, same to embodiment 5。
Spraying protective agent: being 40g/L containing Macrogol 6000, cyclodextrin 4g/L, sodium thiosulfate 20g/L, L-Histidine The aqueous solution (pH value 7.2) of 20g/L.
The stability test and result that adjuvant and spraying protective agent are used cooperatively: by adjuvant each in the present embodiment and spraying guarantor Protecting agent, 1:1 is mixed by volume, and oscillation mixes.Every kind of mixed solution, while 4 DEG C and 37 DEG C are placed in, it is seen whether out in 2 weeks Now precipitate.As a result: it places 2 weeks control adjuvants 2 and spraying protectant mixture for 37 DEG C and turbidity and precipitation phenomenon occurs, remaining adjuvant Protectant mixture appearance is normal by spraying.
Adjuvant and spraying protective agent are used cooperatively to the damage measurement of live vaccine vigor and result: will respectively be helped in the present embodiment 1:1 is mixed by volume with spraying protective agent for agent, and oscillation mixes, and obtains each dilution.It is former that every kind of dilution dissolves 1 bottle of pig branch Body pneumonia live vaccine (168 plants) freeze-dried powder obtains vaccine solution, and the dosage of each dilution is 2mL.Each adjuvant is prepared and is obtained Vaccine solution in 4 DEG C of placement 1h, CCU is then measured by sampling, using PBS buffer solution substitution dilution as control, observation to work The damage of vaccine vigor.As a result: as can be seen from Table 2, mucosal adjuvant 1 of the invention and spraying protectant mixture will not shadows Live vaccine vigor is rung, control adjuvant 2 and spraying protectant mixture can make live vaccine decline about 1 titre, compare adjuvant 3 Or 5 have no significant effect live vaccine vigor with spraying protectant mixture, control adjuvant 6 and spraying protectant mixture meeting So that live vaccine declines about 4 titres.
Each adjuvant of table 2 and spraying protectant mixture damage the vigor of porcine mycoplasmal pneumonia live vaccine
Atomizing effect test and result when adjuvant and spraying protective agent are used cooperatively: by the fine adjuvant of above-mentioned compatibility (mucosal adjuvant 1 of the invention, control adjuvant 2,3,5) 1:1 mix by volume with spraying protective agent respectively, and oscillation mixing obtains To each dilution.In control, mixed using PBS buffer solution substitution adjuvant with spraying protective agent as dilution.Every kind of dilution is equal It dissolves 1 bottle of porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder and obtains vaccine solution, the dosage of each dilution is 2mL.It will be each Adjuvant prepares the vaccine solution obtained and carries out atomization measure, and using ultrasonic atomizer, atomization frequency of oscillation is 1.7MHz, and by spraying 5 After minute, collects aerosol and carry out particle size determination.The results show that mucosal adjuvant of the present invention 1 and the control cooperation spray of adjuvant 2 in table 3 Mist protective agent is in use, atomization is normal, and partial size is without substantially changeing；Compareing adjuvant 3 cooperates spraying protective agent in use, fog-supplying amount subtracts Few, atomization slowly, extends to 2-3 times the time required to atomization, and partial size has and reduces to a certain degree；Control adjuvant 5 cooperates spraying protection Agent is in use, can not be atomized.
3 adjuvant of table cooperates aerosol spray of the spraying protective agent for porcine mycoplasmal pneumonia live vaccine to test
In conclusion the dilution that mucosal adjuvant 1 of the invention is mixed to get with spraying protective agent has good stability, this is dilute It releases liquid and live vaccine is incubated for 1h jointly, do not influence vaccine vigor, while maintaining good atomization.
