CN107375922A - A kind of new Water Soluble Compound immunologic adjuvant and pig circular ring virus disease vaccine - Google Patents

A kind of new Water Soluble Compound immunologic adjuvant and pig circular ring virus disease vaccine Download PDF

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CN107375922A
CN107375922A CN201710597663.XA CN201710597663A CN107375922A CN 107375922 A CN107375922 A CN 107375922A CN 201710597663 A CN201710597663 A CN 201710597663A CN 107375922 A CN107375922 A CN 107375922A
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water soluble
soluble compound
adjuvant
cp974s
pcv2
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CN107375922B (en
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姜平
白娟
朱雪蛟
王先炜
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Nanjing Agricultural University
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Abstract

The present invention discloses a kind of new Water Soluble Compound immunologic adjuvant and pig circular ring virus disease vaccine; the present invention screens and devises tetra- kinds of aqueous adjuvants of CP974S, CP934S, CP974M and CP; prepare PCV2 inactivated vaccines; mouse and pig body immunity test result are shown; CP974S and CP934S adjuvants have stronger immunoadjuvant function to PCV2 inactivation antigens, and CP974S water Adjuvanted vaccines pig bodies immune protection effectiveness is better than PCV2 import ISA206 adjuvant inactivated vaccines.Based on the PCV2Cap recombinant proteins of baculovirus expression, prepare different Adjuvanted vaccines, mouse and pig body immunity test are found, two kinds of aqueous adjuvants of CP974S and CP934S also have stronger immunoadjuvant function to PCV2Cap recombinant proteins, and CP974S Adjuvanted vaccines pig bodies immune effect is better than import PCV2Cap subunit vaccines.CP974S water adjuvants have filled up the blank of China's pig water-soluble adjuvant, and the PCV2 vaccine safeties developed with this are effective, have important application prospect.

Description

A kind of new Water Soluble Compound immunologic adjuvant and pig circular ring virus disease vaccine
Technical field：
The invention belongs to biological immune technical field, specific design a kind of new Water Soluble Compound immunologic adjuvant and pig annulus Virosis vaccine.
Background technology：
Immunologic adjuvant (immunologic adjuvant) is the important composition composition for preparing protide inactivated vaccine, can be bright The faint material induction body of aobvious enhancing polysaccharide or polypeptide etc. antigenicity produces specific immune response；Or resisted with minimal amount of Former and minimum inoculation times stimulate body to produce the antibody of enough immune responses and high titre, and when remaining longer Between, play lasting effect.The mechanism of adjuvant booster immunization reaction is extremely complex, is not yet fully apparent from so far.The effect of adjuvant Mainly include：1. changing normal immunological function, attract a large amount of antigen presenting cell working process antigens；2. change the structure of antigen Type, antigenic substance is degraded and strengthen its immunogenicity；3. extending period of storage of the antigen in tissue, antigen is set slowly to degrade And release, and play the iuntercellular synergy (antigen presenting cell and T cell, T cell and B cell) of immune system.At present, Have been found up to more than hundred kinds of the material of immunological enhancement, it is cytokine class adjuvant, CpG DNA, genetic engineering attenuated The existing numerous studies such as element, immunostimulating complex adjuvant, Liposome Adjuvant, but the vaccine immunity adjuvant of practical application is few. The adjuvant used in the production of China animal vaccine is mainly aluminium salt class adjuvant, oil emu adjuvant and propolis adjuvant etc., pig vaccine Mostly import adjuvant is used, such as French SEPPIC companies Montanide ISA 50V, ISA206, ISA15A and Gel01 adjuvant Typically complex class adjuvant, contain a variety of adjuvant components.For relatively oily adjuvant, water-based class adjuvant side reaction is small, but forms Complicated component, the pig efficient water soluble adjuvant of China's long-term lacking independent intellectual property right.
Porcine circovirus desease is a kind of important immunosupress sexually transmitted disease for endangering world's pig industry, and immunity inoculation is prevention With the most important measure for controlling the disease.Current external commercialization PCV2 vaccines mainly have Merial's BoehringerIngelheim'sInter-vet/Merck's Schering-Plough/Merck'sPCV, Pfizer Animal Health Inc. FosteraTM PCV, They can provide immunoprotection for sow or piglet to varying degrees, mitigate clinical symptoms and viremia virusemia, improve production Efficiency.China have developed 5 kinds of inactivated virus vaccines and 2 kinds of subunit vaccines, realize merchandized handling, but the adjuvant used It is oil adjuvant or the water-soluble adjuvant of import, the water-soluble adjuvant of import is costly, it is therefore desirable to design new water-soluble Property compound immunological adjuvant.
The content of the invention：
It is an object of the invention to provide a kind of Water Soluble Compound immunologic adjuvant.
Another object of the present invention is to provide above-mentioned Water Soluble Compound immunologic adjuvant to prepare pig circular ring virus disease vaccine In application.
The present invention's has a purpose to be to provide the pig circular ring virus disease vaccine for including above-mentioned Water Soluble Compound immunologic adjuvant.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of Water Soluble Compound immunologic adjuvant, carbomer, sodium hydroxide, bacterium are included in the Water Soluble Compound immunologic adjuvant DNA and tocopherol.
The concentration of each component is in the Water Soluble Compound immunologic adjuvant：5~6g/L of carbomer, sodium hydroxide 5%~6% (5 ~6g/100ml), μ g/mL of DNA of bacteria 20~30, tocopherol 0.01%~0.03% (10~30mg/100ml).
