CN111450245A - Hepatitis B treating vaccine and preparation method thereof - Google Patents
Hepatitis B treating vaccine and preparation method thereof Download PDFInfo
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- 239000002502 liposome Substances 0.000 claims abstract description 46
- 101710132601 Capsid protein Proteins 0.000 claims abstract description 41
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- 108091007433 antigens Proteins 0.000 claims abstract description 27
- 239000000427 antigen Substances 0.000 claims abstract description 25
- 238000002156 mixing Methods 0.000 claims abstract description 16
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- 239000002671 adjuvant Substances 0.000 claims abstract description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 9
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims abstract description 8
- 102000004127 Cytokines Human genes 0.000 claims abstract description 6
- 108090000695 Cytokines Proteins 0.000 claims abstract description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 20
- 239000003960 organic solvent Substances 0.000 claims description 20
- 238000002390 rotary evaporation Methods 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
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- 238000000034 method Methods 0.000 claims description 13
- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 claims description 12
- 229940124736 hepatitis-B vaccine Drugs 0.000 claims description 12
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 10
- 235000012000 cholesterol Nutrition 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- 229940067606 lecithin Drugs 0.000 claims description 10
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- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical group [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 102000006992 Interferon-alpha Human genes 0.000 claims description 4
- 108010047761 Interferon-alpha Proteins 0.000 claims description 4
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- 108010074328 Interferon-gamma Proteins 0.000 claims description 4
- 102000000588 Interleukin-2 Human genes 0.000 claims description 4
- 108010002350 Interleukin-2 Proteins 0.000 claims description 4
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 claims description 4
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
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- 208000006454 hepatitis Diseases 0.000 claims 1
- 231100000283 hepatitis Toxicity 0.000 claims 1
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- 229940021747 therapeutic vaccine Drugs 0.000 abstract description 9
- 230000028993 immune response Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
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- -1 aluminum ions Chemical class 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
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- 239000011780 sodium chloride Substances 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
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- 238000004108 freeze drying Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
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- 239000004411 aluminium Substances 0.000 description 1
- 229940024546 aluminum hydroxide gel Drugs 0.000 description 1
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical compound O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
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- 239000011550 stock solution Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- 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
- A61K2039/55505—Inorganic 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
-
- 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
- A61K2039/55522—Cytokines; Lymphokines; Interferons
- A61K2039/55527—Interleukins
- A61K2039/55533—IL-2
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- 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
- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention provides a hepatitis B therapeutic vaccine and a preparation method thereof, which relate to the field of biological products and comprise hepatitis B surface antigen, hepatitis B core antigen, cytokine and aluminum adjuvant, wherein HBsAg plasmid liposome and HBcAg plasmid liposome are respectively contained in the hepatitis B surface antigen and the hepatitis B core antigen, and the vaccine is prepared by mixing the HBsAg plasmid liposome and the HBcAg plasmid liposome after the preparation of the HBsAg plasmid liposome and the preparation of the HBcAg plasmid liposome are finished, sequentially adding the cytokine, phosphate buffer solution, physiological saline and the aluminum adjuvant, and uniformly mixing. The invention has low cost and good immune response effect generated by organisms.
Description
Technical Field
The invention belongs to the field of biological products, and particularly relates to a hepatitis B therapeutic vaccine and a preparation method thereof.
Background
Hepatitis B vaccine is a special medicine for preventing hepatitis B. After vaccination, the vaccine can stimulate the immune system to produce protective antibodies, and once hepatitis B virus appears, the antibodies can play a role to prevent infection, thereby achieving the purpose of preventing hepatitis B infection. Vaccination with hepatitis b vaccine is the most effective method for preventing hepatitis b virus infection.
The hepatitis B vaccine used at present is a genetic engineering vaccine, the main component of which is the surface antigen of hepatitis B virus, namely hepatitis B virus outer coat protein, which is not complete virus. This surface antigen does not contain viral genetic material, is not infectious or pathogenic, but retains immunogenicity, i.e., the ability to stimulate the body to produce protective antibodies. Previously, a hemogenic vaccine was used, the hepatitis B surface antigen of which was prepared from the blood of hepatitis B virus carriers by strict procedures such as purification and inactivation, and the vaccine was eliminated for reasons of safety, source, cost and the like.
