CN101670104A - Oil adjuvant containing panaxoside and preparation method thereof - Google Patents
Oil adjuvant containing panaxoside and preparation method thereof Download PDFInfo
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Abstract
The invention provides an oil adjuvant containing panaxoside and a preparation method thereof. The oil adjuvant is a mixture consisting of panaxoside, adjuvant oil, aluminium stearate and sorbitol mono-oleic acid ester, wherein per milliliter of the oil adjuvant contains 10-100Mug of panaxoside. The preparation method comprises the following steps: heating the oil for preparing the adjuvant to thetemperature of 60-150 DEG C, adding 10-20milligram of aluminium stearate into per milliliter of oil, and mixing evenly; after the temperature of the oil reduces to 50-20 DEG C, adding 5-7% of dehydro-sorbitol mono-oleic acid ester according to volume ratio, stirring evenly, and filtering for standby; and using dimethyl sulfoxide or tween solvent to dissolve the panaxoside into solution; adding 10-100Mug of panaxoside solution into the per milliliter of the prepared standby matter, mixing evenly, and sterilizing for 30min at the temperature of 110 DEG C. Vaccines for immune animals, which is prepared by using oil containing panaxoside as the adjuvant, greatly improve the strength of specificity cells induced by the vaccines and humoral immune response, and reduce local stimulation reactionof injection sites.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of new vaccine adjuvant and preparation method thereof, be specially oily adjuvant of a kind of ginsenoside of containing and preparation method thereof, adopt this adjuvant to prepare the local excitation reaction that vaccine can increase substantially vaccine-induced specific immune response intensity and reduce the injection site.
Background technology
China is that the first in the world is cultured big country, and poultry quantity is huge, and the live pig of delivering for sale every year, beef cattle, sheep are near 900,000,000 (heads).Animal epidemic is of a great variety, after the large-scale cultivation degree improves, and the easier propagation of eqpidemic disease, so the task of animal epidemic control is very arduous.Using vaccine that poultry are carried out compulsory immunization, is one of important measures of eqpidemic disease prevention and control.With cloven-hoofed animal (pig, cattle, sheep etc.) foot and mouth disease is example, China implements 100% and forces planned immunization control policy, need inoculation foot-and-mouth disease vaccine 2～3 times every year, year is 10,000,000,000 milliliters with the vaccine total amount, expense is fully appropriated by country and local finance, only buys annual tens00000000 yuans of the expenditures that just need of vaccine.
Adjuvant is a kind of indispensable composition in the inactivated vaccine, plays a part to strengthen or the reaction of adjusting vaccine immunity.Contain 1 milliliter of adjuvant approximately in every part finished product vaccine, account for 50% of finished product amount of vaccine.The adjuvant of the inactivated vaccines such as foot and mouth disease that China adopts at present is oily adjuvant, mainly is the oily adjuvant (being called for short 206) of homemade mineral oil and French import.But the immune effect of some tank oil Adjuvanted vaccines is not very good at present.Show that such as the investigation of thanking to qin people such as [1] having injected only has behind the foot-and-mouth disease vaccine of oil-containing adjuvant 20.9% piglet to produce enough strong immunoreation, still there is the danger of infection in remaining piglet.People such as Hao Dali find after having analyzed 91 parts of foot-and-mouth disease vaccine piglet serum of having injected the oil-containing adjuvant, only have 31.9% the contained antibody of serum to reach protectiveness level [2].In addition, oily adjuvant mainly promotes humoral immune reaction, and a little less than the pair cell immunoreation.
Summary of the invention
The objective of the invention is to overcome some oil-adjuvant vaccine immune effect differences and the big shortcoming of injection site local excitation, for this reason, the invention provides a kind of new adjuvant, adopt the vaccine of this adjuvant preparation when increasing substantially vaccine-induced cell and humoral immune reaction intensity, to reduce the local excitation reaction of injection site.
A kind of oily adjuvant that contains the ginsenoside of the present invention is a kind of mixture of being made up of ginsenoside, adjuvant oil, aluminium stearate and sorbitol monooleate, contains ginsenoside 10～100 μ g in every milliliter of oily adjuvant.