As a kind of optimal technical scheme, capsular polysaccharide and bacterioprotein are also included in the Water Soluble Compound immunologic adjuvant At least one of OMP.Concentration of the described capsular polysaccharide in the Water Soluble Compound immunologic adjuvant is 20~30 μ g/mL, institute Concentration of the bacterioprotein OMP stated in the Water Soluble Compound immunologic adjuvant is 30~40 μ g/mL.It is further preferred that the water Carbomer, sodium hydroxide, capsular polysaccharide, bacterioprotein OMP, DNA of bacteria and tocopherol are included in dissolubility compound immunological adjuvant；Its The concentration of middle each component is：5~6g/L of carbomer, sodium hydroxide 5%~6% (5~6g/100ml), the μ of capsular polysaccharide 20~30 G/mL, μ g/mL of bacterioprotein OMP 30~40, μ g/mL of DNA of bacteria 20~30, tocopherol 0.01%~0.03% (10~ 30mg/100ml)。
It is further preferred that above-mentioned DNA of bacteria is haemophilus parasuis DNA, above-mentioned capsular polysaccharide is that secondary pig is bloodthirsty Bacillus capsular polysaccharide, above-mentioned bacterioprotein OMP are haemophilus parasuis mycoprotein OMP.People in the art can be used Method known to member prepares haemophilus parasuis DNA, haemophilus parasuis capsular polysaccharide and haemophilus parasuis mycoprotein OMP。
Still more preferably, carbomer, sodium hydroxide, haemophilus parasuis are included in the Water Soluble Compound immunologic adjuvant Capsular polysaccharide, haemophilus parasuis mycoprotein OMP, haemophilus parasuis DNA and tocopherol；The concentration of wherein each component is： 5~6g/L of carbomer, sodium hydroxide 5%~6% (5~6g/100ml), μ g/mL of haemophilus parasuis capsular polysaccharide 20~30, μ g/mL of haemophilus parasuis mycoprotein OMP 30~40, μ g/mL of haemophilus parasuis DNA 20~30, tocopherol 0.01% ~0.03% (10~30mg/100ml).The concentration of each component is still more preferably：Carbomer 5g/L, sodium hydroxide 5% are secondary μ g/mL of haemophilus suis capsular polysaccharide 20, μ g/mL of haemophilus parasuis mycoprotein OMP 30, haemophilus parasuis DNA20 μ G/mL, tocopherol 0.02%.
Above-mentioned Water Soluble Compound immunologic adjuvant is prepared using following methods：Carbomer is added into configuration card ripple in distilled water Nurse jelly；Using the preceding sodium hydroxide solution that sterilizing is slowly added into described carbomer jelly, make described card ripple Nurse jelly is converted into water-soluble character, then adds other components, filtration sterilization, adds sterile purified water constant volume and be sufficiently stirred Mix, 2~8 DEG C of preservations.Specific prepared of the preferable carbomer jelly is referred to as：Weigh carbomer and add to distilled water In, after abundant swelling, mix, after autoclaving, in gelatin, room temperature placement.The autoclaved condition be 110~ 120 DEG C of autoclavings 20~40 minutes.
Most preferably, described carbomer is Carbomer974 or carbomer 934.
Application of the above-mentioned Water Soluble Compound immunologic adjuvant in vaccine is prepared.Described vaccine is preferably pig circular ring virus Vaccine.
A kind of vaccine combination, include above-mentioned Water Soluble Compound immunologic adjuvant and immunogene.Described immunogene is preferred For pig circular ring virus immunogene.Described pig circular ring virus immunogene is the PCV2 antigens or PCV2Cap recombinant proteins of inactivation.
This research is successfully screened and devises 4 kinds of compound water-soluble adjuvants, with the PCV2 antigens and Cap recombinant proteins of inactivation Based on, water-soluble adjuvant vaccine is developed, carries out mouse and pig body immunity test.As a result show, CP974S and CP934S are water-based Adjuvanted vaccines can induce body to produce the special humoral and cellular immune response responses of PCV2, wherein, CP974S water Adjuvanted vaccines lure The horizontal highest of immune response of piglet is led, and the immanoprotection action sick to this can be effectively provided, is opened with important research Hair prospect.
Beneficial effects of the present invention：
This research is successfully screened and devises tetra- kinds of aqueous adjuvants of CP974S, CP934S, CP974M and CP, is prepared PCV2 and is gone out Live vaccine, mouse and pig body immunity test result show that CP974S and CP934S adjuvants have to PCV2 inactivation antigens exempts from more by force Epidemic disease adjuvant effect, CP974S water Adjuvanted vaccines pig bodies immune protection effectiveness are better than PCV2 import ISA206 adjuvant inactivated vaccines.With Based on the PCV2Cap recombinant proteins of baculovirus expression, different Adjuvanted vaccines are prepared, mouse and pig body immunity test are found, Two kinds of aqueous adjuvants of CP974S and CP934S also have stronger immunoadjuvant function, CP974S adjuvant epidemic diseases to PCV2Cap recombinant proteins Seedling pig body immune effect is better than import PCV2Cap subunit vaccines.CP974S water adjuvants have filled up China's pig water-soluble adjuvant Blank, the PCV2 vaccine safeties developed with this are effective, have important application prospect.
Brief description of the drawings：
Fig. 1 is immune mouse PCV2ELISA TPPA results
Fig. 2 is immune mouse PCV2 neutralizing antibody testing results
Fig. 3 is immune piglet PCV2ELISA TPPA results
Fig. 4 is immune piglet PCV2 neutralizing antibody testing results
Fig. 5 is immune mouse PCV2ELISA TPPA results
Fig. 6 is immune mouse PCV2 neutralizing antibody testing results
Fig. 7 is immune mouse lymphocyte proliferation test result
Fig. 8 is immune piglet PCV2ELISA TPPA results
Fig. 9 is immune piglet PCV2 neutralizing antibody testing results
Figure 10 is the testing result of immune piglet lymphproliferation response
Figure 11 is test pig different tissues pathological observation result (HE is dyed, 400X)
Figure 12 is the dynamic change that PCV2 inactivated vaccines induce piglet generation antibody
Figure 13 is 0~25 day pig after attacking poison with respect to daily gain statistical result
Figure 14 is to attack malicious piglet inguinal lymph nodes pathological study result (HE is dyed, 400X)
Figure 15 detects PCV2 antigens (400X) to attack malicious piglet inguinal lymph nodes histogenic immunity groupization
Embodiment：
The development of Part I adjuvant and its immunoadjuvant function to PCV2 inactivation antigens
This research configures tetra- kinds of water-based assistants of CP974S, CP934S, CP974M and CP based on carbomer release polymer Agent, using the PCV2 of inactivation, PCV2 inactivated vaccines are prepared, mouse and pig body immunity test is carried out, as a result shows, CP974S, Two kinds of aqueous adjuvants of CP934S have stronger immunoadjuvant function to PCV2 inactivation antigens, and CP974S water Adjuvanted vaccines pig bodies are immunized Effect is better than PCV2ISA206 adjuvant inactivated vaccines, has important application prospect.