In addition, also carry out this reality to porcine mycoplasmal pneumonia live vaccine mucosal adjuvant 2 (being abbreviated as mucosal adjuvant 2 of the present invention) Apply the experiment in example.Mucosal adjuvant 2 of the present invention is 300 μ g/ml of CTB-P97R1 containing fusion protein, 200 μ g/ of Ploy (I:C) The solution of ml, 62.5 μ g/ml of saponin(e, solvent are PBS buffer solution (10mmol/L, pH7.2-7.4).Experimental result discovery, this hair Bright mucosal adjuvant 2 is similar to 1 effect of mucosal adjuvant of the present invention.Mucosal adjuvant 2 of the invention is mixed to get with spraying protective agent Dilution have good stability, the dilution and live vaccine are incubated for 1h jointly, do not influence vaccine vigor, while maintaining good Atomization.
Embodiment 7: the detection of mucosal adjuvant enhancing porcine mycoplasmal pneumonia live vaccine mucosa-immune Vaccine effectiveness
Vaccine: porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder is 168 plants of mycoplasma hyopneumoniae using routine culture The culture that base culture obtains is lyophilized.Before freeze-drying, there are 168 plants of cultures of 2mL mycoplasma hyopneumoniae per bottled.
Adjuvant: mucosal adjuvant 2 of the present invention is the same as embodiment 6.Control adjuvant 7: being containing 200 μ g/ml Ploy (I:C) and 62.5 μ The solution of g/ml saponin(e, solvent is PBS buffer solution (10mmol/L, pH7.2-7.4), with 1 preparation method of mucosal adjuvant of the present invention It is similar, only lack fusion protein CTB-P97R1 ingredient.Adjuvant 2, control adjuvant 3 are compareed with embodiment 5.
Animal: 7-15 age in days mycoplasma hyopneumoniae antigen and antibody is negative healthy ternary pigs.
Vaccine formulation: by adjuvant each in the present embodiment, 1:1 is mixed by volume with spraying protective agent (with embodiment 6) respectively It closes, prepares dilution.Live vaccine freeze-dried powder, preparation 2.5 × 10 are dissolved respectively with each dilution7The vaccine solution of CCU/ml.Separately Outside, dilution, preparation 2.5 × 10 are substituted with PBS buffer solution (10mmol/L, pH7.2-7.4)7The vaccine solution of CCU/ml.
It is immunized and attacks poison: animal is randomly divided into 7 groups, every group 12.G1 group is negative control group, not immune not attack poison； G2 group is no adjuvant control group, and vaccine solution made of PBS buffer solution is immunized；G3-6 group 2 groups of mucosal adjuvant respectively of the present invention, 3 groups of adjuvant of 7 groups of adjuvant, 2 groups of adjuvant of control and control are compareed, respectively immune mucosal adjuvant 2 of the present invention, control adjuvant 7, control assistant Vaccine solution made of agent 2 and control adjuvant 3.Immunization method is aerosol immunity, specific as follows: one group of piglet is placed in son In pig atomization box, vaccine solution is poured into ultrasonic atomizer and is sprayed, and atomization frequency of oscillation is 1.7MHz, after to be atomized Piglet is allowed to continue to stop 30 minutes in atomization box.The vaccine immunity amount of every pig is 5 × 107CCU.G7 group is to attack malicious control Group, it is not immune only to attack poison.2 months after immune, G2-G7 group attacks poison using mycoplasma hyopneumoniae JS plants, young to G1-G7 group after 28 days Pig carries out dissect.
Immune indexes evaluation: 2 weeks after immune, each group takes 3 animal acquisition anticoagulations and BAL fluid (BALF).Separating peripheral blood mononuclear cells (PBMC) from anticoagulation, with mycoplasma hyopneumoniae whole bacterial protein and P97R1 albumen Stimulation detection lymphopoiesis is horizontal, and specific method refers to Vet J, 2014,199:268-274；Take bronchoalveolar lavage Liquid (BALF), specificity of the detection for the specific sIgA antibody level of the full bacterium of mycoplasma hyopneumoniae and for P97R1 albumen SIgA antibody level, specific method are disclosed in Vaccine, and 2013,31:1305-1311.
