WO2015101271A1 - 一种含人参皂苷的植物油佐剂及其制备方法和应用 - Google Patents

一种含人参皂苷的植物油佐剂及其制备方法和应用 Download PDF

Info

Publication number
WO2015101271A1
WO2015101271A1 PCT/CN2014/095451 CN2014095451W WO2015101271A1 WO 2015101271 A1 WO2015101271 A1 WO 2015101271A1 CN 2014095451 W CN2014095451 W CN 2014095451W WO 2015101271 A1 WO2015101271 A1 WO 2015101271A1
Authority
WO
WIPO (PCT)
Prior art keywords
oil
vaccine
adjuvant
ginsenoside
vegetable oil
Prior art date
Application number
PCT/CN2014/095451
Other languages
English (en)
French (fr)
Inventor
胡松华
张岑容
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Publication of WO2015101271A1 publication Critical patent/WO2015101271A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus

Definitions

  • the invention relates to the technical field of animal vaccines, in particular to a vegetable oil adjuvant containing ginsenosides, a preparation method and application thereof.
  • Adjuvant is a non-specific immunopotentiator, which is an essential component in inactivated vaccine. Adjuvant can enhance the intensity of vaccine immune response and prolong the duration of immune response. Inactivated vaccines such as foot-and-mouth disease and avian influenza vaccine are currently used in China.
  • the adjuvants are all oil adjuvants. In the preparation of vaccines, oil adjuvants account for about 50% of the finished vaccines.
  • the annual production of two kinds of vaccines for swine foot-and-mouth disease vaccine and avian influenza vaccine requires about 3.15 billion milliliters of oil adjuvant, about 3,000 tons. At present, the main raw material for the production of traditional oil adjuvants is mineral oil.
  • the patent document published as CN101474403A discloses an oil adjuvant for inactivating a vaccine, which is subjected to distillation and hydrogenation to remove sulfur, nitrogen, olefins, colloids, heavy metals and most aromatic impurities, and then at a pressure of 4.5 to 10 MPa.
  • the reaction temperature is 150-290 ° C
  • the second hydrogenation reaction is carried out at a volumetric space velocity of 0.1-0.5 h -1 to obtain a de-aromatized white oil.
  • the de-aromatized white oil and the mannitol oleate are 90:10-100:
  • the volume fraction of 8 is mixed.
  • the patent document published as CN1669584A discloses a method for preparing an animal viral and bacterial vaccine using a vegetable oil adjuvant, wherein the vegetable oil adjuvant is vegetable oil. a mixture with a spoon-80, wherein the vegetable oil is one of rapeseed oil, soybean oil, peanut oil, castor oil, and flax oil, and finally the prepared vegetable oil adjuvant is prepared by mixing with the antigen prepared in the literature. vaccine.
  • the vegetable oil adjuvant not only avoids the potential threat of food safety, but also has the performance of mineral oil adjuvant, but the injection of the immune oil emulsion inactivated vaccine has a high viscosity and is difficult to inject and stay in the injection site for a long time. Side effects such as local tissue inflammation, suppuration, necrosis have not been effectively solved.
  • Traditional Chinese medicine is a valuable heritage of China. Compared with synthetic drugs, it has fewer adverse reactions, less toxic side effects, pleiotropic, two-way regulation and no dependence.
  • Some traditional Chinese medicine active ingredients have good immunomodulatory effects, for example, research shows The traditional ginseng ginseng contained in traditional Chinese medicine ginseng can increase macrophage activity, induce interferon production, and stimulate cytotoxic T lymphocyte activity. Therefore, its advantages as a new vaccine adjuvant are very significant and have been paid more and more attention. .
  • the patent document published as CN101670104A discloses an oil adjuvant containing ginsenoside and a preparation method thereof, the oil adjuvant being composed of ginsenoside, adjuvant oil, aluminum stearate and sorbitol monooleate
  • the mixture contains 10 to 100 ⁇ g of ginsenoside per ml of oil adjuvant.
  • the preparation method comprises the following steps: heating the white oil at 60-150 ° C, adding 10-20 mg of aluminum stearate per ml of oil, and mixing; after the oil temperature drops to 50-20 ° C, adding 5-7 according to the volume ratio.
  • ginsenoside is dissolved in dimethyl sulfoxide or Tween solvent to form a solution; ginsenoside solution is added in an amount of 10 to 100 ⁇ g per ml of the prepared preparation.
  • oil adjuvant containing ginsenoside enhances the specific cell- and humoral immune response intensity induced by the vaccine to a certain extent, it is difficult to avoid the residual aryl-containing aromatic hydrocarbon component containing carcinogenicity in white oil. Therefore, the development of new adjuvants with high efficiency and safety has become an important topic in the field of contemporary immunology research.
  • the present invention provides a vegetable oil adjuvant containing ginsenoside, which has an efficient and safe performance.
  • a vegetable oil adjuvant containing ginsenoside containing edible vegetable oil as an adjuvant oil, containing 10 to 100 ⁇ g/mL of ginsenoside.
  • the invention replaces the traditional mineral oil with the vegetable oil, and avoids the condensed aromatic hydrocarbon component containing the carcinogenic effect in the oil adjuvant, so that the vaccine produced by the oil adjuvant can pose a potential threat to food safety after being injected into the animal, and
  • the oil adjuvant of the invention contains ginsenoside, ginsenoside can increase macrophage activity, induce interferon production, stimulate cytotoxic T lymphocyte activity, and ginseng saponin and vegetable oil play a synergistic adjuvant effect, significantly improving the vaccine. Antibody level.
  • the synergistic adjuvant action with the vegetable oil is also different, and preferably, the content of the ginsenoside is 30 to 50 ⁇ g/mL. More preferably, the content of the ginsenoside is 40 to 45 ⁇ g/mL.
  • the present invention can use various types of vegetable oils instead of mineral oils, and both of them can achieve good effects.
  • the edible vegetable oils are sunflower oil, corn oil, olive oil, camellia oil.
  • rapeseed oil More preferably, the edible vegetable oil is rapeseed oil.
  • the invention also provides a preparation method of the above vegetable oil adjuvant, comprising:
  • sorbitan monooleate and the ginsenoside solution are added to the vegetable oil, and the mixture is uniformly mixed.
  • the preparation method of the vegetable oil adjuvant of the invention is simple and convenient, and dimethyl sulfoxide is an important polar aprotic solvent widely used as a solvent and a reaction reagent, and the content of the ginsenoside in the ginsenoside solution is 10 to 60 mg. /mL.
  • the sorbitan monooleate is added in an amount of 10 to 16 mL of sorbitan monooleate per 100 mL of vegetable oil adjuvant.
  • the invention also provides the use of the above vegetable oil adjuvant for preparing a vaccine.
  • the vegetable oil adjuvant of the present invention is widely used, for example, in the preparation of a virus or a bacterial vaccine.
  • the vaccine is a foot-and-mouth disease inactivated vaccine or a Staphylococcus aureus vaccine.
  • Foot-and-mouth disease is a disease caused by foot-and-mouth disease virus infection of cloven-hoofed animals (pig, cattle, sheep, etc.). If the foot-and-mouth disease vaccine for animals is contained in carcinogens, it will bring potential harm to food safety; golden yellow grapes Cocci can cause diseases in many animals.
  • the vegetable oil adjuvant of the present invention is applied to the preparation of a foot-and-mouth disease inactivated vaccine or a Staphylococcus aureus vaccine, respectively, to obtain an efficient and safe vaccine.
  • the present invention uses a vegetable oil containing ginsenoside to prepare a vaccine, thereby greatly improving the safety of the vaccine, and overcoming the potential hazard of conventional mineral oil adjuvant on food hygiene;
  • the vegetable oil adjuvant of the present invention contains an appropriate amount of ginsenoside, wherein ginsenoside and vegetable oil are mixed to exert a synergistic adjuvant effect, and specific antibodies, lymphocyte transformation, serum cytokines and long-lived lymphocytes are significantly improved.
  • the antibody titer is increased by 4.7 times, and the antibody titer of the mineral oil adjuvant containing ginsenoside is only increased by 3.1 times. Therefore, the vegetable oil adjuvant containing ginsenoside of the present invention greatly increases the antibody level of the vaccine;
  • the preparation of the vegetable oil adjuvant of the invention has good fluidity of the vaccine, is convenient for immunization injection, has small local irritation, and reduces the pain of the animal;
  • Figure 1 is a comparison of viscosity results of a foot-and-mouth disease vaccine prepared using GS (ginsenoside) vegetable oil adjuvant and French ISA 206 adjuvant.
  • Figure 2 is a comparison of antibody responses after immunization of mice immunized with foot-and-mouth disease vaccine with and without the addition of GS vegetable oil.
  • Fig. 3 is a comparison diagram of antibody responses after preparing a foot-and-mouth disease vaccine using vegetable oils having different GS contents and preparing mice immunized with foot-and-mouth disease vaccine using French ISA 206 adjuvant.
  • Fig. 4 is a comparison diagram of antibody subclass reactions after preparing a foot-and-mouth disease vaccine using vegetable oils having different GS contents and preparing mice immunized with foot-and-mouth disease vaccine using French ISA 206 adjuvant.
  • Fig. 5 is a graph showing the level of transformation of spleen lymphocytes after preparation of foot-and-mouth disease vaccine with vegetable oil having different GS contents and mice immunized with foot-and-mouth disease vaccine prepared with French ISA 206 adjuvant.