1 materials and methods
1.1 main material
PCV2SH strains virus liquid is built by this laboratory and preserved (similar conventional strain is also suitable).PCV2 monoclonals resist Body 3E5 is prepared by this laboratory and preserved (similar conventional antibody is also suitable)；Goat anti-mouse igg-HRP, SPA-HRP, goat-anti are small Mouse IgG-FITC and DAB colour reagent box is purchased from Wuhan doctor's moral bio-engineering corporation；Other conventional reagents are that analysis is pure.
5 week old cleaning grade ICR mouse, PCV2 negative antibodies are detected through indirect ELISA.20~30 age in days weanling pigs, warp Detection PCV2 and PRRSV antigen-antibody is feminine gender, isolated rearing.
1.2 adjuvant components configure
It is prepared by bacterial eapsular polysaccharide：By haemophilus parasuis (culture presevation numbering CVCC3712, Chinese veterinary medicament supervision Chinese Shou Shou Microbiological Culture Collections administrative center of institute purchases, but not limited to this) inoculation TAB nutrient solutions, 37 DEG C are cultivated 16 hours, Bacteria culture supernatant is taken after centrifugation, using hot phenol method extraction purification capsular polysaccharide, its content is determined with phend-sulphuric acid, Its concentration is 60mg/ml, and it is about 55ku that SDA-PAGE, which determines its molecular mass,.- 70 DEG C of refrigerators save backup.
It is prepared by DNA of bacteria：Haemophilus parasuis bacterium solution, centrifuging and taking thalline, add appropriate lysozyme and SDS cracking thalline. With chloroform-phenol extraction, centrifugation, removing protein is repeated 3 times, RNA is removed with RNase, then with isometric 95% ethanol precipitation DNA, repeats said process 2-3 times, the DNA for precipitating acquisition is dissolved in water, measure DNA concentration is 20mg/ml.- 70 DEG C of ice Case saves backup.
Bacterioprotein OMP is extracted：The extraction of total bacterial protein extracts kit is won using shellfish.Measured according to sample and split in right amount Hydrolysis mother liquor, protease inhibitors Mix and inhibitors of phosphatases Mix and protein stabilized liquid are added, prepare lysate, put standby on ice With.By haemophilus parasuis bacterium solution, thalline is collected by centrifugation, lysate is added after washing thalline with PBS, places 20~30 points on ice Clock.Ultrasonic degradation to bacterium solution becomes clear.Supernatant, as bacterioprotein are taken after centrifugation, protein quantification is carried out using BCA methods, it is dense Spend for 50mg/ml.- 70 DEG C of refrigerators save backup.
1.3 adjuvants configure
CP974S water adjuvants：Configured and formed based on Carbomer974 release polymer, collocation method：Weigh carbomer 50g, add in 9800ml distilled water, after abundant swelling, mix, 115 DEG C of autoclavings 30 minutes, in gelatin configuration card ripple Nurse jelly, room temperature are placed.The sodium hydroxide solution of sterilizing is slowly added to before use makes the final concentration of 5% (5g/ of sodium hydroxide 100ml), it is converted into water-soluble character, add bacterial eapsular polysaccharide, bacterioprotein OMP and DNA of bacteria prepared by step 1.2 Make its final concentration be respectively 20 μ g/mL, 30 μ g/mL and 20 μ g/mL, add the tocopherol aqueous solution make its final concentration of 0.02% (20mg/100ml) (degerming with 0.45 μm of membrane filtration), add sterile purified water to 10000ml, be sufficiently stirred mixing, 2~8 DEG C Preserve, should be no more than 12 months.
CP934S water adjuvants：Configured and formed based on carbomer 934 release polymer, collocation method：As stated above Configure carbomer jelly.The sodium hydroxide solution of sterilizing is slowly added to before use makes the final concentration of 5% (5g/ of sodium hydroxide 100ml), it is converted into water-soluble character, add bacterial eapsular polysaccharide, bacterioprotein OMP and DNA of bacteria prepared by step 1.2 Make its final concentration be respectively 20 μ g/mL, 30 μ g/mL and 20 μ g/mL, add the tocopherol aqueous solution make its final concentration of 0.02% (20mg/100ml) (degerming with 0.45 μm of membrane filtration), add sterile purified water to 10000ml, be sufficiently stirred mixing, 2~8 DEG C Preserve, should be no more than 12 months.
CP974M water adjuvants：Configured and formed based on Carbomer974 release polymer, collocation method：As stated above Configure carbomer jelly.The sodium hydroxide solution of sterilizing is slowly added to before use makes the final concentration of 5% (5g/ of sodium hydroxide 100ml), it is made to be converted into water-soluble character, the bacterioprotein OMP and DNA of bacteria for adding step 1.2 preparation distinguish its final concentration For 30 μ g/mL and 20 μ g/mL, adding the tocopherol aqueous solution makes its final concentration of 0.02% (20mg/100ml) (with 0.45 μm of filter Membrane filtration is degerming), add sterile purified water to be sufficiently stirred mixing to 10000ml, 2~8 DEG C of preservations, should be no more than 12 months.
CP water adjuvants：Configured and formed based on carbomer release polymer, collocation method：Configuration card ripple as stated above Nurse jelly.The sodium hydroxide solution of sterilizing is slowly added to before use makes final concentration of 5% (5g/100ml) of sodium hydroxide, makes It is converted into water-soluble character, and adding DNA of bacteria prepared by step 1.2 makes its final concentration of 20 μ g/mL, adds the tocopherol aqueous solution Make its final concentration of 0.02% (20mg/100ml) (degerming with 0.45 μm of membrane filtration), add sterile purified water to 10000ml, 2 ~8 DEG C of preservations, it should be no more than 12 months.
The preparation of 1.4 vaccines
PCV2 virus liquids are taken, viral level is all higher than 106.0TCID50/ml.It is small with 0.1% β -4 DEG C of propionyl lactone inactivation 24 When, 37 DEG C act on 2 hours, above-mentioned tetra- kinds of adjuvants of CP974S, CP934S, CP974M and CP and ISA206 adjuvants are added, by antigen 4 are pressed with adjuvant:1 ratio mixes (antigen final concentration is identical), is stirred, is sufficiently mixed 10 minutes with 300r/min, configures 5 kinds of vaccines CP974S, CP934S, CP974M, CP and 15A, be stored in 4 DEG C it is standby.