The preparation method of the oily adjuvant of Radix Ginseng Saponin of the present invention is:
(1) oil that will prepare adjuvant heats 60～150 ℃, adds 10～20 milligrams of aluminium stearate, mixing at every milliliter of oil;
(2) treat that oily relaxing the bowels with purgatives of warm nature is reduced to 50～20 ℃ after, add 5～7% sorbitol monooleates that anhydrate by volume, stir, filter, standby;
(3) become solution with dimethyl sulfoxide or tween dissolution with solvents ginsenoside;
(4) add ginsenoside's solution 10～100 μ g in the spares that every milliliter of step 2 makes, mixing was sterilized 30 minutes for 110 ℃, cooling, promptly.
The adjuvant oil of the oily adjuvant of described preparation is MONTANIDE ISA 206 oil or homemade No. 10 white oils.
The oil supply heating is in order to make more good dissolving of aluminium stearate.
The invention has the beneficial effects as follows, be that the vaccine immunity animal that adjuvant is prepared from can increase substantially vaccine-induced specific cell and humoral immune reaction intensity and the local excitation reaction that reduces the injection site with the oil that contains the ginsenoside.In addition, novel oily adjuvant of the present invention is used for production of vaccine, need not to change existing production technology, method is easy.
Description of drawings
Fig. 1: 70 ICR mices are divided into 7 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml: (1) FMDV+ normal saline group; (2) FMDV+10 white oil; (3) FMDV+10 white oil+GSLS (1 μ g); (4) FMDV+10 white oil+GSLS (5 μ g); (5) FMDV+10 white oil+GSLS (10 μ g); (6) FMDV+10 white oil+GSLS (15 μ g); (7) FMDV+10 white oil+GSLS (20 μ g).Every injected in mice 2 times, 3 weeks at interval.Two exempt from back 2 all eye socket venous blood collections, detect the serum IgG level.Two different persons of block diagram subscript letter represent that there were significant differences (P＜0.05).
Fig. 2: 70 ICR mices are divided into 7 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml: (1) FMDV+ normal saline group; (2) FMDV+10 white oil; (3) FMDV+10 white oil+GSLS (2 μ g); (4) FMDV+10 white oil+GSLS (4 μ g); (5) FMDV+10 white oil+GSLS (6 μ g); (6) FMDV+10 white oil+GSLS (8 μ g); (7) FMDV+10 white oil+GSLS (10 μ g).Every injected in mice 2 times, 3 weeks at interval.Two exempt from back 2 all eye socket venous blood collections, detect the serum IgG level.Two different persons of block diagram subscript letter represent that there were significant differences (P＜0.05).
Fig. 3: 40 ICR mices are divided into 5 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml: (1) 1 dose F MDV antigen+normal saline group; (2) 1 dose F MDV antigens+No. 10 white oils; (3) 1 dose F MDV antigens+No. 10 white oil+GSLS; (4) 0.5 dose F MDV antigens+No. 10 white oil+GSLS; (5) 0.25 dose F MDV antigens+No. 10 white oil+GSLS.Every injected in mice 2 times, 3 weeks at interval.Two exempt from back 2 all eye socket venous blood collections, detect the serum IgG level.Two different persons of block diagram subscript letter represent that there were significant differences (P＜0.05).
Fig. 4: 40 ICR mices are divided into 5 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml: (1) 1 dose F MDV antigen+normal saline group; (2) 1 dose F MDV antigen+206 oil; (3) 1 dose F MDV antigen+206 oil+GSLS; (4) 0.5 dose F MDV antigen+206 oil+GSLS; (5) 0.25 dose F MDV antigen+206 oil+GSLS.Every injected in mice 2 times, 3 weeks at interval.Two exempt from back 2 all eye socket venous blood collections, detect the serum IgG level.Two different persons of block diagram subscript letter represent that there were significant differences (P＜0.05).
Fig. 5: 24 ICR mices are divided into 3 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml: (1) FMDV antigen+No. 10 white oils; (2) FMDV antigen+206 oil; (3) FMDV antigen+No. 10 white oil+GSLS.Every injected in mice 2 times, 3 weeks at interval.Two exempt from back 2 all eye socket venous blood collections, detect the serum IgG level.Two different persons of block diagram subscript letter represent that there were significant differences (P＜0.05).