  • Fig. 6 is a graph showing the comparison of serum interferon gamma levels after preparation of foot-and-mouth disease vaccine with vegetable oil having different GS contents and mice immunized with foot-and-mouth disease vaccine prepared with French ISA 206 adjuvant.
  • Fig. 7 is a comparison of serum interleukin-5 levels after preparation of a foot-and-mouth disease vaccine with vegetable oils having different GS contents and mice immunized with a foot-and-mouth disease vaccine prepared with French ISA 206 adjuvant.
  • Figure 8 is a graph showing the comparison of the number of long-lived plasma cells of bone marrow after immunization of mice immunized with foot-and-mouth disease vaccine with and without the addition of GS vegetable oil.
  • Figure 9 is a graph comparing the synergistic adjuvant effects of GS vegetable oil adjuvant and GS mineral oil adjuvant.
  • Figure 10 is a graphical representation of a synergistic adjuvant for a bacterial vaccine in combination of GS and vegetable oil.
  • Figure 11 is a comparison of the effects of various adjuvants on IgG titers.
  • Figure 12 is a graph comparing the effects of various adjuvants on IgG subclass levels.
  • Figure 13 is a comparative diagram of the effects of various adjuvants on the proliferation activity of splenic lymphocytes.
  • Figure 14 is a graph comparing the effects of various adjuvants on the expression levels of serum cytokine IL-5 in mice.
  • Figure 15 is a graph comparing the effects of various adjuvants on the expression levels of serum cytokine IFN- ⁇ in mice.
  • Figure 16 is a comparison of the effect of ZE515 and aluminum gel adjuvant on IgG titers.
  • Figure 17 is a comparison of the effects of ZE515 and aluminum gel adjuvant on IgG subclass levels.
  • DMSO dimethyl
  • Span-80 sorbitan monooleate
  • inactivated Asian type 1 foot-and-mouth disease (FMD) antigen provided by Jinyu Baoling Biopharmaceutical Co., Ltd.
  • FMD inactivated Asian type 1 foot-and-mouth disease
  • antigen 146s content is 5 ⁇ g
  • Tween-80 0.3 mL
  • the phase and water were mixed in equal volume and emulsified with a high speed homogenizer to prepare a FMD rapeseed oil emulsion vaccine.
  • Span-80 0.6 mL of Span-80 was added to 4.4 mL of ISA 206 adjuvant (SEPPIC, France) at 30 ° C, and uniformly mixed to prepare an oil phase.
  • the mixture was mixed in equal volume and emulsified with a high-speed homogenizer to prepare an FMD206 oil emulsion vaccine.
  • a 1 mL oil emulsion vaccine was pipetted with a pipette having an inner diameter of 1.2 mm, and then the pipette was placed in a vertical position, and the emulsion naturally flowed out 0.4 mL, and the required time was recorded.
  • the GS-Rapeseed oil emulsion efflux time was 1.9 seconds, while the 206 oil emulsion vaccine efflux time was 2.8 seconds. See Figure 1.
  • the viscosity of the GS-Rapeseed oil emulsion vaccine was significantly lower than that of the 206 oil emulsion vaccine.
  • Vaccine 1 Prepare a foot-and-mouth disease GS-Rapeseed oil emulsion vaccine according to Example 21, such that the 146s of Asian type 1 FMD antigen per milliliter of vaccine is 0.5 ⁇ g, GS is 40 ⁇ g, rapeseed oil is 44% (volume ratio), Span - 80 is 6% by volume.
  • Vaccine 2 The Asian type 1 FMD rapeseed oil emulsion vaccine was prepared according to the method of Example 21. The preparation process was carried out without adding GS, so that the FMD antigen per ml of the vaccine contained 156s of 0.5 ⁇ g and the rapeseed oil was 44% (volume ratio). -80 is 6% by volume.
  • Vaccine 3 was dissolved in physiological saline, and then the Asian type 1 FMD antigen was diluted with the solution so that the FMD antigen per liter of vaccine contained 0.5 ⁇ g of Fs and GS of 40 ⁇ g.
  • Vaccine 4 Asian type 1 FMD antigen was diluted with physiological saline to give 0.5 ⁇ g of FMD antigen per ml of 146 s.
  • mice Thirty-two ICR mice were randomly divided into 4 groups, 8 in each group, and each group of mice was injected subcutaneously with 0.2 mL of vaccine twice, at intervals of 3 weeks. Each group of mice was vaccinated each time according to the following protocol:
  • Blood samples were taken from the orbital veins 1 and 2 weeks after the second immunization. The blood samples were placed at 4 ° C overnight, centrifuged at 3000 rpm for 10 minutes, and the serum was placed in a centrifuge tube and stored at -20 ° C for use.
  • TMB substrate solution 100 ⁇ L was added to each well. Incubate for 15 minutes at room temperature, stop the reaction by adding 50 ⁇ L of 2 M H 2 SO 4 to each well, and mix gently by shaking. The OD value at a wavelength of 450 nm was measured with a microplate reader in 15 minutes.
  • the level of FMD antibody in group 2 was significantly higher than that in group 3 and group 4. There was no statistical difference in antibody levels between the other groups, but the antibody level in group 1 was detected twice after the second treatment. The median was significantly higher than the other groups, as shown in Figure 2, indicating that the combination of GS and rapeseed oil played a synergistic adjuvant effect, significantly increasing the level of FMD antibodies.
  • rapeseed oil FMD emulsion vaccines with different GS contents were prepared according to Example 21.
  • An ISA 206 oil adjuvant FMD vaccine was prepared as in Example 21, which contained 0.5 ⁇ g of Asian Type 1 FMD antigen 146s per ml of vaccine, 44% (by volume) of ISA 206 oil, and 6% (volume ratio) of Span-80.
  • mice Fifty ICR mice were randomly divided into 7 groups, 8 in each group. Each group of mice was subcutaneously injected with 0.2 mL of vaccine twice a day for 3 weeks. Each group of mice was vaccinated each time according to the following protocol:
  • Blood was collected from the eyelids 1 week after the first immunization and 1-6 weeks after the second exemption, and serum was prepared to detect anti-FMD-specific IgG, IgG subclasses and serum cytokines in the serum.
  • the number of long-lived plasma cells was determined by separating the bone marrow plasma cells from the femur at the 6th week after the second immunization.
  • the method is the same as in Example 22.
  • the detection steps are as follows:
  • mice Two weeks after the second immunization, the spleens of the mice were aseptically isolated, washed with PBS solution, sieved to prepare a single cell suspension, centrifuged at 380 ⁇ g for 10 min at 4 ° C, resuspended in red blood cell lysate, washed twice with PBS, RPMI 1640 (including 10 After resuspension of % fetal bovine serum, 100 IU/mL penicillin, 100 ⁇ g/mL streptomycin, trypan blue staining counts live cells (live cells >95%) wrong! No bookmarks were specified. error! No bookmarks were specified. .
  • the concentration of IFN- ⁇ in serum was determined using Mouse IFN- ⁇ Sunny ELISA kit (purchased from Lianke Biotechnology Co., Ltd.), and IL-5 concentration was detected by Mouse IL-5 ELISA kit (eBioscience Inc, San Diego, USA).
  • the concentration of cytokines is calculated from the cytokine standard curve.
  • the thigh femur and tibia of the mouse were aseptically isolated, and the single cell suspension was prepared by blasting the bone marrow with a syringe, centrifuged at 380 ⁇ g for 10 min at 4 ° C, resuspended by adding red blood cell lysate, washed twice with PBS, and RPMI 1640 (containing 10% fetal). After resuspending bovine serum, 100 IU/mL penicillin, 100 ⁇ g/mL streptomycin, the cells were counted and adjusted to a cell concentration of 2.4 ⁇ 10 7 cells/mL, and the enzyme-linked immunospot assay reference was used.
  • 2.4 ⁇ 10 6 cells/well cells were added to HA-96 well plates coated with 7 ⁇ g/mL FMDV antigen, incubated for 5 h at 37 ° C in a 5% CO 2 incubator, washed three times with PBS, and HRP-labeled goat anti-mouse IgG was added. H+L) 4 ° C overnight.
  • PBST was washed five times to add AEC coloring solution, 100 ⁇ L of deionized water was added to each well to stop color development, the plate was stored in the dark, and the data was processed by enzyme-linked immunospotting software.
  • mice injected with GS-Rapeseed oil FMD were significantly higher than those in the control group.
  • the levels of serum IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) in the GS-rapeseed oil FMD vaccine mice were significantly higher than those in the unadjuvanted control group, but not significantly compared with the ISA 206 adjuvant group. Difference; serum IgG subclasses were highest when GS 4 ⁇ g (20 ⁇ g GS/mL) was contained in a single injection of FMD vaccine (0.2 mL).
  • mice injected with GS-Rapeseed oil FMD vaccine were significantly higher than that of the control group.
  • the level of lymphocyte transformation in mice injected with GS-Rapeseed oil FMD vaccine was significantly higher than that in the control group without adjuvant, but there was no significant difference compared with the ISA 206 adjuvant group;
  • the level of lymphocyte transformation was highest when GS4 ⁇ g (20 ⁇ g GS/mL) was contained in 0.2mL).
  • mice injected with GS-Rapeseed oil FMD vaccine was significantly higher than that of the control group.
  • the serum levels of IFN- ⁇ and IL-5 in the GS-rapeseed oil FMD vaccine (4 ⁇ g containing GS per vaccine) were significantly higher than those in the unadjuvanted control group, but There was no significant difference compared with the ISA 206 adjuvant group.
  • the number of long-lived plasma cells in the GS-rapeseed oil FMD vaccine (4 ⁇ g of GS per vaccine) was significantly higher than that of the unadjuvanted control group.
  • the preparation method was the same as in Example 21.
  • mice Forty ICR mice were randomly divided into 5 groups, 8 in each group, and each group of mice was injected subcutaneously with 0.2 mL of vaccine twice, at intervals of 3 weeks. Each group of mice was vaccinated each time according to the following protocol:
  • Blood samples were taken from the orbital veins 1 and 2 weeks after the second immunization. The blood samples were placed at 4 ° C overnight, centrifuged at 3000 rpm for 10 minutes, and the serum was placed in a centrifuge tube and stored at -20 ° C for use.
  • TMB substrate solution 100 ⁇ L was added to each well. Incubate for 15 minutes at room temperature, stop the reaction by adding 50 ⁇ L of 2 M H 2 SO 4 to each well, and mix gently by shaking. The OD value at a wavelength of 450 nm was measured with a microplate reader in 15 minutes.
  • the OD value is greater than the cutoff value (the OD value of the unimmunized mouse serum treated by the above method, and then multiplied by 2.1) is positive, and the maximum dilution factor in a series of positive holes of one serum is the effect of the serum. price.
  • the antibody titers of rapeseed oil and ISA 206 oil emulsion FMD vaccine were 1:452 and 1:477, respectively, without adding ginsenoside; GS-rapese oil and GS-ISA after adding ginsenoside
  • the antibody titers of the 206 oil emulsion FMD vaccine were 1:2152 and 1:1505, respectively.
  • the antibody titer was increased by 4.7 times after adding ginsenoside to rapeseed oil, and the antibody titer was increased by 3.1 times after adding ginsenoside to ISA 206.
  • the synergistic adjuvant effect produced by the combination of GS and rapeseed oil is much higher than the synergistic effect produced by the combination of GS and ISA 206.
  • a GS-Rapeseed oil adjuvant was prepared as in Example 10.
  • the following experimental vaccines were prepared according to the adjuvant 100 ⁇ L and the S. aureus toxin antigen 50 ⁇ L ratio:
  • Staphylococcus aureus toxin antigen 50 ⁇ L + rapeseed oil 100 ⁇ L
  • mice 35 ICR female mice were randomly divided into 5 groups of 7 each. Five groups of mice were immunized with subcutaneous injection of the above five experimental vaccines, and the two immunizations were separated by 2 weeks. Blood samples were collected from the eyelids at 1, 2, 3, and 4 weeks after the second immunization. The blood samples were placed in a refrigerator at 4 ° C overnight and then centrifuged (4000 rpm) for 10 min to prepare serum, which was stored at -20 ° C after storage and set aside. 1.3 Detection of specific IgG levels
  • Example 26 Adjuvant effect of the present invention on a CP5 type Staphylococcus aureus vaccine
  • Aluminum Glue Adjuvant (including 20% Al(OH) 3 ), Zhongmu Industrial Co., Ltd. Jiangxi Biopharmaceutical Products; HRP-labeled goat anti-mouse IgG antibody, IgG1 antibody, IgG2a antibody, Santa Cruz product; mouse IFN- ⁇ ELISA Test kit, mouse IL-5 ELISA test kit, Lianke Biotechnology Co., Ltd. products.
  • CP5 type capsular polysaccharide Staphylococcus aureus (HZ016 strain) was isolated from clinical mastitis cases in dairy farms in the suburbs of Hangzhou, inoculated with broth culture medium, cultured at 37 ° C for 20 h, and added 40% per 100 mL of bacterial liquid.
  • S. aureus aluminum gel vaccine containing 15% Al(OH) 3 , containing bacteria ⁇ 1.5 ⁇ 10 7 / mL, toxin 150 ⁇ g / mL
  • Sa-Ag and ZE515 adjuvant were prepared by emulsification at a ratio of 1:2 to prepare a S. aureus vegetable oil emulsion vaccine (containing 1.5 ⁇ 10 7 bacteria) /mL, toxin 150 ⁇ g / mL).
  • Ginsenoside GS-R is a product of Jilinzhou Hongjiu Biotechnology Co., Ltd.
  • mice Female ICR mice, 19-22 g, were purchased from Slack Co., Ltd., Shanghai Experimental Animal Center, Chinese Academy of Sciences. To study the adjuvant effect of ZE515, animals were randomly divided into 5 groups, 8 in each group. Sa-Ag, Sa-Ag+VO, Sa-Ag+GS-R, Sa-Ag+ZE515 and saline were injected subcutaneously into the abdomen. . Each mouse was injected with 0.2 mL and immunized twice, at intervals of two weeks. Three weeks after the second immunization, blood was collected, and the cells were allowed to stand at 37 ° C for 1 h, and then centrifuged at 3,600 rpm for 8 min at 4 ° C overnight. The serum was separated and stored in a 0.2 mL centrifuge tube and stored at -20 ° C. The mice were sacrificed after blood collection, and the spleens were isolated to prepare lymphocytes for lymphocyte proliferation test.
  • An indirect ELISA method was employed. 96-well plates (antigen concentration 13.5 ⁇ g/mL) were added with toxin antigen, serum to be tested (1:200) was added, and IgG was detected by HRP-labeled goat anti-mouse IgG antibody (1:2000). HRP-labeled goat anti-mouse The IgG antibody subclass was detected by IgG1 (1:1000) and IgG2a antibody (1:1000), and the coloration time was 10 min.
  • the spleen was aseptically isolated three weeks after the second administration.
  • the spleen was ground and washed with sterile Hanks solution, filtered through a 200 mesh copper mesh, and the filtrate was centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the red blood cells were lysed with red blood cell lysate, then centrifuged at 1500 rpm for 10 min, the supernatant was discarded, and the Hanks solution was used. After washing twice, the cell concentration was adjusted to 5 ⁇ 10 6 /mL in a cell culture solution (RPMI 1640 medium + 10% fetal bovine serum, 100 ⁇ g/mL streptomycin, 100 IU/mL penicillin).
  • the serum IL-5 and IFN- ⁇ were detected by ELISA kit according to the instructions.
  • mice were randomly divided into 4 groups: group 1, Sa-Ag + ZE515; group 2, Sa-Ag + aluminum gel; group 3, Sa-Ag; group 4: normal saline.
  • group 1 Sa-Ag + ZE515
  • group 2 Sa-Ag + aluminum gel
  • group 3 Sa-Ag
  • group 4 normal saline.
  • Each mouse was injected subcutaneously with 0.2 mL twice a day for two weeks.
  • three weeks after the second immunization each mouse was intraperitoneally injected with 5 ⁇ 10 7 CFU of CP5-type Staphylococcus aureus (HZ016 strain), and the clinical manifestations, morbidity and mortality of the mice were observed.
  • Figure 11 shows that after Sa-Ag and VO or GS-R were mixed, the IgG titers were 1:422 and 1:184, respectively, which were 4 times and 2 times that of the control group (1:95), but no significant difference. (P>0.05); however, VO and GS-R were mixed to form ZE515 and then mixed with Sa-Ag to form an emulsion. The IgG titer increased to 1:2558, which was 27 times higher than that of the control group, which was significantly higher than other groups (P ⁇ 0.05).
  • Figure 12 shows that VO, GS-R and ZE515 have different degrees of enhancement of IgG1 and IgG2a levels, of which ZE515 has a stronger effect on IgG subclass than the other groups, and there is a significant difference compared with the unadjuvanted control group ( P ⁇ 0.05).
  • Figure 13 shows that VO, GS-R and ZE515 promote lymphocyte proliferation activity induced by Con A, LPS and Sa-Ag.
  • the lymphocyte proliferation index of mice immunized with VO, GS-R or ZE515 vaccine was significantly higher than that of the control group.
  • the lymphocyte proliferation index was from high to low, followed by ZE515 group, VO group, GS-R group and no adjuvant control group.
  • Figures 14 and 15 show the levels of serum cytokines IL-5 and IFN- ⁇ in each group of mice. Serum cytokine levels were high to low, followed by ZE515 group, VO group, GS-R group and no adjuvant control group. Serum levels of IL-5 and IFN- ⁇ in the ZE515 group were 2-fold and 6-fold, respectively, compared with the unadjuvanted control group (P ⁇ 0.05).
  • Figure 16 shows that both ZE515 and aluminum gel adjuvants significantly promoted antibody production, but ZE515 promoted significantly higher than aluminum gel adjuvant (P ⁇ 0.05).
  • Figure 17 shows that both ZE515 and aluminum hydroxide adjuvants have a significant effect on the production of IgG subclasses. There was no significant difference in the effect of ZE515 and aluminum gel on IgG1 (P>0.05), but the promotion of IgG2a was significantly higher than that of aluminum hydroxide adjuvant (P ⁇ 0.05).
  • mice showed mental depression after 24 hours of attack, the hair was rough, the body was thin, they did not like activities, they were afraid of cold, and they were slow to respond to external stimuli.
  • ZE515 had the least number of illnesses, followed by aluminum gel, and only one of the control group did not have disease, and all of the saline group were sick.
  • the mice in the ZE515 group only died without disease.
  • the aluminum gel group died only on the 6th day after the challenge, and the control mice died after 3 days of challenge.
  • the mice in the saline group were attacked. Three died within 72 hours, and one died on the fourth and seventh days after the attack.
  • mice in the ZE515 group were able to find that the mice were active and sensitive after 48 hours.
  • the activity frequency of the mice increased after 3 days in the aluminum gel group, and half of the mice were weak after the tolerance, and the movement was slow, and one died.
  • the mice in the control group improved after 4 days, and the exercise was weak.
  • the mice in the saline group gradually recovered their exercise ability after 4 days.
  • the protection rate of the aluminum hydroxide adjuvant vaccine was 50%, which was not significant compared with the control group; the protection rate of the ZE515 adjuvant vaccine was 75%, which was significantly different from the control group (P ⁇ 0.01). ).
  • Th1 lymphocytes secrete cytokines such as IFN- ⁇ to promote the production of IgG2a.
  • IFN- ⁇ activates cytotoxic T lymphocytes and helps to remove intracellular bacteria.
  • Immunization of mice with ZE515 and Sa-Ag-containing S. aureus vaccine resulted in a significant increase in IgG1 and IgG2a levels ( Figure 12), which promoted lymphocyte proliferative responses induced by Con A, LPS and Sa-Ag ( Figure 13).
  • Significantly increased the production of IL-5 and IFN- ⁇ ( Figures 14 and 15), indicating that the ZE515-containing adjuvant can simultaneously promote Th1 and Th2-type immune responses and produce adjuvant effects on bacterial vaccines.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)