1.5 mouse immuning test
70 cleaning grade mouse are randomly divided into 7 groups, 1-5 groups are helped for ISA206, CP974S, CP934S, CP974M, CP Vaccinating agent inoculation group, the 6th group is PCV2 antigen control groups, and the 7th group is PBS control group.Mouse carries out head after adapting to 2 days and exempted from, and exempts from Epidemic disease method is dorsal sc multi-point injection, and vaccine immunity dosage is 200 μ L.Blank control group does not make any processing.Head exempts from rear 14d Booster immunization is carried out, the dosage and method of booster immunization are as before.After head exempts from 14d, all mouse are subjected to docking blood sampling, Separation serum is used for ELISA antibody tests.After head exempts from 28d, 42d, every group takes 5 eyeball of mouse blood samplings at random, separates serum, point Ce Ding not ELISA antibody and neutralizing antibody titers；It is sterile to win mouse spleen, lymphocyte is separated after adding sterilizing PBS grindings, is surveyed Determine lymphproliferation response.
1.6 piglet immunologicals are tested
25 first 30 age in days weanling pigs are taken, PCV2 and PRRSV antigen-antibodies are feminine gender after testing, are randomly divided into 5 groups, often Group 5.1-4 groups are ISA206 (French SEPPIC companies), CP974S, CP934S, SMI3115 (French SEPPIC companies) epidemic disease Seedling inoculation group, the 5th group is PCV2 antigen immune control groups, the 1-5 immune group pig musculi colli injecting immune, 1ml/ heads, 3 weeks Booster immunization is once afterwards.6th group is blank control group.Each group isolated rearing observation after immune, the 21st, 35 after first immunisation Its blood sampling, separates serum, determines PCV2ELISA antibody and neutralizing antibody.Take a blood sample within 35 days after first immunisation, separate lymphocyte, Carry out lymphocyte proliferation assay.
1.7EL ISA antibody tests
Envelope antigen is the CapC that this laboratory prepares purification storage, and this albumen is by Bacillus coli expression.With pH9.6 carbonic acid Salt buffer is diluted to final concentration of 5ug/ml, coated elisa plate, 100 μ L/ holes, and 4 DEG C of coatings are overnight after 37 DEG C of effect 2h；Washing 3 times, each 3-5min；200 μ L 0.15%BSA confining liquids are added to close plank, 37 DEG C of effect 2h per hole；Washing；By blood to be checked It is clear to use PBS doubling dilutions, each sample a line, 100 μ L, 37 DEG C of effect 1h are added per hole；Washing；Then enzyme target SPA (1 is added: 10000 times of dilutions) (during detection mice serum, using enzyme mark sheep anti-mouse igg instead), 100 μ L/ holes, 37 DEG C of effect 1h；Washing；Add bottom Thing liquid TMB develops the color, finally with 2mM H2SO4Terminating reaction.Result judgement：Serum OD to be checked450Value/negative serum OD450Value >= 2.1 be the positive.
1.8 neutralizing antibodies detect
Serum to be checked is inactivated in 56 DEG C of water-bath 30min, after 12000rpm centrifugations 10min is degerming, suctions out supernatant to going out In the EP pipes of bacterium.The serum handled well is subjected to doubling dilution with maintaining liquid again, is 1: 4,1: 8,1: 16,1: 32 ... ... successively At the same time, the PCV2 of propagation is diluted to 200TCID with maintaining liquid50/ 0.1mL, then from the body such as the serum of different dilution factors Product mixing, 37 DEG C of water-baths act on 1h.The nutrient solution in 96 orifice plates with PK15 cells is discarded, cell one is washed with pure DMEM Time, then serum-virus mixture after water-bath is acted on is added in hole, and each serum dilution sets 3 repetitions, and 100 μ L are every Hole, 37 DEG C of culture 72h.Negative serum control, negative serum virus control and blank control are set simultaneously.With exempting from indirectly after 72h Epidemic disease fluorescence method (IFA) determine serum neutralize antibody titers, using can suppress 50%PCV2 infection serum greatest dilution as The neutralize antibody titers of serum to be checked.
1.9 data statistic analysis
Statistical analysis is carried out to experimental data using SPSS softwares, and compares the difference of each group numerical value, wherein P<0.05 table Show significant difference, P<0.01 represents that difference is extremely notable.
2 results
2.1 vaccine physical behaviors
Tetra- kinds of Adjuvanted vaccines of CP974S, CP934S, CP974M and CP are in semi-transparent clear suspension, in the aqueous solution. ISA206 Adjuvanted vaccines are creamy white, and vaccine is dripped in cold water surface for few drops, spread in cloud.
2.2 mouse immuning test results
ELISA antibody test results are shown in Fig. 1.CP974S, CP934S and CP974M Adjuvanted vaccines first immunisation 14 days Induction produces PCV2 antibody, and head exempts from latter 28-35 days, CP974S, CP934S, CP974M Adjuvanted vaccines immune group ELISA antibody water Put down apparently higher than PCV2CP Adjuvanted vaccines immune groups (P<0.05).Wherein, CP974S, CP934S vaccine immunity group antibody level are bright It is aobvious to be higher than ISA206 commodity adjuvant groups (P<0.05), meanwhile, negative control group is then not detected by PCV2 specific antibodies.
Neutralizing antibody testing result such as Fig. 2.Head exempts from latter 28-35 days, and CP974S, CP934S and CP974M Adjuvanted vaccines are immunized PCV2 neutralizing antibodies level is organized apparently higher than CP vaccine immunity groups (P<0.05), wherein, CP974S vaccine immunity group antibody levels Highest, (P similar to ISA206 commodity adjuvant groups>0.05), meanwhile, negative control group is then not detected by PCV2 neutralizing antibodies.
The above results show that CP974S and CP934S adjuvants have stronger immunoadjuvant function to PCV2 antigens, wherein, CP974S immunoadjuvant functions are most strong.The PCV2 inactivated vaccines prepared with this have preferable immunogenicity, can induce compared with Gao Shui Flat humoral immune response.