Fig. 6: 48 ICR mices are divided into 6 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml: (1) FMDV antigen+normal saline group; (2) commodity FMDV vaccine; (3) FMDV antigen+No. 10 white oil; (4) FMDV antigen+No. 10 white oil+GSLS (4 μ g); (5) FMDV antigen+206 oil; (6) FMDV antigen+206 oil+GSLS (4 μ g).Every injected in mice 2 times, 3 weeks at interval.Two mice dislocation put to death after exempting from after, put in 75% ethanol and soaked 15 minutes, whole skin of abdomen is cut from mice pubis leading edge with eye scissors, be immersed in and fix in the neutral formalin more than 24 hours, tissue is completely fixed.After skin blotted surface liquid with absorbent paper, measure skin thickness with slide gauge.Two different persons of block diagram subscript letter represent that there were significant differences (P＜0.05).
The specific embodiment
The explanation of the used main agents of embodiment:
1, aluminium stearate is a Wenzhou City chemistry materials factory product, white powder, and purity is with Al
2O
3Demarcate 8.4～9.4%.
2, the sorbitol monooleate that anhydrates is Wenzhou clear and bright chemical industry company limited product, and its technical specification is as follows
Fatty acid %....................................................... ... ... ... ... ... 71-75
Polyhydric alcohol %....................................................... ... ... ... ... ... ..29.5-33.5
Acid number (mgKOH/g) ... ... ... ... ... ... ... ... ... ... ... ... ..≤8
Saponification number (mgKOH/g) ... ... ... ... ... ... ... ... ... ... ... .145-160
Hydroxyl value (mgKOH/g) ... ... ... ... ... ... ... ... ... ... ... ... .193-210
Moisture %....................................................... ... ... ... ... ... ...≤2.0
3, white oil is a slab oil, the Hangzhou Refinery product.It is through the mineral oil behind the special deep refining.White oil is colourless, tasteless, chemical inertness, light stability can be good, basic composition is saturated hydrocarbon structure, and materials such as aromatic hydrocarbon, nitrogenous, oxygen, sulfur are similar to zero.Because this super refining depth in actual manufacturing process, is difficult to heavy fraction is implemented, so the molecular weight of white oil is usually all within the 250-450 scope.Have good oxidative stability, chemical stability, light stability, colourless, tasteless, do not corrode fiber textile.As adjuvant is No. 10 white oils.
4, tween is available from Wenzhou clear and bright chemical industry company limited product, specification:
Acid number (mg KOH/g) ... ... ... ... ... ... ... ... ... ... ...≤2.2
Saponification number (mg KOH/g) ... ... ... ... ... ... ... ... ... ... .45-60
Hydroxyl value (mg KOH/g) ... ... ... ... ... ... ... ... ... ... ... 65-80
Iodine number (gl/100g) ... ... ... ... ... ... ... ... ... ... ... .18-24
PH value ... ... ... ... ... ... ... ... ... ... ... ... ... .5.0-8.0
Proportion ... ... ... ... ... ... ... ... ... ... ... ... ... ..1.06-1.09
Viscosity (mm
2/ S, 25 ℃) ... ... ... ... ... ... ... ... ... ... 350-550
Moisture %....................................................... ... ... ... ...≤3.0
Ignition residue %....................................................... ... ... ... ..≤0.2
Heavy metal (Pb meter) %....................................................... ... .≤0.001
Refrigeration test ... ... ... ... ... ... ... ... ... ... ... ... ..5 ± 2 ℃/24hr
Embodiment 1: the oily adjuvant that contains the ginsenoside with homemade No. 10 white oils preparation
1. measure No. 10 white oils of 1000ml (Hangzhou Refinery product) with graduated cylinder, put in the aluminum pot.
2. with electric furnace heating white oil, add the 20g aluminium stearate in white oil, the limit adds, and stir on the limit, and it is dissolved fully.
3. treat that the white oil temperature descends slightly, in white oil, add class of 60ml department, stir, filter, get limpid oil with the aseptic hospital gauze of multilamellar, standby.