The genetic engineering hepatitis B vaccine is produced by adopting the genetic engineering technology, namely constructing a genetic recombinant plasmid containing hepatitis B surface antigen (HBsAg), and then transfecting corresponding host cells, such as yeast and CHO cells (Chinese hamster ovary cells) to produce hepatitis B surface antigen protein. The recombinant yeast hepatitis B vaccine is produced by using the recombinant yeast, the recombinant CHO hepatitis B vaccine is produced by using the CHO cells, and the purity of the antigen protein of the product can reach more than 99 percent through cell disruption, a series of microfiltration, ultrafiltration, chromatography and the like after fermentation, and the dosage is 5 micrograms or 10 micrograms per bottle. They can be used for the prevention of all known subtypes of hepatitis B virus.
In hepatitis B vaccines, the purified antigen is co-precipitated with aluminum hydroxide as an adjuvant, and during co-precipitation, the antigen is adsorbed on the aluminum hydroxide. Vaccines are generally less stable and can be stored for 12 months at 2-8℃, but their efficacy decreases rapidly as the temperature increases, and many vaccines are stable for days or hours at 37℃, which is not conducive to transport at room temperature. In order to improve the stability of the vaccine, the vaccine can be dried by a freeze-drying method, but the freeze-drying method destroys the gel structure of aluminum hydroxide, so that the hepatitis B vaccine cannot be freeze-dried.
Chinese patent CN201110126060.4 discloses a method for preparing hepatitis B vaccine containing aluminum adjuvant Al (OH)3Is generated by an on-line reaction, namely PBS buffer solution, KAl (SO4)2Mixing the solution and hepatitis B surface antigen stock solution, adding NaOH solution into the mixture, and continuously generating Al (OH)3While the adjuvant continuously wraps and adsorbs hepatitis B surface antigen;
chinese patent CN201710992798.6 discloses a bivalent hepatitis B vaccine and a preparation method thereof, which comprises recombinant wild type hepatitis B virus surface antigen (recombinant w-HBsAg) and recombinant mutant hepatitis B virus surface antigen (recombinant m-HBsAg) with the concentration of 10-60 mu g/ml and aluminum hydroxide gel solution, wherein the recombinant w-HBsAg contains HBsAg or HBsAg amino acid fragment of wild type HBV, the recombinant m-HBsAg contains mutant HBsAg or mutant HBsAg amino acid fragment, and the mutant site of the mutant HBsAg is L21S, I126S and G145R.
The invention provides a hepatitis B therapeutic vaccine with low cost and good effect of stimulating organism to generate immune response and a preparation method thereof.
Disclosure of Invention
The invention aims to provide a hepatitis B therapeutic vaccine and a preparation method thereof aiming at the defects of the existing hepatitis B therapeutic vaccine and the preparation method thereof.
The invention provides the following technical scheme:
a vaccine for treating hepatitis B is composed of hepatitis B surface antigen, hepatitis B core antigen, cytokine and aluminium adjuvant, and the HBsAg plasmid liposome and HBcAg plasmid liposome are respectively contained in said hepatitis B surface antigen and hepatitis B core antigen.
Preferably, the cytokine is interleukin-2, interferon- α, interferon-gamma, and the aluminum adjuvant is one of aluminum hydroxide or aluminum phosphate.
A preparation method of a hepatitis B therapeutic vaccine comprises the following steps:
s1, preparing HBsAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, adding HBsAg plasmid solution, heating in water bath, rotary evaporation, and filtering to obtain HBsAg plasmid liposome;
s2, preparing HBcAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, adding HBcAg plasmid solution, heating in water bath, rotary evaporation, and filtering to obtain HBcAg plasmid liposome;
s3, mixing the HBsAg plasmid liposome and the HBcAg plasmid liposome uniformly to obtain a mixed solution of hepatitis B surface antigen and hepatitis B core antigen, adding cell factors, mixing uniformly, adding phosphate buffer solution and normal saline into a container, mixing uniformly, filtering for sterilization, and adding sterile aluminum hydroxide or aluminum phosphate to obtain the vaccine.
Preferably, the HBsAg plasmid solution in step S1 has a concentration of 0.300-0.800mg/ml and a pH of 7.10-7.50.
Preferably, the HBcAg plasmid solution in step S2 has a concentration of 0.300-0.800mg/ml and a pH of 7.10-7.50.
Preferably, the temperature of the water bath in steps S1 and S2 is 34-40 ℃.