Abstract

本发明公开了一种含人参皂苷的植物油佐剂及其制备方法和应用，以使用植物油作为佐剂油，含有10-100µg/mL的人参皂苷。

Description

一种含人参皂苷的植物油佐剂及其制备方法和应用 技术领域
本发明涉及动物疫苗技术领域，具体涉及一种含人参皂苷的植物油佐剂及其制备方法和应用。
背景技术
佐剂是非特异性免疫增强剂，是灭活疫苗中的一种必要成分，佐剂可增强疫苗免疫反应的强度和延长免疫反应的持续时间，我国目前采用的口蹄疫等灭活疫苗和禽流感疫苗的佐剂均为油佐剂。在疫苗制备中，油佐剂约占成品疫苗量的50％，全国每年用于生产猪口蹄疫疫苗和禽流感疫苗两种疫苗需用油佐剂约31.5亿毫升，约3000吨。目前生产传统油佐剂的主要原料是矿物质油，难以避免会残留稠环芳香烃类成分，有致癌作用，对食品安全构成潜在威胁。同时国内部分油乳剂灭活疫苗粘度较大、注射困难，注射后不易吸收，在注射部位长时间滞留，引起局部组织炎症、化脓、坏死等副作用，造成动物痛苦。因此，一些发达国家对油佐剂疫苗的使用进行了限制，例如早在20多年以前美国政府严禁动物上市前42天之内注射油佐剂疫苗。
公开号为CN101474403A的专利文献公开了一种灭活疫苗的油佐剂，将白油经蒸馏、加氢去除硫、氮、烯烃、胶质、重金属和大部分芳烃杂质，再在压力4.5～10MPa，反应温度150～290℃，体积空速0.1～0.5h-1下进行第二次加氢反应得到脱芳白油，最后将脱芳白油和甘露醇油酸脂按90:10～100:8的体积份混合而成。虽然该专利降低了油佐剂的芳烃含量，粘度降低，减小了动物防疫注射时推针时的阻力，但是该佐剂的制备方法复杂繁琐，而且在佐剂中仍然会残留有致癌作用稠环芳香烃类成分，对食品安全构成潜在威胁。
为了避免佐剂中含有稠环芳香烃类等致癌物质，公开号为CN1669584A的专利文献公开了一种利用植物油佐剂制备动物病毒性、细菌性疫苗的方法，该文献中的植物油佐剂为植物油与司盘-80的混合物，其中植物油为菜籽油、豆油、花生油、蓖麻油、胡麻油中的一种，最后将制备得到的植物油佐剂与该文献中制备得到的抗原按比例混匀制备得到疫苗。植物油佐剂既避免了引起食品安全的潜在威胁，又具有矿物质油佐剂的性能，但是对于佐剂的免疫油乳剂灭活疫苗粘度较大而造成的注射困难以及在注射部位长时间滞留，引起局部组织炎症、化脓、坏死等副作用仍然未能有效地解决。
中药是我国的宝贵遗产，与合成药物相比，具有不良反应少、毒副作用小，多效性、双向调节性和无依赖性等特点，一些中药活性成分具有良好的免疫调剂作用，例如研究表明，传统的补益类中药人参中所含的人参皂苷可提高巨噬细胞活性，诱导干扰素生产，刺激细胞毒性T淋巴细胞活性，因此作为新型疫苗佐剂其优势十分显著，已经越来越受到重视。
公开号为CN101670104A的专利文献公开了一种含人参皂苷的油佐剂及其制备方法，该油佐剂是一种由人参皂苷、佐剂油、硬脂酸铝和山梨醇单油酸酯组成的混合物，每毫升油佐剂中含人参皂苷10～100μg。其制备方法为：将白油加热60～150℃，在每毫升油中加入10～20毫克硬脂酸铝，混匀；待油温下降至50～20℃后，按体积比加入5～7％去山梨醇单油酸酯，搅拌均匀，过滤，备用；用二甲基亚砜或吐温溶剂溶解人参皂苷成溶液；在每毫升制得的备用物中加入人参皂苷溶液10～100μg。该含有人参皂苷的油佐剂虽然在一定程度上提高了疫苗诱导的特异性细胞和体液免疫反应强度，但是由于白油中难以避免会残留含有致癌作用稠环芳香烃类成分。因此研制具有高效免疫性能以及安全的新型佐剂已成为当代免疫学研究领域的一个重要课题。
发明内容
本发明提供了一种含人参皂苷的植物油佐剂，该含人参皂苷的油佐剂具有高效、安全的性能。
一种含人参皂苷的植物油佐剂，以食用植物油作为佐剂油，含有10～100μg/mL的人参皂苷。
本发明以植物油替代传统的矿物质油，避免了油佐剂中含有致癌作用的稠环芳香烃类成分，使该油佐剂生产的疫苗免疫注射动物后可对食品安全构成潜在威胁，而且本发明的油佐剂中含有人参皂苷，人参皂苷可提高巨噬细胞活性，诱导干扰素生产，刺激细胞毒性T淋巴细胞活性，人参皂苷与植物油混合后发挥了协同佐剂作用，显著提高了疫苗的抗体水平。
当油佐剂中人参皂苷的含量不同时，与植物油所起的协同佐剂作用也不同，作为优选，所述人参皂苷的含量为30～50μg/mL。作为更优选，所述人参皂苷的含量为40～45μg/mL。
植物油的种类有多种，本发明可以使用多种类型的植物油替代矿物质油，均可起到很好的效果，作为优选，所述食用植物油为葵花籽油、玉米油、橄榄油、山茶油或菜籽油。作为更优选，所述食用植物油为菜籽油。
本发明还提供了上述植物油佐剂的制备方法，包括：
(1)将人参皂苷加入二甲基亚砜中进行溶解，混匀得到人参皂苷溶液；
(2)将所述人参皂苷溶液加入植物油中直接混合均匀；
或
植物油加热至20～40℃后，在植物油中加入去水山梨醇单油酸酯和所述人参皂苷溶液，混合均匀。
本发明的植物油佐剂的制备方法简单方便，二甲基亚砜是一种重要的极性非质子溶剂，广泛用作溶剂和反应试剂，所述人参皂苷溶液中人参皂苷的含量为10～60mg/mL。
所述去水山梨醇单油酸酯的添加量为：每100mL植物油佐剂中加入10～16mL去水山梨醇单油酸酯。
本发明还提供了上述植物油佐剂在制备疫苗中的应用。
将本发明的植物油佐剂应用于疫苗的制备之中，可得到流动性较好疫苗，具有较低的粘滞度，便于免疫注射，同时具有增强疫苗免疫作用。
本发明的植物油佐剂应用范围较广，例如可应用于病毒或是细菌类疫苗的制备之中，作为优选，所述疫苗为口蹄疫灭活疫苗或金黄色葡萄球菌疫苗。口蹄疫是一种由口蹄疫病毒感染偶蹄类动物(猪、牛、羊等)所产生的疾病，若给动物注射的口蹄疫疫苗中含有致癌物质，那么将会给食品安全带来潜在危害；金黄色葡萄球菌可引起许多动物的疾病。将本发明的植物油佐剂应用于口蹄疫灭活疫苗或金黄色葡萄球菌疫苗的制备，分别得到一种高效且安全的疫苗。
与现有技术相比，本发明的有益效果体现在：
(1)本发明用含人参皂苷的植物油来制备疫苗，使疫苗的安全性大幅度提高，克服了常规矿物质油佐剂对食品卫生潜在的危害；
(2)本发明的植物油佐剂中含有适量的人参皂苷，其中人参皂苷与植物油混合后发挥了协同佐剂作用，特异性抗体、淋巴细胞转化、血清细胞因子和长寿型淋巴细胞等均显著提高，抗体效价提高了4.7倍，而含有人参皂苷的矿物质油佐剂的抗体效价仅提高了3.1倍，因此本发明含有人参皂苷的植物油佐剂大幅度提高了疫苗的抗体水平；
(3)本发明的植物油佐剂制备得到疫苗流动性好，方便免疫注射，局部刺激性小，减轻了动物痛苦；
(4)将本发明的植物油佐剂在用于疫苗生产时，无需改变现有生产工艺，方法简 便。
附图说明
图1为用GS(人参皂苷)植物油佐剂和法国ISA 206佐剂制备口蹄疫疫苗的粘滞度结果对比图。
图2为添加和不添加GS植物油制备口蹄疫疫苗免疫小鼠后的抗体反应对比图。
图3为用GS含量不同的植物油制备口蹄疫疫苗和用法国ISA 206佐剂制备口蹄疫疫苗免疫小鼠后的抗体反应对比图。
图4为用GS含量不同的植物油制备口蹄疫疫苗和用法国ISA 206佐剂制备口蹄疫疫苗免疫小鼠后的抗体亚类反应对比图。
图5为用GS含量不同的植物油制备口蹄疫疫苗和用法国ISA 206佐剂制备口蹄疫疫苗免疫小鼠后的脾淋巴细胞转化水平对比图。
图6为用GS含量不同的植物油制备口蹄疫疫苗和用法国ISA 206佐剂制备口蹄疫疫苗免疫小鼠后的血清干扰素γ水平对比图。
图7为用GS含量不同的植物油制备口蹄疫疫苗和用法国ISA 206佐剂制备口蹄疫疫苗免疫小鼠后的血清白介素5水平对比图。
图8为添加和不添加GS植物油制备口蹄疫疫苗免疫小鼠后的骨髓长寿型浆细胞数量对比图。
图9为GS植物油佐剂和GS矿物质油佐剂协同佐剂作用的比较图。
图10为GS和植物油组合对细菌性疫苗的协同佐剂用图。
图11为各种佐剂对IgG效价影响的对比图
图12为各种佐剂对IgG亚类水平影响的对比图。
图13为各种佐剂对脾淋巴细胞增殖活性影响的对比图。
图14为各种佐剂对小鼠血清细胞因子IL-5表达水平影响的对比图。
图15为各种佐剂对小鼠血清细胞因子IFN-γ表达水平影响的对比图。
图16为ZE515和铝胶佐剂对IgG效价影响的对比图。
图17为ZE515和铝胶佐剂对IgG亚类水平影响的对比图。
具体实施方式
实施例1含人参茎叶皂苷的葵花籽油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)在1000mL葵花籽油(上海嘉里食品工业有限公司产品)中加入1mL上述人参茎叶皂苷溶液，充分混均匀，即得每毫升含人参茎叶皂苷43μg的植物油佐剂。
实施例2含人参茎叶皂苷的玉米油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)在1000mL玉米油(上海嘉里食品工业有限公司产品)中加入1mL上述人参茎叶皂苷溶液，充分混均匀，即得每毫升含人参茎叶皂苷43μg的植物油佐剂。
实施例3含人参茎叶皂苷的橄榄油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)在1000mL橄榄油油(上海嘉里食品工业有限公司产品)中加入1mL上述人参茎叶皂苷溶液，充分混均匀，即得每毫升含人参茎叶皂苷3μg的植物油佐剂。
实施例4含人参茎叶皂苷的山茶油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)在1000mL山茶油(浙江莱博士食品科技有限公司产品)中加入1mL上述人参茎叶皂苷溶液，充分混均匀，即得每毫升含人参茎叶皂苷43μg的植物油佐剂。