4. take by weighing 4g stem and leaf of Radix Ginseng saponin (the grand Pharmaceutical product of a specified duration in Jilin), be added in the 10ml dimethyl sulfoxide (DMSO), mixing, dissolving, standby.
5. get stem and leaf of Radix Ginseng saponin solution 100 microlitres that step 4 makes and add in the 1000ml oil adjuvant that step 2 makes, abundant mixing, 110 ℃, sterilization in 30 minutes, cooling promptly gets every milliliter of oily adjuvant that contains ginsenoside 40 μ g.
Embodiment 2: French MONTANIDE ISA 206 oil are used to prepare the oily adjuvant that contains the ginsenoside
1. measure 1000ml MONTANIDE ISA 206 adjuvants (French SEPPIC company) with graduated cylinder, put in the aluminum pot.
2. with electric furnace heating white oil, add the 16g aluminium stearate in white oil, the limit adds, and stir on the limit, and it is dissolved fully.
3. treat that the white oil temperature descends slightly, in white oil, add 60ml Span-80, stir, filter, get limpid oil with the aseptic hospital gauze of multilamellar, standby.
4. take by weighing 10g stem and leaf of Radix Ginseng saponin (the grand Pharmaceutical product of a specified duration in Jilin), be added in the 10ml dimethyl sulfoxide (DMSO), mixing, dissolving, standby.
5. get stem and leaf of Radix Ginseng saponin solution 100 microlitres that step 4 makes and add in the 1000ml oil adjuvant that step 2 makes, abundant mixing, 110 ℃, sterilization in 30 minutes, cooling promptly gets every milliliter of oily adjuvant that contains ginsenoside 100 μ g.
Embodiment 3: ginsenoside's different content is to one of influence of immune effect of vaccine in the oil seepage
1. the preparation of oily adjuvant
(1) measures No. 10 white oils of 1000ml (Hangzhou Refinery product) with graduated cylinder, put in the aluminum pot.
(2) with electric furnace heating white oil, add the 20g aluminium stearate in white oil, the limit adds, and stir on the limit, and it is dissolved fully.
(3) treat that the white oil temperature descends slightly, in white oil, add 60ml Span-80, stir, filter, get limpid oil with the aseptic hospital gauze of multilamellar, standby.
(4) take by weighing 0.5g stem and leaf of Radix Ginseng saponin (GSLS) (the grand Pharmaceutical product of a specified duration in Jilin), be added in the test tube that contains 10ml dimethyl sulfoxide (DMSO), mixing, dissolving, standby.
(5) get 5 in 200ml beaker, in 5 beakers, add 100 milliliters of the oil that step 3 makes respectively, add stem and leaf of Radix Ginseng saponin solution 25 microlitres, 125 microlitres, 250 microlitres, 375 microlitres, 500 microlitres that step 4 makes then respectively.Abundant mixing, 110 ℃, sterilization in 30 minutes, cooling promptly gets every milliliter and contains the oily adjuvant that the ginsenoside is respectively 10 micrograms, 50 micrograms, 100 micrograms, 150 micrograms and 200 micrograms.
2.FMDV the preparation of experimental vaccine
In deactivation O type foot and mouth disease virus (FMDV) 10ml, add 0.3ml tween 80, mixing.1ml FMDV antigen is joined in the 1ml oil phase, and the limit adds, and stir on the limit, treat that water all adds after, the reuse high speed agitator continued 10 minutes, promptly formed the oil-adjuvant vaccine of water-in-oil type.
3. laboratory animal and grouping
70 ICR mices are divided into 7 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml:
(1) FMDV+ normal saline group
(2) FMDV+10 white oil
(3) FMDV+10 white oil+GSLS (1 μ g)
(4) FMDV+10 white oil+GSLS (5 μ g)
(5) FMDV+10 white oil+GSLS (10 μ g)
(6) FMDV+10 white oil+GSLS (15 μ g)
(7) FMDV+10 white oil+GSLS (20 μ g)
Every injected in mice 2 times, 3 weeks at interval.
4. sampling
2 all eye socket venous blood collections behind the booster immunization, blood sample is put 4 ℃ and is spent the night, and 3000 left the heart 10 minutes, and get serum and put in the centrifuge tube ,-20 ℃ of preservations, standby.