2.3 pig body immunity test results
PCV2ELISA Antibody Results are shown in that Fig. 3, head exempt from latter 28-35 days, CP974S and CP934S Adjuvanted vaccines immune groups are produced Raw PCV2 antibody, antibody level are higher than ISA206 import Adjuvanted vaccines immune groups (P>0.05), and SMI3115 Adjuvanted vaccines are immunized Group antibody level is relatively low.Meanwhile negative control group is then not detected by PCV2 neutralizing antibodies.
Neutralizing antibody result is shown in Fig. 4.Head exempts from latter 28-35 days, CP974S and CP934S Adjuvanted vaccines groups are produced in PCV2 And antibody, antibody level (P similar with ISA206 import Adjuvanted vaccines groups>0.05), wherein, CP974S vaccine immunity group antibody water Flat highest.Meanwhile negative control group is then not detected by PCV2 neutralizing antibodies.
The above results show that CP974S and CP934S adjuvants can strengthen PCV2 inactivation antigen Humorals, wherein, CP974S immunoadjuvant functions are most strong, better than the ISA206 adjuvants of commercialization.
Immunoadjuvant function of the novel aqueous adjuvant of Part II to PCV2cap albumen
This research uses above-mentioned tetra- kinds of aqueous adjuvants of CP974S, CP934S, CP974M and CP, with baculovirus expression Based on Cap recombinant proteins, prepare PCV2 subunit vaccines, carry out mouse and pig body immunity test, as a result show, CP974S and Two kinds of aqueous adjuvants of CP934S have stronger immunoadjuvant function to PCV2Cap recombinant proteins, and CP974S Adjuvanted vaccines pig bodies are exempted from Epidemic disease effect is better than import PCV2Cap subunit vaccines.
1 materials and methods
1.1 main material
The recombinant baculovirus rBcapkm of expression Cap protein is built by this laboratory and preserves (similar strain or antigen It is applicable).High Five cells (U.S. ATCC) purchase preservation by this laboratory；3E5 is by this laboratory system for PCV2 monoclonal antibodies It is standby and preserve (comparator antibody is also suitable)；Goat anti-mouse igg-HRP, SPA-HRP, goat anti-mouse igg-FITC and DAB colour developing examinations Agent box is purchased from Wuhan doctor's moral bio-engineering corporation；Other conventional reagents are that analysis is pure.
5 week old cleaning grade ICR mouse 70, PCV2 negative antibodies are detected through indirect ELISA；30 age in days weanling pigs 25 Head, PCV2 and PRRSV antigen-antibody is feminine gender after testing, isolated rearing.
Adjuvant：CP974S water adjuvant, CP934S water adjuvant, CP974M water adjuvant, CP water adjuvants, the step of with Part I 1.3 prepare.
The preparation of 1.2 vaccines
Take recombinant baculovirus rBcapkm to be inoculated with Sf9/High Five insect cells, collect generation within 96 hours after infection The cell of lesion.Inhale first and abandon cells and supernatant, it is molten that every bottle of cell (75cm2) adds 1mL 0.01mol/L pH 7.2PBS Liquid, cell is blown down, then ultrasonic treatment is carried out to cell suspension, obtain the cell pyrolysis liquid containing Porcine circovirus type 2 Cap.Will be above-mentioned Tetra- kinds of adjuvants of CP974S, CP934S, CP974M and CP and ISA 15A adjuvants mix with isometric cell pyrolysis liquid respectively, will Antigen and adjuvant press 4:1 ratio mixes, and is stirred, is sufficiently mixed 10 minutes with 300r/min, 5 kinds of CP974S, CP934S of configuration, CP974M, CP and 15A Adjuvanted vaccines, be stored in 4 DEG C it is standby.
1.3 mouse immuning test
70 cleaning grade mouse are randomly divided into 7 groups, 1-5 groups are CP974S, CP934S, CP974M, CP and 15A adjuvant Vaccine, the 6th group is recombinant protein antigen control group, and the 7th group is PBS control group.Mouse is immunized after adapting to 2 days, and side is immunized Method is dorsal sc multi-point injection, and vaccine immunity dosage is 200 μ L.Blank control group does not make any processing.Head exempts from rear 14d and carried out Booster immunization, the dosage and method of booster immunization are as before.
After head exempts from 14d, all mouse are subjected to docking blood sampling, separation serum is used for ELISA antibody tests.Head exempt from 28d, After 42d, every group takes 5 eyeball of mouse blood samplings at random, separates serum, determines ELISA antibody and neutralizing antibody titers respectively；It is sterile Mouse spleen is won, lymphocyte is separated after adding sterilizing PBS grindings, determines lymphproliferation response.
1.4 piglet immunologicals are tested
25 first 30 age in days weanling pigs are screened, PCV2 and PRRSV antigen-antibodies are feminine gender after testing, are randomly divided into 5 groups, Every group 5.1-3 groups are CP974S, CP934S and 15A Adjuvanted vaccines inoculation group；4th group is that commercialization subunit vaccine compares Group, the 5th group is PBS control group.Immune group pig musculi colli injecting immune, 1ml/ heads, booster immunization is once after 3 weeks.5th group is Blank control group.Each group isolated rearing observation after immune, take a blood sample within the 21st, 35 day after first immunisation, separate serum, measure PCV2ELISA antibody and neutralizing antibody.Take a blood sample within 35 days after first immunisation, separate lymphocyte, carry out lymphocyte proliferation assay.
1.5ELISA antibody test
Ibid.
1.6 neutralizing antibodies detect
Ibid.
1.7 lymphocyte proliferation assay
Ibid.
1.8 data statistic analysis
Statistical analysis is carried out to experimental data using SPSS softwares, and compares the difference of each group numerical value, wherein P<0.05 table Show significant difference, P<0.01 represents that difference is extremely notable.
2 results
2.1 vaccine physical behaviors
CP974S, CP934S, CP974M and CP Adjuvanted vaccines are in semi-transparent clear suspension, in the aqueous solution.15A adjuvants Vaccine is creamy white, and vaccine is dripped in cold water surface for few drops, spread in cloud.