实施例5含人参茎叶皂苷的菜籽油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)在1000mL菜籽油(上海嘉里食品工业有限公司产品)中加入1mL上述人参茎叶皂苷溶液，充分混均匀，即得每毫升含人参茎叶皂苷43μg的植物油佐剂。
实施例6含人参茎叶皂苷的葵花籽油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)将860mL葵花籽油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参茎叶皂苷溶液，再充分混均匀，即得每毫升含人参茎叶皂苷40μg的植物油佐剂。
实施例7含人参茎叶皂苷的玉米油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)将860mL玉米油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参茎叶皂苷溶液，再充分混均匀，即得每毫升含人参茎叶皂苷40μg的植物油佐剂。
实施例8含人参茎叶皂苷的橄榄油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)将860mL橄榄油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参茎叶皂苷溶液，再充分混均匀，即得每毫升含人参茎叶皂苷40μg的植物油佐剂。
实施例9含人参茎叶皂苷的山茶油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)将860mL山茶油(浙江莱博士食品科技有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参茎叶皂苷溶液，再充分混均匀，即得每毫升含人参茎叶皂苷40μg的植物油佐剂。
实施例10含人参茎叶皂苷的菜籽油佐剂的制备
(1)称取人参茎叶皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂苷的溶液，备用。
(2)将860mL菜籽油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参茎叶皂苷溶液，再充分混均匀，即得每毫升含人参茎叶皂苷40μg的植物油佐剂。
实施例11含人参皂苷的葵花籽油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)在1000mL葵花籽油(上海嘉里食品工业有限公司产品)中加入1mL上述人参皂苷溶液，充分混均匀，即得每毫升含人参皂苷43μg的植物油佐剂。
实施例12含人参皂苷的玉米油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参茎叶皂甙的溶液，备用。
(2)在1000mL玉米油(上海嘉里食品工业有限公司产品)中加入1mL上述人参皂苷溶液，充分混均匀，即得每毫升含人参皂苷43μg的植物油佐剂。
实施例13含人参皂苷的橄榄油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)在1000mL橄榄油油(上海嘉里食品工业有限公司产品)中加入1mL上述人参皂苷溶液，充分混均匀，即得每毫升含人参皂苷43μg的植物油佐剂。
实施例14含人参皂苷的山茶油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)在1000mL山茶油(浙江莱博士食品科技有限公司产品)中加入1mL上述人参皂苷溶液，充分混均匀，即得每毫升含人参皂苷43μg的植物油佐剂。
实施例15含人参皂苷的菜籽油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)在1000mL菜籽油(上海嘉里食品工业有限公司产品)中加入1mL上述人参皂苷溶液，充分混均匀，即得每毫升含人参皂苷43μg的植物油佐剂。
实施例16含人参皂苷的葵花籽油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)将860mL葵花籽油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参皂苷溶液，再充分混均匀，即得每毫升含人参皂苷40μg的植物油佐剂。
实施例17含人参皂苷的玉米油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)将860mL玉米油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参皂苷溶液，再充分混均匀，即得每毫升含人参皂苷40μg的植物油佐剂。
实施例18含人参皂苷的橄榄油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)将860mL橄榄油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参皂苷溶液，再充分混均匀，即得每毫升含人参皂苷40μg的植物油佐剂。
实施例19含人参皂苷的山茶油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)将860mL山茶油(浙江莱博士食品科技有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参皂苷溶液，再充分混均匀，即得每毫升含人参皂苷40μg的植物油佐剂。
实施例20含人参皂苷的菜籽油佐剂的制备
(1)称取人参皂苷(吉林宏久药业产品)0.50克，加入至10mL二甲基亚砜(DMSO)中，混匀，溶解，制成每毫升含50mg人参皂苷的溶液，备用。
(2)将860mL菜籽油(上海嘉里食品工业有限公司产品)加热至30℃，然后在油中加入140mL去水山梨醇单油酸酯(Span-80)，混合均匀后加入0.8mL上述人参皂苷溶液，再充分混均匀，即得每毫升含人参皂苷40μg的植物油佐剂。
实施例21含人参皂苷(GS)的菜籽油佐剂疫苗的粘滞度的测定
1.方法
1.1GS-菜籽油佐剂疫苗的制备：
将50mg溶解于1mL二甲基亚(DMSO)，制备成50mg/mL的GS溶液，在4.4mL30℃的菜籽油中加入0.6mL去水山梨醇单油酸酯(Span-80)，充分混匀，再在其中加入人参皂苷DMSO溶液4μL，混匀，制成每毫升菜籽油含GS 40μg油相。在4.7mL灭活亚洲1型口蹄疫(FMD)抗原(金宇保灵生物药品有限公司提供)(抗原146s含量为5μg)中加入0.3mL吐温-80，充分混匀，制成水相，将油相和水相等体积混合，用高速匀浆器乳化，制成FMD菜籽油乳剂疫苗。
1.2 MONTANIDE ISA 206油佐剂疫苗的制备：
在4.4mL 30℃的ISA 206佐剂(法国SEPPIC公司)中加入0.6mL Span-80，混合均匀，制成油相。在4.7mL灭活亚洲1型FMD抗原(金宇保灵生物药品有限公司提供)(抗原146s含量为5μg)中加入0.3mL吐温-80，充分混匀，制成水相，将油相和水相等体积混合，用高速匀浆器乳化，制成FMD206油乳剂疫苗。
1.3疫苗的黏滞性测定：
用内径为1.2mm的吸管吸取1mL油乳剂疫苗，然后置吸管于垂直位置，让乳剂自然流出0.4mL，记录所需时间。
2.结果
GS-菜籽油乳剂疫苗的流出时间为1.9秒，而206油乳剂疫苗的流出时间为2.8秒，见图1，GS-菜籽油乳剂疫苗的粘滞度显著低于206油乳剂疫苗。
实施例22菜籽油和人参皂苷(GS)协同发挥佐剂作用
1.方法
1.1试验疫苗的制备
疫苗1：按照实施例21制备口蹄疫GS-菜籽油乳剂疫苗，使每毫升疫苗含亚洲1型FMD抗原146s为0.5μg，GS为40μg，菜籽油为44％(体积比)，司盘-80为6％(体积比)。
疫苗2：按照实施例21方法制备亚洲1型FMD菜籽油乳剂疫苗，制备过程不加GS，使每毫升疫苗含FMD抗原146s为0.5μg，菜籽油为44％(体积比)，司盘-80为6％(体积比)。
疫苗3：将GS溶解在生理盐水，然后用该溶液稀释亚洲1型口FMD抗原，使每毫升疫苗含FMD抗原146s为0.5μg和GS为40μg。
疫苗4：用生理盐水稀释亚洲1型FMD抗原，使每毫升含FMD抗原146s为0.5μg。
1.2动物分组和免疫
将32只ICR小鼠随机分成4组，每组8只，每组小鼠两次皮下注射0.2mL疫苗，间隔3周。各组小鼠每次按照以下方案注射疫苗：
(1)FMD抗原0.1μg+GS 4μg+菜籽油；
(2)FMD抗原0.1μg+菜籽油；
(3)FMD抗原0.1μg+GS 4μg；
(4)生理盐水
二免后1、2周眼眶静脉采血，血样置4℃过夜，3000转离心10分钟，取血清置离心管中，-20℃保存，备用。
1.3 FMD特异性IgG检测
(1)在ELISA板上每孔加入50μL经碳酸盐缓冲液(pH9.6)稀释的牛抗亚洲1型FMD抗体(1:1000)(中国农科院兰州兽医研究所)，封板，4℃过夜。
(2)用洗涤液洗涤5次，每次300μL，拍干。每孔加入300μL磷酸盐缓冲液(含5％脱脂奶+0.05％吐温-20)封闭，37℃孵育2小时。
(3)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入50μL经1:3稀释的O型口蹄疫病毒抗原，振荡混匀，4℃孵育2小时。
(4)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入50μL经磷酸盐缓冲液(含0.05％吐温-20)1:50稀释的待检血清，37℃孵育1小时。