5.FMDV specific IgG detects
(1) every hole adds the cattle resisting O-type foot and mouth disease virus antibody (1: 1000) (Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science) of 50 μ l through carbonate buffer solution (pH9.6) dilution on elisa plate, shrouding, and 4 ℃ are spent the night.
(2) with cleaning mixture washing 5 times, each 300 μ l pat dry.Every hole adds 300 μ l phosphate buffers (containing 5% defatted milk+0.05% tween 20) sealing, hatches 2 hours for 37 ℃.
(3) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the O type foot-and-mouth disease virus antigen of 50 μ l through dilution in 1: 3, and the vibration mixing was hatched 2 hours for 4 ℃.
(4) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the to be checked serum of 50 μ l through phosphate buffer (containing 0.05% tween 20) 1: 50 dilution, hatches 1 hour for 37 ℃.
(5) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the anti-Cavia porcellus IgG of goat (h+l) antibody (U.S. Betheyl Laboratory company) through dilution in 1: 1000.Hatched 1 hour for 37 ℃.
(6) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds through 1: 10000 anti-goat IgG FC-HRP of rabbit, hatches 1 hour for 37 ℃.With cleaning mixture washing 5 times, each 300 μ l pat dry.
(7) every hole adds 100 μ l tmb substrate solution.Incubated at room 15 minutes, every hole add 50 μ l 2M H
2SO
4Cessation reaction, and the mixing that vibrates gently.In 15 minutes, be determined at the OD value of 450nm wavelength with microplate reader.
6. result
The FMDV specific IgG level that contains ginsenoside's 1 to 10 μ g oil-adjuvant vaccine group is higher than the matched group that does not contain the ginsenoside,
Wherein contain ginsenoside's 5 μ g oil-adjuvant vaccine group IgG levels and be significantly higher than the matched group (P＜0.05) that does not add the ginsenoside.
Embodiment 4: ginsenoside's different content is to two of the influence of immune effect of vaccine in the oil seepage
1. the preparation of oily adjuvant
Step (1) to (4) is with example 3.
(5) get 5 in 200ml beaker, in 5 beakers, add 100 milliliters of the oil that step 3 makes respectively, add stem and leaf of Radix Ginseng saponin solution 50 microlitres, 100 microlitres, 150 microlitres, 200 microlitres, 250 microlitres that step 4 makes then respectively.Abundant mixing, 110 ℃, sterilization in 30 minutes, cooling promptly gets every milliliter and contains the oily adjuvant that ginsenoside (GSLS) is respectively 20 micrograms, 40 micrograms, 60 micrograms, 80 micrograms and 100 micrograms.
2.FMDV the preparation of experimental vaccine
With example 3.
3. laboratory animal and grouping
70 ICR mices are divided into 7 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml:
(1) FMDV+ normal saline group
(2) FMDV+10 white oil
(3) FMDV+10 white oil+GSLS (2 μ g)
(4) FMDV+10 white oil+GSLS (4 μ g)
(5) FMDV+10 white oil+GSLS (6 μ g)
(6) FMDV+10 white oil+GSLS (8 μ g)
(7) FMDV+10 white oil+GSLS (10 μ g)
Every injected in mice 2 times, 3 weeks at interval.
4. sampling
With example 3.
5.FMDV specific IgG detects
With example 3.
6. result
The FMDV specific IgG level that contains ginsenoside's 2 to 10 μ g oil-adjuvant vaccine groups is higher than the matched group that does not contain the ginsenoside, wherein contains ginsenoside 5 μ g and 6 μ g oil-adjuvant vaccine group IgG levels are significantly higher than the matched group (P＜0.05) that does not add the ginsenoside.
Embodiment 5: ginsenoside's oil adjuvant (homemade No. 10 oil) is to the influence of different antigenic content immune effect of vaccine
1. the preparation of oily adjuvant
Step (1) to (4) is with example 3.
(5) oil that adding step 3 makes in the 200ml beaker is 100 milliliters, adds stem and leaf of Radix Ginseng saponin solution 100 microlitres that step 4 makes then, abundant mixing, and 110 ℃, sterilization in 30 minutes, cooling promptly gets every milliliter and contains ginsenoside (GSLS) 40 micrograms oil adjuvant.