2.2 mouse immuning test results
2.2.1 humoral immune response
ELISA antibody test results are shown in Fig. 5.CP974S, CP934S, CP974M and CP Adjuvanted vaccines first immunisation 14 days is i.e. It can induce and produce PCV2 antibody, head exempts from latter 28-42 days, CP974S, CP934S, CP974M and CP Adjuvanted vaccines immune group ELISA Antibody level is apparently higher than Cap protein immune group (P<0.05).Wherein, CP974S vaccine immunities group antibody level highest, and Apparently higher than 15A adjuvant groups (P<0.05), meanwhile, negative control group is then not detected by PCV2 specific antibodies.
Neutralizing antibody testing result such as Fig. 6.Head exempts from latter 28-42 days, CP974S, CP934S, CP974M and CP Adjuvanted vaccines Immune group PCV2 neutralizing antibodies level is apparently higher than Cap protein immune group (P<0.05), wherein, CP974S vaccine immunity group antibody Horizontal highest, and apparently higher than 15A adjuvant groups (P<0.05), meanwhile, negative control group is then not detected by PCV2 neutralizing antibodies.
2.2.2 cellullar immunologic response
As a result Fig. 7 is seen.After head exempts from 28 days, vaccine immunity group lymphocyte proliferation assay reaction unobvious, but head exempts from 42 days Afterwards, CP974S, CP934S and CP974M immune group PCV2SI values are apparently higher than PBS groups (* P<0.05), show with CP974S, Recombinant baculovirus Porcine circovirus type 2 Cap prepared by CP934S and CP974M adjuvants can induce mouse and produce the specific lymphs of PCV2 Cell proliferative response.
The above results show that CP974S, CP934S and CP974M adjuvant have relatively strong immune assistant to PCV2Cap recombinant proteins Agent acts on, wherein, CP974S adjuvant effects are most strong.The recombinant baculovirus Porcine circovirus type 2 Cap subunit vaccine prepared with this has There is preferable immunogenicity, be capable of the humoral immune response and cellullar immunologic response of induced higher levels.
2.3 pig body immunity test results
2.3.1ELISA antibody
As a result Fig. 8 is seen.Head exempts from latter 21-35 days, and CP974S, CP934S and 15A Adjuvanted vaccines immune group produce PCV2 and resisted Body, antibody level (P similar with import vaccine immunity group>0.05).Meanwhile negative control group is then not detected by PCV2 and resisted Body.
2.3.2 neutralizing antibody
As a result Fig. 9 is seen.Head exempts from latter 21-35 days, and CP974S, CP934S and 15A adjuvant group produce PCV2 neutralizing antibodies, resists Horizontal (the P similar with import vaccine immunity group of body>0.05).Meanwhile negative control group is then not detected by PCV2 neutralizing antibodies.
2.3.3 cellullar immunologic response
35 days collection blood after head exempts from, carries out lymphocyte proliferation experiment, as a result as shown in Figure 10, CP974S, CP934S With import vaccine immunity group SI values apparently higher than PBS groups (* P<0.05) CP974S and CP934S adjuvants prepared by this research, are shown Recombinant Cap protein vaccine can induce piglet and produce cellullar immunologic response.
The above results show, CP974S and CP934S adjuvants can strengthen PCV2Cap recombinant protein body fluid immune effects and Cellullar immunologic response, it is sub- single with import Cap with the recombinant baculovirus Porcine circovirus type 2 Cap subunit vaccine immune effect that this is prepared Position vaccine is similar.
The novel aqueous adjuvant inactivated vaccine securities of Part III PCV2 and immune protection effectiveness
This research uses CP974S aqueous adjuvants, prepares PCV2 inactivated vaccines, carries out pig body vaccine safety and immune guarantor Potency test is protected, is as a result shown, PCV2CP974S adjuvant inactivated vaccines have preferable security, can effectively inducing mouse and pig Body produces humoral immune response, and it is possible to effectively provide the protective effect to PCV2 attacks, immune effect is better than PCV2 commodity Change inactivated vaccine.
1 materials and methods
1.1 main material
PCV2SH strains virus liquid is built and preserved by this laboratory.PCV2 monoclonal antibodies 3E5 is prepared by this laboratory And preserve.
CP974S water adjuvants：Configured and formed based on Carbomer974 release polymer, collocation method：Weigh carbomer 50g, add in 9800ml distilled water, after abundant swelling, mix, 115 DEG C of autoclavings 30 minutes, in gelatin configuration card ripple Nurse jelly, room temperature are placed.The sodium hydroxide solution of sterilizing is slowly added to before use makes the final concentration of 5% (5g/ of sodium hydroxide 100ml), it is converted into water-soluble character, add bacterial eapsular polysaccharide, bacterioprotein OMP and DNA of bacteria prepared by step 1.2 Make its final concentration be respectively 20 μ g/mL, 30 μ g/mL and 20 μ g/mL, add the tocopherol aqueous solution make its final concentration of 0.02% (20mg/100ml) (degerming with 0.45 μm of membrane filtration), add sterile purified water to 10000ml, be sufficiently stirred mixing, 2~8 DEG C Preserve, should be no more than 12 months.
1.2 vaccine formulation
3 batches of PCV2 virus liquids are taken, viral level is all higher than 106.0TCID50/ml.With 0.1% β -4 DEG C of propionyl lactone inactivation 24 Hour, 37 DEG C are acted on 2 hours, add above-mentioned CP974S adjuvants, and antigen and adjuvant are pressed into 4:1 ratio mixes, and is stirred with 300r/min Mix, be sufficiently mixed 10 minutes, configure 3 kinds of CP974S Adjuvanted vaccines (1001,1002,1003), be stored in 4 DEG C it is standby.
1.3 vaccine safeties are tested
21 age in days piglet 24 is taken, is divided into 4 groups, every group 6,3 batches of CP974S inactivated vaccines of the 1st~3 group of intramuscular injection (1001,1002,1003), dosage of inoculation 4ml/ heads；4th group is blank control group, isolated rearing 30 after cutting overbit and weighing My god, observation piglet whether there is clinical symptoms.Body temperature (rectal temperature) is measured daily within 1~7 day after inoculation.Weighed during off-test. Every group of random taking-up 2 in the 7th day, carries out pathological anatomy, and take injection site musculature, lungs and lymph node group after inoculation Knit, histotomy, tissues observed lesion is made after fixing in formaldehyde.