(5)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入经1：1000稀释的山羊抗豚鼠IgG抗体(美国Betheyl Laboratory公司)，37℃孵育1小时。
(6)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入经1:10000兔抗山羊IgGFC-HRP，37℃孵育1小时。用洗涤液洗涤5次，每次300μL，拍干。
(7)每孔加入100μL TMB底物溶液。室温孵育15分钟，每孔加入50μL 2 M H2SO4终止反应，并轻轻振荡混匀。在15分钟内，用酶标仪测定在450nm波长的OD值。
2.结果
除了二免后2周，组2的FMD抗体水平显著高于组3和组4外，其余各组间的抗体水平均无统计学差异，但组1的抗体水平在二免后的两次检测中均显著高于其它各组，见图2，说明GS和菜籽油混合后发挥了协同佐剂作用，显著提高了FMD抗体水平。
实施例23不同含量人参皂苷的菜籽油对FMD疫苗的免疫佐剂作用
1.方法
1.1实验疫苗的制备
按照实施例21制备四种GS含量不同的菜籽油FMD乳剂疫苗，每毫升疫苗含亚洲1型FMD抗原146s为0.5μg，菜籽油为44％(体积比)，司盘-80为6％(体积比)，GS含量分别为0μg，20μg，40μg和60μg。按照实施例21制备ISA 206油佐剂FMD疫苗,每毫升疫苗含亚洲1型FMD抗原146s为0.5μg，ISA 206油为44％(体积比)，司盘-80为6％(体积比)。
1.2动物分组和免疫
将56只ICR小鼠随机分成7组，每组8只，每组小鼠两次腹部皮下注射疫苗0.2mL，间隔3周。各组小鼠每次按照以下方案注射疫苗：
(1)FMD抗原0.1μg+GS 2μg+菜籽油；
(2)FMD抗原0.1μg+GS 4μg+菜籽油；
(3)FMD抗原0.1μg+GS 6μg+菜籽油；
(4)FMD抗原0.1μg+菜籽油；
(5)FMD抗原0.1μg+ISA 206；
(6)FMD抗原0.1μg+生理盐水；
(7)生理盐水
于一免后1周、二免后1-6周眼眶采血，制备血清，检测血清中抗FMD特异性IgG,IgG亚类和血清细胞因子。于二免后第6周股骨分离骨髓浆细胞测定长寿型浆细胞数量。
1.3 FMDV特异性抗体检测
1.3.1 IgG的检测
方法同实施例22。
1.3.2 IgG亚类的检测
检测步骤如下：
(1)96孔板包被50μL 0.05M碳酸盐缓冲液稀释的兔抗FMD亚洲1型抗体(1：800)4℃过夜，PBST洗涤五次；
(2)每孔加入300μL 5％脱脂乳37℃封闭2h，PBST洗涤5次；
(3)每孔加入50μL FMD亚洲I型抗原(1：8)4℃孵育2h，PBST洗涤5次；
(4)每孔加入50μL 5％脱脂乳稀释的血清(1：200)37℃孵育1h，PBST洗涤5次；
(5)每孔加入50μL羊抗鼠IgG1，IgG2a，IgG2b或者IgG3(1：1000)37℃孵育1h，PBST洗涤5次；
(6)每孔加入100μL TMB底物37℃显色15min，再加入50μL 2M硫酸终止反，用酶标仪在450nm波长测OD值。
1.4淋巴细胞转化的测定
二免后2周无菌分离小鼠脾脏，PBS溶液洗涤后，过筛制备单细胞悬液，4℃380×g离心10min，红细胞裂解液重悬离心，PBS洗涤2次，RPMI 1640(含10％胎牛血清、100IU/mL青霉素,100μg/mL链霉素)重悬后，台盼蓝染色计数活细胞(活细胞＞95％)错误！未指定书签。错误！未指定书签。。在96孔细胞板上加入200μl终浓度5×106个/mL脾淋巴细胞，分别使用终浓度ConA(5μg/mL)、LPS(8μg/mL)、FMD(10μg/mL)和培养基刺激，培养板置于37℃含5％CO2培养箱孵育48h。孵育结束前4h每孔加入50μl MTT溶液(2mg/mL)，离心培养板(1400g，5min)并小心倒掉细胞培养液。每孔加入150μl DMSO(含4％的1M HCl)溶解结晶染料，15min后置于490nm测OD值，刺激指数(SI)＝(刺激孔OD-空白孔OD)/(未刺激孔OD-空白孔OD)。
1.5血清细胞因子的测定
血清中IFN-γ的浓度测定运用Mouse IFN-γSunny ELISA试剂盒(购自联科生物科技有限公司)，IL-5浓度的检测用Mouse IL-5 ELISA试剂盒(eBioscience Inc，San Diego,USA)，细胞因子的浓度根据细胞因子标准曲线来计算。
1.6.长寿型淋巴细胞的测定
无菌分离小鼠大腿股骨、胫骨，用注射器吹出骨髓过筛制备单细胞悬液，4℃380×g离心10min，加入红细胞裂解液重悬离心，PBS洗涤2次，RPMI 1640(含10％胎牛血清、100IU/mL青霉素,100μg/mL链霉素)重悬后，细胞计数并调整细胞浓度为2.4×107个/mL，酶联免疫斑点试验参考文献。2.4×106个/孔细胞被加入包被有7μg/mL FMDV抗原的HA-96孔板，37℃含5％CO2培养箱孵育5h，PBS洗涤三次，加入HRP标记的羊 抗鼠IgG(H+L)4℃过夜.PBST洗涤五次加入AEC显色液，每孔加入100μL去离子水终止显色，将板避光晾干计数，应用酶联免疫斑点软件处理数据。
2.结果
2.1.注射GS-菜籽油FMD疫苗小鼠的IgG和IgG亚类水平显著高于对照组
如图3所示，注射GS-菜籽油FMD疫苗小鼠血清IgG水平在二免后1-6周始终显著高于无佐剂的对照组，但和ISA 206佐剂组比较无显著差异；当一次注射量的FMD疫苗(0.2mL)中含有GS 4μg(20μg GS/mL)时I血清IgG水平最高。
如图4所示，注射GS-菜籽油FMD疫苗小鼠血清IgG亚类(IgG1,IgG2a,IgG2b和IgG3)水平显著高于无佐剂的对照组，但和ISA 206佐剂组比较无显著差异；当一次注射量的FMD疫苗(0.2mL)中含GS 4μg(20μg GS/mL)时血清IgG亚类的水平最高。
2.2注射GS-菜籽油FMD疫苗小鼠淋巴细胞转化水平显著高于对照组
如图5所示，注射GS-菜籽油FMD疫苗小鼠的淋巴细胞转化水平显著高于无佐剂的对照组，但和ISA 206佐剂组比较无显著差异；当一次注射量的疫苗(0.2mL)中含GS4μg(20μg GS/mL)时淋巴细胞转化水平最高。
2.3注射GS-菜籽油FMD疫苗小鼠血清细胞因子水平显著高于对照组
如图6和图7所示，注射GS-菜籽油FMD疫苗(每次注射的疫苗中含GS 4μg)小鼠血清IFN-γ和IL-5水平显著高于无佐剂的对照组，但和ISA 206佐剂组比较无显著差异。
2.4注射GS-菜籽油FMD疫苗小鼠骨髓长寿型浆细胞数量显著高于对照组
如图8所示，注射GS-菜籽油FMD疫苗(每次注射的疫苗中含GS 4μg)小鼠长寿型浆细胞的数量显著高于无佐剂的对照组。
实施例24 GS-植物油和GS-矿物油协同佐剂作用的比较
1.方法
1.1 GS-菜籽油和GS-ISA 206油乳剂疫苗的制备
制备方法同实施例21。
1.2动物分组和免疫
将40只ICR小鼠随机分成5组，每组8只，每组小鼠两次皮下注射0.2mL疫苗，间隔3周。各组小鼠每次按照以下方案注射疫苗：
(1)生理盐水
(2)FMD抗原0.1μg+菜籽油；
(3)FMD抗原0.1μg+菜籽油+GS 4μg；
(4)FMD抗原0.1μg+ISA 206；
(5)FMD抗原0.1μg+ISA 206+GS 4μg。
二免后1、2周眼眶静脉采血，血样置4℃过夜，3000转离心10分钟，取血清置离心管中，-20℃保存，备用。
1.3 FMD特异性抗体效价检测
(1)在ELISA板上每孔加入50μL经碳酸盐缓冲液(pH9.6)稀释的牛抗亚洲1型FMD抗体(1:1000)(中国农科院兰州兽医研究所)，封板，4℃过夜。
(2)用洗涤液洗涤5次，每次300μL，拍干。每孔加入300μL磷酸盐缓冲液(含5％脱脂奶+0.05％吐温-20)封闭，37℃孵育2小时。
(3)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入50μL经1:3稀释的O型口蹄疫病毒抗原，振荡混匀，4℃孵育2小时。
(4)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入50μL经磷酸盐缓冲液(含0.05％吐温-20)1:50稀释的待检血清，37℃孵育1小时。
(5)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入经1：1000稀释的山羊抗豚鼠IgG抗体(美国Betheyl Laboratory公司)，37℃孵育1小时。
(6)用磷酸盐缓冲液洗涤5次，每次300μL，拍干。每孔加入经1:10000兔抗山羊IgGFC-HRP，37℃孵育1小时。用洗涤液洗涤5次，每次300μL，拍干。
(7)每孔加入100μL TMB底物溶液。室温孵育15分钟，每孔加入50μL 2 M H2SO4终止反应，并轻轻振荡混匀。在15分钟内，用酶标仪测定在450nm波长的OD值。
(8)OD值大于cutoff值(未免疫小鼠血清经上述方法处理所测的OD值，然后乘以2.1)的为阳性，一个血清一系列阳性孔中的最大稀释倍数即为该血清的效价。
2.结果
如图9所示，不添加人参皂苷时，菜籽油和ISA 206油乳剂FMD疫苗的抗体效价分别为1：452和1：477；添加人参皂苷以后，GS-菜籽油和GS-ISA 206油乳剂FMD疫苗的抗体效价分别为1：2152和1：1505。菜籽油添加人参皂苷之后抗体效价提高了4.7倍，而ISA 206添加人参皂苷之后抗体效价提高了3.1倍。GS和菜籽油组合产生的协同佐剂作用大大高于GS和ISA 206组合产生的协同作用。
实施例25 GS和植物油组合对金黄色葡萄球菌疫苗的免疫增强作用