2.FMDV the preparation of experimental vaccine
With example 3.
3. laboratory animal and grouping
40 ICR mices are divided into 5 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml:
(1) 1 dose F MDV antigen+normal saline group
(2) 1 dose F MDV antigens+No. 10 white oils
(3) 1 dose F MDV antigens+No. 10 white oil+GSLS
(4) 0.5 dose F MDV antigens+No. 10 white oil+GSLS
(5) 0.25 dose F MDV antigens+No. 10 white oil+GSLS
Every injected in mice 2 times, 3 weeks at interval.
4. sampling
With example 3.
5.FMDV specific IgG detects
(1) every hole adds the cattle resisting O-type foot and mouth disease virus antibody (1: 1000) (Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science) of 50 μ l through carbonate buffer solution (pH9.6) dilution on elisa plate, shrouding, and 4 ℃ are spent the night.
(2) with cleaning mixture washing 5 times, each 300 μ l pat dry.Every hole adds 300 μ l phosphate buffers (containing 5% defatted milk+0.05% tween 20) sealing, hatches 2 hours for 37 ℃.
(3) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the O type foot-and-mouth disease virus antigen of 50 μ l through dilution in 1: 3, and the vibration mixing was hatched 2 hours for 4 ℃.
(4) with phosphate buffer washing 5 times, each 300 μ l pat dry.Add the to be checked serum of 50 μ l in every row's first hole, make doubling dilution then through phosphate buffer (containing 0.05% tween 20) 1: 20 dilution.Hatched 1 hour for 37 ℃.
(5) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the anti-Cavia porcellus IgG of goat (h+l) antibody (U.S. Betheyl Laboratory company) through dilution in 1: 1000.Hatched 1 hour for 37 ℃.
(6) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds through 1: 10000 anti-goat IgG FC-HRP of rabbit, hatches 1 hour for 37 ℃.With cleaning mixture washing 5 times, each 300 μ l pat dry.
(7) every hole adds 100 μ l tmb substrate solution.Incubated at room 15 minutes, every hole add 50 μ l 2M H
2SO
4Cessation reaction, and the mixing that vibrates gently.In 15 minutes, be determined at the OD value of 450nm wavelength with microplate reader.
(8) the maximum dilution multiple in OD value positive greater than cutoff value (immune serum is handled the OD value surveyed through said method, multiply by 2.1 then), a series of positive hole of serum is tiring of this serum.
6. result
Adopting the inventive method is the new adjuvant that feedstock production contains the ginsenoside with homemade No. 10 white oils, with this adjuvant configuration FMDV vaccine.When antigen dose equated, the antibody titer (1: 373) of new Adjuvanted vaccines was significantly higher than tire (1: 109) (P＜0.05) of former Adjuvanted vaccines.The reduce by half antibody titer (1: 202) of vaccine of new adjuvant and antigen dose is higher than tire (109) of former Adjuvanted vaccines, but no difference of science of statistics (P＞0.05).The antibody titer (1: 50) of new adjuvant and 1/4 antigen dose vaccine is lower than tire (1: 109) of former Adjuvanted vaccines, but no difference of science of statistics (P＞0.05).Illustrate that new adjuvant that to adopt the inventive method be feedstock production with homemade No. 10 white oils can reduce the antigen consumption and immune effect is constant.
Embodiment 6: ginsenoside's oil adjuvant (ISA 206 oil) is to the influence of different antigenic content immune effect of vaccine
1. the preparation of oily adjuvant
(1) measures 1000ml MONTANIDE ISA 206 adjuvants (French SEPPIC company) with graduated cylinder, put in the aluminum pot.
(2) with electric furnace heating white oil, add the 20g aluminium stearate in white oil, the limit adds, and stir on the limit, and it is dissolved fully.
(3) treat that the white oil temperature descends slightly, in white oil, add 60ml Span-80, stir, filter, get limpid oil with the aseptic hospital gauze of multilamellar, standby.