1.4 pig body Immunoprotection tests
30 first 30 age in days weanling pigs are taken, PCV2 and PRRSV antigen-antibodies are feminine gender after testing, are randomly divided into 6 groups, often Group 5, each group isolated rearing.1-3 groups are inoculated with 3 batches of CP974S inactivated vaccines (1001,1002,1003), the 4th group of inoculation PCV2SH strain ISA206 adjuvant inactivated vaccines, the 5th group is not inoculated with as malicious control group is attacked, and the 6th group is blank control group.One exempts from Booster immunization is carried out within 21 days afterwards, notices whether observation immune group piglet has adverse reaction after immune.Two exempt from latter 14 days to immune group Attack poison with poison group piglet is attacked, every piglet collunarium attack malicious 2mL, intramuscular injection 2mL (PCV2SH, 105TCID50/mL), attack 4th day and 7 days injection hemocyanin, 2mL every (1mg/mL) after poison, while thioglycollate medium is injected intraperitoneally, 10mL is every Head, attack the 14th day and 21 days intraperitoneal injection thioglycollate medium of poison, the every heads of 10mL.Every morning measures all piglets after attacking poison Body temperature, and observe the clinical manifestation of piglet.Take a blood sample within 21 days and 35 days after exempting from one, separation serum is used for ELISA antibody with Detected with antibody test, PCV2 viremia virusemias.Attack after poison 0 day and 28 natural gift also known as measure every pig body weight, calculate relative daily gain (RDWG).Attack after poison to cut open for 28 days and kill all piglets, observe the organ disease situation such as every pig lungs and lymph node, take lymph node, Lungs are fixed in 4% paraformaldehyde, make Frozen tissue section, for pathological observation and PCV2 immunohistochemical assays, together When, take inguinal lymph nodal tissue to carry out PCV2Real-time PCR detections.
1.5ELISA antibody test
Carry out as stated above.
1.6 neutralizing antibodies detect
Carry out as stated above.
1.7 pathological examination
Pathological anatomy is carried out according to a conventional method, observes internal organs pathological change, and gather groin and the good fortune of lymphoglandulae tracheales 4% After your Malin fixes, paraffin section, HE dyeing, microscope tissues observed lesion are prepared.
1.8 ImmunohistochemistryMethods Methods detect lymph node tissue PCV2 antigens
Slide is handled according to a conventional method, prepares inguinal lymph nodes paraffin section, and PCV2 is detected with ImmunohistochemistryMethods Methods Antigen：(1) paraffin section routinely dewaxes into water.(2) 3%H2O2Deionized water is incubated 5~10min, distillation washing 3 times.(3) resist Former hot repair is answered：0.01M citrate buffers (pH6.0) are immersed into section, micro-wave oven is heated to low fiery 20min after boiling.It is cold But washed 1~2 time with PBS (0.01M, pH7.2~7.6) afterwards.(4) 5% sheep blood serum confining liquid, 37 DEG C of incubations are added dropwise 30min, get rid of surplus liquid.(5) the anti-PCV2 monoclonal antibodies (1 of mouse are added dropwise:100), 37 DEG C of incubation 4h, 2min is washed with PBS × 3 times.(6) goat anti-mouse igg of HRP marks is added dropwise, 37 DEG C of incubation 1h, 2min × 3 time are washed with PBS.(7) DAB develops the color：Use DAB colour reagent boxes, about 50ul reagents 1 are added in 1mL reagents 2 (DAB substrate solutions), well mixed to add to section, room temperature shows Color, between controlling 5~20min of the reaction time under mirror, untill thering is cell dye in brown, distillation water washing.(8) haematoxylin is light Degree is redyed, mounting.(9) micro- sem observation, brown dye cell number >=10% and judges PCV2 antigen positives in lymph follicle.
1.9 Real-time PCR detect lymph node tissue PCV2 contents
1.9.1 sample DNA extracts：200 μ l serum or lymph node tissue suspension are taken, adds 400 μ lPBS, final concentration 1% SDS and the μ g/mL of final concentration 50 Proteinase K, 56 DEG C of water-bath 30min add isometric phenol, and vibration mixes, 12000rpm from Heart 10min；Careful supernatant of drawing is managed to new EP, adds isometric phenol/chloroform, and vibration mixes, 12000rpm centrifugations 10min；Careful supernatant of drawing is managed to new EP, adds isometric chloroform, and vibration mixes, 12000rpm centrifugations 10min；Carefully Draw supernatant to manage to new EP, add the 3M sodium acetates (pH5.2) of 1/10 volume and the absolute ethyl alcohol of the precooling of 2.5 times of volumes ,- 20 DEG C of overnight precipitation DNA；12000rpm centrifuges 15min, abandons supernatant, and precipitation washed once with 75% ethanol, drying at room temperature；Finally 20 μ L aseptic double-distilled water dissolving DNAs are added, -20 DEG C save backup.
1.9.2 Real-time PCR：Reaction system is：μ L, the 2 × Power SYBR Green PCR of DNA 2 Each 1 μ L of μ L, primers F/R of MasterMix (TOYOBO companies) 10 (final concentration 400nM, F:5’-CCAGGAGGGCGTTCTGACT- 3’；R:5 '-CGTTACCGCTGGAGAAGGAA-3 '), aseptic double-distilled water supplies volume to 20 μ L.Put ABI 7300real time Carried out in PCR instrument, response procedures are：Pre-degeneration 95 DEG C of 2min, 95 DEG C of 15s, 60 DEG C of 1min, carry out 40 circulations.Concurrently set Negative control and by the use of 10 times of doubling dilutions of positive plasmid template pT-SH as template, operates, it is bent to draw standard in the same way Line, corresponding copy number is calculated according to sample Ct values and standard curve.
1.10 data statistic analysis
Statistical analysis is carried out to experimental data using SPSS softwares, and compares the difference of each group numerical value, wherein P<0.05 table Show significant difference, P<0.01 represents that difference is extremely notable.