The invention has the beneficial effects that: the method is simple, and can effectively induce organism to generate immune response and generate more protective antibodies.
Detailed Description
Example 1
The preparation method of the hepatitis B therapeutic vaccine is characterized by comprising the following steps:
s1, preparing HBsAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, adding HBsAg plasmid solution with concentration of 0.300mg/ml and pH value of 7.10, heating in water bath, rotary evaporation, and filtering to obtain HBsAg plasmid liposome, wherein the temperature of the water bath is 34 deg.C;
s2, preparing HBcAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, adding HBcAg plasmid solution, wherein the concentration of the HBcAg plasmid solution is 0.300mg/ml, the pH value is 7.10, heating in a water bath, carrying out rotary evaporation, and filtering to obtain HBcAg plasmid liposome, wherein the temperature of the water bath is 34 ℃;
s3, pouring the hepatitis B surface antigen plasmid liposome and the hepatitis B core antigen plasmid liposome into a container, mixing, adding interleukin-2, mixing uniformly, adding phosphate buffer solution and normal saline into the container, filtering and sterilizing after mixing uniformly, and adding sterile aluminum hydroxide to obtain the vaccine, wherein the final concentrations of the hepatitis B surface antigen plasmid liposome and the hepatitis B core antigen plasmid liposome are both 20ug/ml, the final concentration of the interleukin-2 is 200U/ml, the final concentration of aluminum ions is 1.0mg/ml, the final concentration of phosphate buffer solution (pH7.10) is 20mmol/ml, and the final concentration of sodium chloride is 150 mmol/ml.
Example 2
The preparation method of the hepatitis B therapeutic vaccine is characterized by comprising the following steps:
s1, preparing HBsAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, pouring HBsAg plasmid solution with concentration of 0.500mg/ml and pH value of 7.30, heating in water bath, rotary evaporating, and filtering to obtain HBsAg plasmid liposome, wherein the temperature of the water bath is 37 deg.C;
s2, preparing HBcAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, pouring into HBcAg plasmid solution with concentration of 0.500mg/ml and pH value of 7.30, heating in water bath, rotary evaporation, and filtering to obtain HBcAg plasmid liposome, wherein the temperature of the water bath is 37 ℃;
s3, pouring the hepatitis B surface antigen plasmid liposome and the hepatitis B core antigen plasmid liposome into a container, adding interferon- α, mixing uniformly, adding phosphate buffer solution and normal saline into the container, filtering for sterilization after mixing uniformly, and adding sterile aluminum hydroxide to obtain the vaccine, wherein the final concentrations of the hepatitis B surface antigen plasmid liposome and the hepatitis B core antigen plasmid liposome are both 20ug/ml, the final concentration of the interferon- α is 100U/ml, the final concentration of aluminum ions is 1.0mg/ml, the final concentration of the phosphate buffer solution (pH7.30) is 20mmol/ml, and the final concentration of sodium chloride is 150 mmol/ml.
Example 3
The preparation method of the hepatitis B therapeutic vaccine is characterized by comprising the following steps:
s1, preparing HBsAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, pouring HBsAg plasmid solution with concentration of 0.800mg/ml and pH value of 7.50, heating in water bath, rotary evaporation, and filtering to obtain HBsAg plasmid liposome, wherein the temperature of the water bath is 40 deg.C;
s2, preparing HBcAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, pouring into HBcAg plasmid solution with concentration of 0.800mg/ml and pH value of 7.50, heating in water bath, rotary evaporation, and filtering to obtain HBcAg plasmid liposome, wherein the temperature of the water bath is 40 deg.C;
s3, pouring the hepatitis B surface antigen plasmid liposome and the hepatitis B core antigen plasmid liposome into a container, adding interferon-gamma, mixing uniformly, adding phosphate buffer solution and normal saline into the container, filtering for sterilization after mixing uniformly, and adding sterile aluminum phosphate to prepare the vaccine, wherein the final concentrations of the hepatitis B surface antigen plasmid liposome and the hepatitis B core antigen plasmid liposome are both 20ug/ml, the final concentration of the interferon-gamma is 100U/ml, the final concentration of aluminum ions is 1.0mg/ml, the final concentration of the phosphate buffer solution (pH7.50) is 20mmol/ml, and the final concentration of sodium chloride is 150 mmol/ml.