1.方法
1.1实验疫苗的制备
按照实施例10制备GS-菜籽油佐剂。按照佐剂100μL和金黄色葡萄球菌毒素抗原50μL比例制成如下实验疫苗：
(1)金黄色葡萄球菌毒素抗原50μL+菜籽油100μL
(2)金黄色葡萄球菌毒素抗原50μL+菜籽油100μL+GS 4μg
(3)金黄色葡萄球菌毒素抗原50μL+生理盐水100μL+GS 4μg
(4)金黄色葡萄球菌毒素抗原50μL+生理盐水100μL
(5)生理盐水150μL
1.2动物分组和免疫
将35只ICR雌性小鼠随机分成5组，每组7只。用上述五种实验疫苗分别二次皮下注射免疫5组小鼠，两次免疫间隔2周。第二次免疫后1、2、3、4周眼眶采血，血样置4℃冰箱过夜后离心(4000rpm)10min，制备血清，分装后置-20℃保存，备用。1.3特异性IgG水平的检测
(1)在96孔酶标板中每孔加入100μl金葡菌毒素蛋白包被液(以0.05mol/L碳酸盐缓冲液1:10稀释)，4℃过夜。
(2)每孔加入300μl的PBST洗涤液，洗涤3次，每次3min。
(3)每孔加入300μl的5％的脱脂乳，37℃封闭2h。
(4)洗涤三次，同第二步，然后每孔加入100μl的稀释血清(以5％的脱脂乳1:200稀释)，37℃孵育1h。
(5)洗涤三次，同第二步，然后每孔加入100μl的HRP标记的山羊抗小鼠的IgG抗体(以5％的脱脂乳1:2000稀释)，37°孵育1h。
(6)洗涤三次，同第二步，然后每孔加入100μl的TMB显色液，37℃孵育20min。
(7)每孔加入50μL的2M的H2SO4终止反应。
(8)酶标仪测定OD450
2.结果
如图10所示，第二次免疫后4次抗体检测结果表明，GS和菜籽油组合组的抗体水平高于其它各组，说明两者发挥的协同佐剂作用。
实施例26本发明对CP5型金黄色葡萄球菌疫苗的佐剂作用
1.方法
1.1试剂和药品
铝胶佐剂(含20％Al(OH)3)，中牧实业股份有限公司江西生物药厂产品；HRP标记山羊抗小鼠IgG抗体、IgG1抗体、IgG2a抗体，Santa Cruz产品；小鼠IFN-γELISA检测试剂盒，小鼠IL-5 ELISA检测试剂盒，联科生物技术有限公司产品。
1.2金黄色葡萄疫苗的制备
CP5型荚膜多糖的金黄色葡萄球菌(HZ016株)分离自杭州市郊区奶牛场的临床型乳房炎病例，接种肉汤培养液，于37℃震荡培养20h，每100mL菌液中加入40％福尔马林1mL(相当于甲醛浓度0.4％)，灭活24h，5000rpm离心20min，无菌PBS洗沉淀三次后用PBS调节至一定浓度，即为菌体抗原；肉汤培养上清液与饱和硫酸铵等体积混合，4℃静置过夜，8000rpm离心30min，弃上清，沉淀溶于无菌PBS，透析72h，0.22μm滤膜无菌过滤，用PBS调节至一定浓度，即为毒素抗原；将菌体抗原与毒素抗原等体积混合成细菌抗原(Sa-Ag)。
铝胶佐剂与细菌抗原(Sa-Ag)无菌匀速搅拌过夜，制备成金黄色葡萄球菌铝胶疫苗(含15％Al(OH)3，含菌量1.5×107个/mL，毒素150μg/mL)；Sa-Ag与ZE515佐剂(含人参皂甙GS-R和植物油VO，按实施例10制备)按照比例1:2乳化制备成金黄色葡萄球菌植物油乳剂疫苗(含菌量1.5×107个/mL，毒素150μg/mL)。人参皂甙GS-R，为吉林省宏久生物科技股份有限公司产品。
1.3动物分组及处理
雌性ICR小鼠，19～22g，购自中国科学院上海实验动物中心斯莱克有限公司。为研究ZE515的佐剂作用，将动物随机分成5组，每组8只，腹部皮下分别注射Sa-Ag、Sa-Ag+VO，Sa-Ag+GS-R,Sa-Ag+ZE515和生理盐水。每只小鼠注射0.2mL，免疫两次，间隔两周。二免后三周采血，于37℃静置1h，4℃过夜后3600rpm离心8min，分离血清，分装于0.2mL离心管，-20℃保存。采血后处死小鼠，分离脾脏制备淋巴细胞进行淋巴细胞增殖试验。
1.4血清IgG及亚类检测
采用间接ELISA方法。用毒素抗原包96孔板(抗原浓度为13.5μg/mL)，加入待测血清(1:200)，用HRP标记山羊抗小鼠IgG抗体(1:2000)检测IgG，HRP标记山羊抗小鼠IgG1(1:1000)、IgG2a抗体(1:1000)检测IgG抗体亚类，显色时间为10min。
1.5淋巴细胞增殖试验
于二免后三周无菌分离脾脏。脾脏磨碎后用无菌Hanks溶液冲洗，经200目铜网过滤，滤液于1500rpm，10min离心，弃上清液，用红细胞裂解液裂解红细胞，然后1500rpm离心10min，弃上清，再用Hanks溶液洗涤两次，然后将细胞浓度在细胞培 养液(RPMI 1640培养液+10％胎牛血清，100μg/mL链霉素，100 IU/mL青霉素)中调整至5×106个/mL。在96孔板中加入100μl Con A(6μg/mL)，LPS(10μg/mL)或者细菌毒素抗原(30μg/mL)，100μL淋巴细胞悬液。细胞置于37℃，5％的CO2细胞培养箱中培养48h。在结束培养前4h加入50μL的MTT(2mg/mL)。培养板经1500rpm离心10min，弃上清液，每孔加入150μL的DMSO，避光振荡15min，直至孔中蓝紫色颗粒完全溶解，测量OD570，计算淋巴细胞刺激指数：SI＝(刺激孔OD-空白孔OD)/(未刺激孔OD-空白孔OD)。
1.7细胞因子IL-5和IFN-γ检测
ELISA试剂盒检测小鼠血清IL-5和IFN-γ，按照说明书进行。
1.8小鼠攻毒保护试验
前期实验证明HZ016株金黄色葡萄球菌分离菌对小鼠的LD50为5×107CFU。本试验将32只ICR小鼠随机分成4组：组1，Sa-Ag+ZE515；组2，Sa-Ag+铝胶；组3，Sa-Ag；组4：生理盐水。每只小鼠2次腹部皮下注射0.2mL，间隔两周。二免后三周每只小鼠腹腔注射CP5型金黄色葡萄球菌(HZ016株)活菌液5×107CFU，观察小鼠的临床表现、发病及死亡情况。计算保护率，保护率＝{(攻毒生理盐水组阳性率-疫苗免疫组阳性率)/攻毒生理盐水组阳性率}×100％，阳性率＝(患病个体数/个体总数)×100％。
2.结论
图11表明，Sa-Ag和VO或者GS-R混合之后，IgG效价分别是1:422和1:184，分别是对照组(1:95)的4倍和2倍，但无显著性差异(P>0.05)；但VO和GS-R混合形成ZE515后再和Sa-Ag混合形成乳剂，IgG效价升高至1:2558，是对照组的27倍，显著高于其它各组(P<0.05)。
图12表明，VO、GS-R和ZE515对IgG1和IgG2a水平有不同程度的增强作用,其中ZE515对IgG亚类的增强作用高于其它各组，和无佐剂的对照组比较有显著差异(P<0.05)。
图13表明，VO、GS-R和ZE515促进了由Con A、LPS和Sa-Ag诱导的淋巴细胞增殖活性。含VO、GS-R或ZE515疫苗免疫后小鼠的淋巴细胞增殖指数显著高于对照组。淋巴细胞增殖指数由高到低，依次为ZE515组、VO组、GS-R组和无佐剂对照组。
图14和15表明，各组小鼠血清细胞因子IL-5和IFN-γ的水平。血清细胞因子水平由高到低，依次为ZE515组、VO组、GS-R组和无佐剂对照组。ZE515组血清IL-5和IFN-γ的含量分别是无佐剂对照组的2倍和6倍(P<0.05)。
图16表示ZE515和铝胶佐剂对抗体产生均有显著促进作用，但ZE515促进作用显著高于铝胶佐剂(P<0.05)。
图17表示ZE515和氢氧化铝佐剂对IgG亚类的产生均有显著促进作用。ZE515和铝胶对IgG1的影响无显著差异(P>0.05)，但对IgG2a产生的促进作用，ZE515显著高于氢氧化铝佐剂(P<0.05)
免疫小鼠在攻毒24h后均表现精神沉郁，被毛粗乱，体态瘦弱，不喜活动，畏冷抱团，并且对外界刺激反应迟钝。根据患病数统计，ZE515患病数量最少，铝胶其次，对照组仅有1只没有患病，生理盐水组全部患病。根据死亡率统计，ZE515组小鼠仅有发病没有死亡，铝胶组仅在攻毒后第6天死亡1只，对照组小鼠攻毒3天后陆续死亡3只，生理盐水组小鼠攻毒后72h内死亡3只，攻毒后第4天和第7天各死亡1只。根据病愈时间统计，ZE515组小鼠最早，48h后就能发现小鼠行动活跃，反应灵敏；铝胶组3天后小鼠活动频率增加，半数小鼠耐过后体态瘦弱，行动迟缓，1只死亡；对照组小鼠4天后状态好转，运动力很弱，生理盐水组小鼠4天后渐渐恢复运动能力。
表1疫苗免疫对金黄色葡萄球菌的攻毒保护作用(8个小鼠/组)
Figure PCTCN2014095451-appb-000001
*和对照组比较
从表1可以看出，氢氧化铝佐剂疫苗的保护率为50％，和对照组比较无显著性；ZE515佐剂疫苗的保护率为75％，和对照组比较差异极显著(P<0.01)。
以上实验证明，植物油(VO)或者人参根皂甙(GS-R)本身不会显著增强金黄色葡萄球菌抗原(Sa-Ag)诱导的免疫应答，但当VO和GS-R混合形成ZE515后可发挥佐剂作用，显著增强Sa-Ag诱导的免疫反应(图11-15)；(2)ZE515对Sa-Ag的佐剂作用显著强于常规铝胶佐剂(图16-17)，含ZE515的金黄色葡萄球菌疫苗对金葡菌强度攻毒的保护率为75％，而含铝胶佐剂的疫苗保护率只有50％(表1)。
Th1类淋巴细胞分泌IFN-γ等细胞因子，促进IgG2a的产生。IFN-γ激活细胞毒性T淋巴细胞，有助于清除胞内的细菌。含ZE515和Sa-Ag的金黄色葡萄球菌疫苗免疫小鼠后，使IgG1和IgG2a水平显著升高(图12)，促进了Con A、LPS和Sa-Ag诱导的淋巴细胞增殖反应(图13)，显著增加IL-5和IFN-γ的产生(图14和15)，说明含ZE515佐剂可同时促进Th1和Th2类免疫反应，对细菌性疫苗产生佐剂作用。