2 results
2.1 vaccine safety
21 age in days piglet overdose 3 batches of CP974S Adjuvanted vaccines of intramuscular injection, isolated rearing are observed 30 days, and all piglets are equal Without clinical abnormal symptom.Rectal temperature is measured daily within 1~7 day after inoculation, be 39.3~39.6 DEG C.7th day every group after inoculation It is random to take out 2, pathological anatomy is carried out, without obvious Pathologic changes, takes injection site musculature, lungs and lymph node group Knit, tissues observed lesion, (see Figure 11) without exception, show that the Adjuvanted vaccines have higher-security.
2.2 immune swine PCV2 antibody tests
As a result Figure 12 is seen.7-14 days after 3 batches of vaccine immunities, immune group can detect PCV2 antibody；35 days after head is immune, Vaccine immunity group ELISA antibody mean titre is up to 1:More than 2080, antibody level is substantially similar to commercialized vaccine.
2.3 immune swines attack clinical symptoms after poison
Attack it is nonimmune after poison attack in malicious control group and vaccine immunity group, there is body temperature rise in 1~3 day in only indivedual pigs, But Non Apparent Abnormality clinical manifestation, appetite is normal, but vaccine immunity group pig attacks malicious control with respect to daily gain apparently higher than nonimmune Group (P<0.05), but to not attacking the similar (P of blank control group no significant difference of poison>0.05) (Figure 13).
2.4 pathological change
Attack after poison the 25th day, slaughter all test pigs, carry out pathological anatomy and histopathological examination, as a result show, attack The malicious pig of control group 3/5 has slight naked eyes lesion, shows as pig swollen lymph node, lungs elasticity step-down, in 5/5 pig lymph node tissue Lymphocyte is reduced, 5/5 pig macrophages infiltration；3/5 pig lung tissue has inflammatory cell infiltration.CP974S Adjuvanted vaccines and SH All only have in 1 pig lymph node tissue in commercially available vaccine immune group and lymphocyte reduction and macrophages infiltration, other pigs occur The equal Non Apparent Abnormality of lymph node tissue (Figure 14).
PCV2 is detected in 2.5 histoorgans
Attack after poison 25 days, slaughter all test pigs, take lymph node tissue, prepare histotomy, carry out SABC detection PCV2 antigens, (Figure 15) as a result is shown, it is nonimmune to attack malicious control group and have the lymph node tissue PCV antigen positives of 4/5 pig, and own PCV2 antigens are not detected in vaccine immunity group.
PCV2 content detections in 2.6 blood and lymph node tissue
Attack after poison 25 days, slaughter all test pigs, take blood and lymph node tissue, extract viral DNA respectively, use Real- Time PCR methods detect PCV2 contents, the results are shown in Table 1.Vaccine immunity group blood and lymph node tissue, PCV2 contents are notable Malicious control group (P is attacked less than nonimmune<0.05), without significant difference (P between CP974S Adjuvanted vaccines and SH commercially available vaccine immune groups <0.05) pig body PCV2 infection can effectively be reduced after, showing vaccine immunity.
Table 1. attacks PCV2Real-time PCR testing results in malicious pig blood and lymph node tissue

Claims (10)

  1. A kind of 1. Water Soluble Compound immunologic adjuvant, it is characterised in that：Carbomer, hydrogen-oxygen are included in the Water Soluble Compound immunologic adjuvant Change sodium, DNA of bacteria and tocopherol.
  2. 2. Water Soluble Compound immunologic adjuvant according to claim 1, it is characterised in that：In the Water Soluble Compound immunologic adjuvant The concentration of each component is：5~6g/L of carbomer, sodium hydroxide 5%~6%, μ g/mL of DNA of bacteria 20~30, tocopherol 0.01%~0.03%.
  3. 3. Water Soluble Compound immunologic adjuvant according to claim 1 or 2, it is characterised in that：The immune assistant of the Water Soluble Compound At least one of capsular polysaccharide and bacterioprotein OMP are also included in agent.
  4. 4. Water Soluble Compound immunologic adjuvant according to claim 3, it is characterised in that：Described capsular polysaccharide is water-soluble at this Concentration in property compound immunological adjuvant is 20~30 μ g/mL, and described bacterioprotein OMP is in the Water Soluble Compound immunologic adjuvant Concentration be 30~40 μ g/mL.
  5. 5. according to any described Water Soluble Compound immunologic adjuvant in Claims 1 to 4, it is characterised in that：Described DNA of bacteria For haemophilus parasuis DNA, described capsular polysaccharide is haemophilus parasuis capsular polysaccharide, and described bacterioprotein OMP is pair Haemophilus suis mycoprotein OMP.
  6. 6. according to any described Water Soluble Compound immunologic adjuvant in Claims 1 to 5, it is characterised in that：The Water Soluble Compound Carbomer, sodium hydroxide, haemophilus parasuis capsular polysaccharide, haemophilus parasuis mycoprotein OMP, pair are included in immunologic adjuvant Haemophilus suis DNA and tocopherol；The concentration of wherein each component is：5~6g/L of carbomer, sodium hydroxide 5%~6%, secondary pig μ g/mL of haemophilus capsular polysaccharide 20~30, μ g/mL of haemophilus parasuis mycoprotein OMP 30~40, haemophilus parasuis μ g/mL of DNA 20~30, tocopherol 0.01%~0.03%.
  7. 7. according to any described Water Soluble Compound immunologic adjuvant in claim 1~6, it is characterised in that：The Water Soluble Compound Immunologic adjuvant is prepared using following methods：Carbomer is added carbomer jelly is configured in distilled water；Using preceding to described The sodium hydroxide solution of sterilizing is slowly added in carbomer jelly, described carbomer jelly is converted into water-soluble character, Then other components are added, filtration sterilization, adds sterile purified water constant volume and is sufficiently stirred mixing, 2~8 DEG C of preservations.
  8. 8. according to any described Water Soluble Compound immunologic adjuvant in claim 1~7, it is characterised in that：Described carbomer For Carbomer974 or carbomer 934.
  9. 9. application of any described Water Soluble Compound immunologic adjuvant in vaccine is prepared in claim 1~8.
  10. 10. a kind of vaccine combination, it is characterised in that include the immune assistant of any described Water Soluble Compound in claim 1~7 Agent and immunogene.
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