Animal experiments
40 Kunming mice at 8 weeks are selected and randomly divided into a blank control group, an example 1 group, an example 2 group and an example 3 group, each group comprises 10 mice, the blank control group is injected with a proper amount of physiological saline, the example 1 group is injected with the vaccine prepared according to the method of the example 1, the example 2 group is injected with the vaccine prepared according to the method of the example 2, the example 3 group is injected with the vaccine prepared according to the method of the example 3, the corresponding vaccines are respectively injected at 0 th, 1 st and 2 nd weeks, the dosage is 0.5ml, 3 times, the inner canthus blood taking is respectively carried out at 4 th, 8 th, 12 th, 24 th and 48 th weeks, the antibody content in the serum is detected by using a hepatitis B antibody detection kit, and the experimental results are shown in tables 1-2:
TABLE 1 hepatitis B vaccine induced hepatitis B antibody (HBsAb) in mice
| Time (week) | Blank group | EXAMPLE 1 group | EXAMPLE 2 group | EXAMPLE 3 group |
| 4 | <1:5 | 1:8100 | 1:10100 | 1:7100 |
| 8 | <1:5 | 1:11700 | 1:12800 | 1:11750 |
| 12 | <1:5 | 1:10500 | 1:11800 | 1:11500 |
| 24 | <1:5 | 1:9900 | 1:10100 | 1:10920 |
| 48 | <1:5 | 1:3500 | 1:5600 | 1:4280 |
TABLE 2 hepatitis B vaccine induced hepatitis B antibody (HBcAb) in mice
| Time (week) | Blank group | EXAMPLE 1 group | EXAMPLE 2 group | EXAMPLE 3 group |
| 4 | <1:5 | 1:7200 | 1:13200 | 1:8700 |
| 8 | <1:5 | 1:16900 | 1:18200 | 1:15750 |
| 12 | <1:5 | 1:13300 | 1:15800 | 1:14800 |
| 24 | <1:5 | 1:7600 | 1:12700 | 1:9920 |
| 48 | <1:5 | 1:4200 | 1:6200 | 1:5920 |
The results show that the mice injected with hepatitis B vaccines produced under different production conditions all produce corresponding antibodies against hepatitis B surface antigen and hepatitis B core antigen within 4-48 weeks, and reach the highest level by 12 weeks and can still maintain a certain amount by 48 weeks.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A vaccine for treating hepatitis B is characterized by comprising hepatitis B surface antigen, hepatitis B core antigen, cell factor and aluminum adjuvant, wherein HBsAg plasmid liposome and HBcAg plasmid liposome are respectively contained in the hepatitis B surface antigen and the hepatitis B core antigen.
2. The therapeutic hepatitis B vaccine of claim 1, wherein the cytokine is one of interleukin-2, interferon- α, interferon- γ, and aluminum adjuvant is aluminum hydroxide or aluminum phosphate.
3. A process for the preparation of a vaccine for the treatment of hepatitis b according to any one of claims 1 to 2, characterized in that it comprises the following steps:
s1, preparing HBsAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, adding HBsAg plasmid solution, heating in water bath, rotary evaporation, and filtering to obtain HBsAg plasmid liposome;
s2, preparing HBcAg plasmid liposome: dissolving lecithin and cholesterol with organic solvent, removing the organic solvent by rotary evaporation, adding HBcAg plasmid solution, heating in water bath, rotary evaporation, and filtering to obtain HBcAg plasmid liposome;
s3, mixing the HBsAg plasmid liposome and the HBcAg plasmid liposome uniformly, standing for 2h to obtain a mixed solution of hepatitis B surface antigen and hepatitis B core antigen, adding a cytokine, mixing uniformly, adding a phosphate buffer solution and normal saline into a container, mixing uniformly, filtering for sterilization, and adding a sterile aluminum adjuvant to obtain the vaccine.
4. The method of claim 3, wherein the HBsAg plasmid solution in step S1 has a concentration of 0.300-0.800mg/ml and a pH of 7.10-7.50.
5. The method of claim 3, wherein the HBcAg plasmid solution in the step S2 has a concentration of 0.300-0.800mg/ml and a pH of 7.10-7.50.
6. The method for preparing the vaccine for treating hepatitis B according to claim 3, wherein the temperature of the water bath in steps S1 and S2 is 34-40 ℃.
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