(4) take by weighing 0.5g stem and leaf of Radix Ginseng saponin (the grand Pharmaceutical product of a specified duration in Jilin), be added in the test tube that contains 10ml dimethyl sulfoxide (DMSO), mixing, dissolving, standby.
(5) oil that adding step 3 makes in the 200ml beaker is 100 milliliters, adds stem and leaf of Radix Ginseng saponin solution 100 microlitres that step 4 makes then, abundant mixing, and 110 ℃, sterilization in 30 minutes, cooling promptly gets every milliliter and contains ginsenoside's 40 micrograms oil adjuvant.
2.FMDV the preparation of experimental vaccine
With example 3.
3. laboratory animal and grouping
40 ICR mices are divided into 5 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml:
(1) 1 dose F MDV antigen+normal saline group
(2) 1 dose F MDV antigen+206 oil
(3) 1 dose F MDV antigen+206 oil+GSLS
(4) 0.5 dose F MDV antigen+206 oil+GSLS
(5) 0.25 dose F MDV antigen+206 oil+GSLS
Every injected in mice 2 times, 3 weeks at interval.
4. sampling
With example 3.
5.FMDV specific IgG detects
(1) every hole adds the cattle resisting O-type foot and mouth disease virus antibody (1: 1000) (Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science) of 50 μ l through carbonate buffer solution (pH9.6) dilution on elisa plate, shrouding, and 4 ℃ are spent the night.
(2) with cleaning mixture washing 5 times, each 300 μ l pat dry.Every hole adds 300 μ l phosphate buffers (containing 5% defatted milk+0.05% tween 20) sealing, hatches 2 hours for 37 ℃.
(3) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the O type foot-and-mouth disease virus antigen of 50 μ l through dilution in 1: 3, and the vibration mixing was hatched 2 hours for 4 ℃.
(4) with phosphate buffer washing 5 times, each 300 μ l pat dry.Add the to be checked serum of 50 μ l in every row's first hole, make doubling dilution then through phosphate buffer (containing 0.05% tween 20) 1: 20 dilution.Hatched 1 hour for 37 ℃.
(5) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the anti-Cavia porcellus IgG of goat (h+l) antibody (U.S. Betheyl Laboratory company) through dilution in 1: 1000.Hatched 1 hour for 37 ℃.
(6) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds through 1: 10000 anti-goat IgG FC-HRP of rabbit, hatches 1 hour for 37 ℃.With cleaning mixture washing 5 times, each 300 μ l pat dry.
(7) every hole adds 100 μ l tmb substrate solution.Incubated at room 15 minutes, every hole add 50 μ l 2M H
2SO
4Cessation reaction, and the mixing that vibrates gently.In 15 minutes, be determined at the OD value of 450nm wavelength with microplate reader.
(8) the maximum dilution multiple in OD value positive greater than cutoff value (immune serum is handled the OD value surveyed through said method, multiply by 2.1 then), a series of positive hole of serum is tiring of this serum.
6. result
Adopt the inventive method with French ISA 206 oil preparation novel adjuvants, with the FMDV vaccine of new adjuvant configuration, when antigen dose equated, the antibody titer (1: 549) of new Adjuvanted vaccines was significantly higher than tire (1: 174) (P＜0.05) of former Adjuvanted vaccines.The reduce by half antibody titer (1: 254) of vaccine of new adjuvant and antigen dose is higher than tire (174) of former Adjuvanted vaccines, but no difference of science of statistics (P＞0.05).The antibody titer (1: 160) of new adjuvant and 1/4 antigen dose vaccine is lower than tire (1: 174) of former Adjuvanted vaccines, but no difference of science of statistics (P＞0.05).Illustrate that new adjuvant that to adopt the inventive method be feedstock production with French ISA 206 oil can reduce the antigen consumption and immune effect is constant.
Embodiment 7: new adjuvant that homemade No. 10 white oils are made and French ISA 206 adjuvants are relatively
1. the preparation of oily adjuvant
The preparation of No. 10 white oils is with example 5.The preparation of France's 206 oil is with example 6.
2.FMDV the preparation of experimental vaccine
With example 3.