Claims (11)

  1. 一种含人参皂苷的植物油佐剂，其特征在于，以食用植物油作为佐剂油，含有10～100μg/mL的人参皂苷。
  2. 如权利要求1所述的植物油佐剂，其特征在于，所述人参皂苷的含量为30～50μg/mL。
  3. 如权利要求2所述的植物油佐剂，其特征在于，所述人参皂苷的含量为40～45μg/mL。
  4. 如权利要求1所述的植物油佐剂，其特征在于，所述食用植物油为葵花籽油、玉米油、橄榄油、山茶油或菜籽油。
  5. 如权利要求4所述的植物油佐剂，其特征在于，所述食用植物油为菜籽油。
  6. 如权利要求1～5任一所述的植物油佐剂的制备方法，包括：
    (1)将人参皂苷加入二甲基亚砜中进行溶解，混匀得到人参皂苷溶液；
    (2)将所述人参皂苷溶液加入植物油中直接混合均匀；
    或
    植物油加热至20～40℃后，在植物油中加入去水山梨醇单油酸酯和所述人参皂苷溶液，混合均匀。
  7. 如权利要求6所述的制备方法，其特征在于，所述人参皂苷溶液中人参皂苷的含量为10～60mg/mL。
  8. 如权利要求6所述的制备方法，其特征在于，所述去水山梨醇单油酸酯的添加量为：每100mL植物油佐剂中含10～16mL去水山梨醇单油酸酯。
  9. 如权利要求1～5任一所述的植物油佐剂在制备疫苗中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述疫苗为病毒疫苗或细菌疫苗。
  11. 如权利要求10所述的应用，其特征在于，所述疫苗为口蹄疫灭活疫苗或金黄色葡萄球菌疫苗。
PCT/CN2014/095451 2013-12-31 2014-12-30 一种含人参皂苷的植物油佐剂及其制备方法和应用 WO2015101271A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310754903.4A CN103751775A (zh) 2013-12-31 2013-12-31 一种含人参皂苷的植物油佐剂及其制备方法和应用
CN201310754903.4 2013-12-31

Publications (1)

Publication Number Publication Date
WO2015101271A1 true WO2015101271A1 (zh) 2015-07-09

Family

ID=50519200

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/095451 WO2015101271A1 (zh) 2013-12-31 2014-12-30 一种含人参皂苷的植物油佐剂及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN103751775A (zh)
WO (1) WO2015101271A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116983396A (zh) * 2023-08-09 2023-11-03 浙江省农业科学院 兔支气管败血波氏杆菌灭活疫苗乳剂及其制备和应用

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103751775A (zh) * 2013-12-31 2014-04-30 浙江大学 一种含人参皂苷的植物油佐剂及其制备方法和应用
CN104383534A (zh) * 2014-10-24 2015-03-04 威海芝恩药业股份有限公司 一种含有岩藻聚糖硫酸酯的免疫佐剂及其应用
CN104857511B (zh) * 2015-02-13 2018-03-30 浙江大学 含人参皂甙的疫苗稀释剂
CN108478789B (zh) * 2018-03-22 2021-09-28 胡松华 促进抗体生成的疫苗稀释剂及其制备方法和用途
CN108853493A (zh) * 2018-07-26 2018-11-23 中国人民解放军陆军军医大学 麦冬皂苷d及其纳米乳在制备疫苗佐剂中的应用
CN110179975B (zh) * 2019-06-15 2021-01-08 浙江大学 植物油疫苗佐剂及其制备方法和用途
CN112294954B (zh) * 2019-07-31 2024-04-12 洛阳赛威生物科技有限公司 一种禽用佐剂组合物及其制备方法和应用
CN111358944B (zh) * 2020-02-29 2021-12-31 浙江大学 复合植物油疫苗佐剂及其用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101670104A (zh) * 2009-09-24 2010-03-17 浙江大学 一种含人参皂甙的油佐剂及其制备方法
WO2013149017A1 (en) * 2012-03-28 2013-10-03 Kansas State University Research Foundation Vaccine adjuvant
CN103751775A (zh) * 2013-12-31 2014-04-30 浙江大学 一种含人参皂苷的植物油佐剂及其制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1323718C (zh) * 2004-03-15 2007-07-04 中国农业科学院兰州兽医研究所 一种利用植物油佐剂制备动物病毒性、细菌性疫苗的方法
CN101185757B (zh) * 2007-10-09 2011-04-06 浙江大学 一种含复合佐剂的疫苗及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101670104A (zh) * 2009-09-24 2010-03-17 浙江大学 一种含人参皂甙的油佐剂及其制备方法
WO2013149017A1 (en) * 2012-03-28 2013-10-03 Kansas State University Research Foundation Vaccine adjuvant
CN103751775A (zh) * 2013-12-31 2014-04-30 浙江大学 一种含人参皂苷的植物油佐剂及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, KAINIANG ET AL.: "Formulating Newcastle disease and avian influenza oil-emulsion vaccines by replacing mineral oil with animal and vegetable oil", POULTRY INDUSTRY SCIENCE AND TECHNOLOGY: MEDICINE AND PHARMACOLOGY, vol. 10, no. 2, 31 December 1994 (1994-12-31), pages 46 - 47 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116983396A (zh) * 2023-08-09 2023-11-03 浙江省农业科学院 兔支气管败血波氏杆菌灭活疫苗乳剂及其制备和应用
CN116983396B (zh) * 2023-08-09 2024-05-07 浙江省农业科学院 兔支气管败血波氏杆菌灭活疫苗乳剂及其制备和应用

Also Published As

Publication number Publication date
CN103751775A (zh) 2014-04-30

Similar Documents

Publication Publication Date Title
WO2015101271A1 (zh) 一种含人参皂苷的植物油佐剂及其制备方法和应用
Sun et al. Immunologic enhancement of compound Chinese herbal medicinal ingredients and their efficacy comparison with compound Chinese herbal medicines
Fan et al. Effect of epimedium polysaccharide-propolis flavone immunopotentiator on immunosuppression induced by cyclophosphamide in chickens
Guo et al. Astragalus polysaccharide and sulfated epimedium polysaccharide synergistically resist the immunosuppression
Song et al. Ginseng stem–leaf saponins (GSLS) and mineral oil act synergistically to enhance the immune responses to vaccination against foot-and-mouth disease in mice
Rivera et al. Ginseng and aluminium hydroxide act synergistically as vaccine adjuvants
Kong et al. Chinese herbal ingredients are effective immune stimulators for chickens infected with the Newcastle disease virus
Xiao et al. Improvement of a commercial foot-and-mouth disease vaccine by supplement of Quil A
CN103110942B (zh) 一种灭活鸡新城疫疫苗的制备方法
Qiu et al. Immunopotentiating effects of four Chinese herbal polysaccharides administered at vaccination in chickens
Fan et al. Microemulsion can improve the immune-enhancing activity of propolis flavonoid on immunosuppression and immune response
CN102743750B (zh) 一种复方免疫增强剂、禽用疫苗及其制备方法
Zhang et al. Rapeseed oil and ginseng saponins work synergistically to enhance Th1 and Th2 immune responses induced by the foot-and-mouth disease vaccine
WO2015103723A1 (zh) 中药桑叶多糖和杜仲多糖免疫增强剂及其应用
Liu et al. Immunopotentiation of Polysaccharides of Atractylodes macrocephala Koidz-loaded nanostructured lipid carriers as an adjuvant
Yang et al. Co-adjuvant effects of plant polysaccharide and propolis on chickens inoculated with Bordetella avium inactivated vaccine
CN101766815A (zh) 紫杉醇及多西紫杉醇的用途
CN105833267A (zh) 一种副猪嗜血杆菌病灭活疫苗新佐剂的制备方法
Yang et al. The Taishan Robinia pseudoacacia polysaccharides enhance immune effects of rabbit haemorrhagic disease virus inactivated vaccines
Maqbool et al. Ginseng stem‐leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine
Xiao et al. Enhancement of serological immune responses to foot-and-mouth disease vaccine by a supplement made of extract of cochinchina momordica seeds
Pomorska-Mól et al. The effect of doxycycline treatment on the postvaccinal immune response in pigs
Fan et al. The adjuvanticity of ophiopogon polysaccharide liposome against an inactivated porcine parvovirus vaccine in mice
Chen et al. Adjuvanticity of compound astragalus polysaccharide and sulfated epimedium polysaccharide per os
CN107158374B (zh) 一种免疫增强剂、口蹄疫灭活疫苗及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14877456

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14877456

Country of ref document: EP

Kind code of ref document: A1