It attacks poison protection evaluation: attacking after poison 28 days, each group puts to death remaining 9 animals, referring to MADEC and KOBISCH (1982) method (Journees Rech.Porcine en France, 1982,14:405-412) reported is to experiment pig lung Damage score, calculate average value.The left lobus cardiacus (LCL) of entire lungs point, left sharp leaf (LAL), left lobus diaphragmaticus (LDL), the right heart Leaf (RCL), right sharp leaf (RAL), right lobus diaphragmaticus (RDL) and accessory lobes (IL), totally 7 lobes of the lung.The injury scores of each lungs are above-mentioned 7 The sum of dorsal surface and facies ventralis lesion scores of a lobe of the lung, total score are 28 points.Each lobe of the lung beats 0-4 according to the area of damage respectively Point, wherein not damaged is 0 point, the damage of 1-25% area is 1 point, and the damage of 26-50% area is 2 points, and the damage of 51-75% area is 3 points, the damage of 76-100% area is 4 points.Attack malicious protective rate calculation method (see Vet J, 2014,199:268-274) are as follows: (attack Malicious control group scoring mean value-immune group scoring mean value)/(attacking malicious control group scoring mean value-negative control group scoring mean value) × 100%.
Test result:
As shown in Figure 4 and Figure 6, mucosal adjuvant 2 of the present invention can significantly increase in BALF mycoplasma hyopneumoniae sIgA antibody and Lymphopoiesis response in blood all has statistical difference with no adjuvant control group ratio, and it is excellent that immunostimulation enhances ability In 3 control adjuvants.Meanwhile Fig. 5 and Fig. 7 shows that mucosal adjuvant 2 of the present invention can also enhance in BALF for P97R1 antigen Lymphopoiesis response in sIgA antibody level and blood, effect are also better than 3 control adjuvants.The above results suggest that Mucosal adjuvant 2 of the present invention specific can enhance local mucosal immune response and cellullar immunologic response, have good immune thorn The ability of swashing, and can further strengthen the immune response for important protective antigens P97R1.
As shown in figure 8, the lung lesion scoring of 2 groups of animals of mucosal adjuvant of the present invention is 2.44 points after attacking poison, malicious protection is attacked Rate is 91.34%；The lung lesion scoring of no adjuvant control group animal is 9.67 points, and attacking malicious protective rate is only 55.95%, explanation Mucosal adjuvant 2 of the present invention can significantly improve the Vaccine effectiveness of live vaccine.And remaining 3 control adjuvant group scorings and no vehicle control Group compares no difference of science of statistics, may be deposited in adjuvant component, compatibility or atomization with mucosal adjuvant 2 of the present invention due to it Lead to the aerosol immunity effect for failing to significantly increase porcine mycoplasmal pneumonia live vaccine in certain gap.
Embodiment 8: porcine mycoplasmal pneumonia live vaccine and existing porcine mycoplasmal pneumonia live vaccine containing mucosal adjuvant 2 of the present invention The comparison of Vaccine effectiveness
Animal: 7-15 age in days mycoplasma hyopneumoniae antigen and antibody is negative healthy ternary pigs.
It is immunized and attacks poison: animal is randomly divided into 4 groups, every group 5.G1 group is negative control group, not immune not attack poison；G2 Group (applying the technology of the present invention immune group) carries out aerosol immunity, specific method with live vaccine prepared by mucosal adjuvant 2 of the present invention It sees below.G3 group (applying prior art immune group) carries out the aerosol immunity of live vaccine with the prior art.G4 group is to attack malicious control Group, it is not immune only to attack poison.Poison is attacked with JS plants of mycoplasma hyopneumoniae within 2 months after immune, attack 28 days dissects after poison.Poison protection is attacked to comment Valence: with embodiment 7.