3. laboratory animal and grouping
24 ICR mices are divided into 3 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml:
(1) FMDV antigen+No. 10 white oil
(2) FMDV antigen+206 oil
(3) FMDV antigen+No. 10 white oil+GSLS
Every injected in mice 2 times, 3 weeks at interval.
4. sampling
With example 3.
5.FMDV specific IgG detects
With example 3.
6. result
10 times of the prices of France's ISA 206 oil are to homemade No. 10 white oils.Adopt method of the present invention in homemade No. 10 white oils, to add the adjuvant properties that the ginsenoside can improve state's produce oil significantly.The vaccine that is higher than French ISA 206 oil configurations with the immune effect of vaccine of this adjuvant configuration.
Embodiment 8: stem and leaf of Radix Ginseng saponin is to the partial abirritate effect of inoculation
1. the preparation of oily adjuvant
The preparation of No. 10 white oils is with example 5.The preparation of France's 206 oil is with example 6.
2.FMDV the preparation of experimental vaccine
With example 3.
3. laboratory animal and grouping
48 ICR mices are divided into 6 groups at random, respectively the following experimental vaccine of subcutaneous injection 0.2ml:
(1) FMDV antigen+normal saline group
(2) commodity FMDV vaccine
(3) FMDV antigen+No. 10 white oil
(4) FMDV antigen+No. 10 white oil+GSLS (4 μ g)
(5) FMDV antigen+206 oil
(6) FMDV antigen+206 oil+GSLS (4 μ g)
Every injected in mice 2 times, 3 weeks at interval.
4. skin collection
After mice dislocation execution, the labelling group number is put then in 75% ethanol and was soaked 15 minutes, whole skin of abdomen is cut from mice pubis leading edge with eye scissors, is immersed in and fixes in the neutral formalin more than 24 hours, and tissue is completely fixed.
5. skin thickness is measured
After fixed skin blotted surface liquid with absorbent paper, measure skin thickness with slide gauge, record data.
6. result
Each skin thickness of organizing injected in mice foot-and-mouth disease vaccine position is followed successively by:＞No. 10 white oil oil seepage group＞ISA 206 oil of commercial seedling group＞ISA 206 oil seepages group+＞No. 10 white oil+GSLS oil seepages of GSLS oil seepage group.The adding stem and leaf of Radix Ginseng saponin prepares the vaccine immunity animal in ISA 206 oil and No. 10 white oils, and the skin thickness of injection site significantly reduces (P＜0.05), illustrates that novel adjuvant of the present invention can alleviate the local irritation of oil-adjuvant vaccine.
Claims (3)
1, a kind of oily adjuvant that contains the ginsenoside, this oil adjuvant is applied to vaccine production, it is characterized in that: this oil adjuvant is a kind of mixture of being made up of ginsenoside, adjuvant oil, aluminium stearate and sorbitol monooleate, contains ginsenoside 10～100 μ g in every milliliter of oily adjuvant.
2, a kind of according to claim 1 preparation method that contains ginsenoside's oily adjuvant is characterized in that following steps:
(1) oil that will prepare adjuvant heats 60～150 ℃, adds 10～20 milligrams of aluminium stearate, mixing at every milliliter of oil;
(2) treat that oily relaxing the bowels with purgatives of warm nature is reduced to 50～20 ℃ after, add 5～7% sorbitol monooleates that anhydrate by volume, stir, filter, standby;
(3) make solution with dimethyl sulfoxide or tween dissolution with solvents ginsenoside;
(4) add ginsenoside's solution 10～100 μ g in the spares that every milliliter of step 2 makes, mixing was sterilized 30 minutes for 110 ℃, cooling, promptly.
3, as a kind of preparation method that contains ginsenoside's oily adjuvant as described in the claim 2, it is characterized in that: the adjuvant oil of the oily adjuvant of described preparation is MONTANIDE ISA 206 oil or homemade No. 10 white oils.
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| CN103751775A (en) * | 2013-12-31 | 2014-04-30 | 浙江大学 | Ginsenoside-containing vegetable oil adjuvant and preparation method and application thereof |
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| US9101607B2 (en) | 2010-03-31 | 2015-08-11 | Stabilitech Ltd. | Method for preserving alum adjuvants and alum-adjuvanted vaccines |
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