It is as follows using the live vaccine and aerosol immunity method of mucosal adjuvant 2 of the present invention preparation in the present embodiment: this is sent out 1:1 is mixed bright mucosal adjuvant 2 (with embodiment 6) by volume with spraying protective agent (with embodiment 6), obtains dilution.With dilute Liquid dissolution porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder is released, obtains 2.5 × 107The vaccine solution of CCU/ml.Utilize ultrasound Atomizer is sprayed, and atomization frequency of oscillation is 1.7MHz, and the immunizing dose of every pig is 5 × 107CCU。
A kind of existing porcine mycoplasmal pneumonia live vaccine aerosol immunity method (patent " porcine mycoplasmal pneumonia atomization live vaccine And its preparation and method of inspection ZL201210275639.1 "): take porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder with containing Deionized water solution (pH value 7.5) dissolution of 50mg/ml glycerol, 1mg/ml polyvinylpyrrolidone, obtain 2.5 × 107The vaccine solution of CCU/ml.It is sprayed using medical lower respiratory tract appositional pattern sprayer, air pressure pump pressure is 0.2M Pa, the immunizing dose of every pig are 5 × 107CCU。
Test result: as shown in figure 9, pressing above-mentioned side using the porcine mycoplasmal pneumonia live vaccine containing mucosal adjuvant 2 of the present invention Method carries out aerosol immunity, Lung score 3, protective rate 87.13%.And use existing porcine mycoplasmal pneumonia live vaccine gas molten Glue immunological technique is immune, Lung score 10.4, protective rate 50.50%.This example demonstrates that using mucosal adjuvant 2 of the present invention The live vaccine significant effect of preparation is better than existing public technology.
Embodiment 9: the porcine mycoplasmal pneumonia live vaccine mucosa-immune protection period evaluation containing mucosal adjuvant 2 of the present invention
Vaccine: porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder.
Mucosal adjuvant 2 of the present invention is the same as embodiment 6.
Animal: 7-15 age in days mycoplasma hyopneumoniae antigen and antibody is negative healthy ternary pigs.
Vaccine formulation: with mucosal adjuvant 2 of the present invention, 1:1 is mixed by volume with spraying protective agent (with embodiment 6), is obtained Dilution；Porcine mycoplasmal pneumonia live vaccine (168 plants) freeze-dried powder is dissolved using the dilution, obtains 2.5 × 107The epidemic disease of CCU/ml Seedling solution.It is substituted with PBS buffer solution and dissolves live vaccine after mucosal adjuvant 2 of the present invention is mixed with spraying protective agent, preparation 2.5 × 107CCU/ml without vehicle control vaccine solution.
It is immunized and attacks poison: animal is randomly divided into 4 groups, every group 12.G1 group is negative control group, not immune not attack poison； G2 group is mucosal adjuvant group of the present invention, the vaccine solution that immune mucosal adjuvant 2 of the present invention is prepared, the same embodiment of immunization method 7, the vaccine immunity amount of every pig is 5 × 107CCU.G3 group is no adjuvant control group, is immunized without vehicle control vaccine solution, exempts from Epidemic disease method is the same as G2 group.G4 group is to attack malicious control group, not immune only to attack poison.Poison is attacked within 9 months after immune, attacks 28 days dissects after poison.It attacks Poison protection evaluation: with embodiment 7.
Test result: as shown in Figure 10, no adjuvant control group, Lung score 12.67, protective rate only has 39.15%.This The scoring of invention mucosal adjuvant group animal lung lesion is 3.5, attacks malicious protective rate and reaches 85%, still be can provide after 9 months immune It is good to attack malicious protecting effect, illustrate that the live vaccine containing mucosal adjuvant 2 of the present invention is reachable through aerosol mucosa-immune post protection period By 9 months.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications
<130> 20180930
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<170> PatentIn version 3.3
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<213> artificial
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ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc 120
attactttta agaatggtgc aatttttcaa gtagaagtac caggtagtca acatatagat 180
tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 240
gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 300
agtatggcaa atgctagctt acctcagccg ccagcagcta aacctgaagc agcaaaacca 360
gtagcagcta aacctgaagc agcaaaacca gtagcagcta aacctgaagc agcaaaacca 420
gtagcagcta aacctgaagc agcaaaacca gtagcggcta aacctgaagc agctaaacca 480
gtagcagcta aaccagttgc tactaatact aatactaata ctggcttttc acttacaaat 540
aaaccaaaag aagactattt cccaatggct ctcgagcacc accaccacca ccactga 597
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Met Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr
1 5 10 15
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20 25 30
Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile
35 40 45
Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys
50 55 60
Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu
65 70 75 80
Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala
85 90 95
Ile Ala Ala Ile Ser Met Ala Asn Ala Ser Leu Pro Gln Pro Pro Ala
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115 120 125
Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys
130 135 140
Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro
145 150 155 160
Val Ala Ala Lys Pro Val Ala Thr Asn Thr Asn Thr Asn Thr Gly Phe
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Claims (10)

1. a kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, it is characterised in that the mucosal adjuvant is containing cholera toxin The solution of the recombinant protein c TB-P97R1 of B subunit and the expression of mycoplasma hyopneumoniae P 97 R 1 Antigen Fusion, the adjuvant contain 0.2-2 mg/ml recombinant protein c TB-P97R1.
2. according to porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants described in right 1, it is characterised in that the recombinant protein c TB- The amino acid sequence of P97R1 is as shown in SEQ ID NO:2.
3. porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants according to claim 1 or claim 2, it is characterised in that the recombination egg White CTB-P97R1 is prepared with the following method: by the area the R1 Gene Fusion of mycoplasma hyopneumoniae adhesion factor P97 to cholera toxin The C-terminal of B subunit gene is fitted into construction recombination plasmid pET28a-CTB-P97R1 in pET-28a (+) plasmid；By the recombination matter Grain imports Escherichia coli, obtains recombinant bacterium；CTB-P97R1 is expressed using IPTG induction recombinant bacterium, collects cellular lysate liquid supernatant, Purifying, obtains recombinant protein c TB-P97R1.
4. porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants according to claim 3, it is characterised in that the mucosa-immune Adjuvant also contains 150-300 μ g/ml Poly(I:C) and 50-150 μ g/ml saponin(e.
5. porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants according to claim 4, it is characterised in that the saponin(e is Quillaja saponin, ginsenoside or notoginsenoside.
6. the preparation method of porcine mycoplasmal pneumonia live vaccine mucosal adjuvant described in claim 1, it is characterised in that prepare 10mg/ml Then recombinant protein c TB-P97R1 mother liquor is diluted using solvent, the solvent is phosphate buffer.
7. the preparation method of porcine mycoplasmal pneumonia live vaccine mucosal adjuvant according to claim 6, it is characterised in that prepare The Poly(I:C of 1mg/ml) mother liquor and 1mg/ml saponin(e mother liquor, solvent be phosphate buffer；By the recombinant protein c TB- P97R1 mother liquor, Poly(I:C) mother liquor and saponin(e mother liquor mixing after, diluted using solvent.
8. porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants are in terms of preparing porcine mycoplasmal pneumonia live vaccine described in claim 1 Application.
9. applying according to claim 8, it is characterised in that by adjuvant described in claim 1 and spraying protective agent according to volume Than being mixed for 1:1, i (mycoplasma hyopneumoniae) vaccine strain culture or freeze-dried powder is then added, is uniformly mixed, obtains porcine mycoplasmal Pneumonia live vaccine solution.
10. applying according to claim 9, it is characterised in that the spraying protective agent is 35-45 containing Macrogol 6000 The aqueous solution of g/L, cyclodextrin 3-5 g/L, sodium thiosulfate 18-22 g/L and L-Histidine 18-22g/L, pH 7.0- 7.5。
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