CN107007831A - The immunologic adjuvant of hepatitis B DNA vaccine - Google Patents
The immunologic adjuvant of hepatitis B DNA vaccine Download PDFInfo
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- CN107007831A CN107007831A CN201710455438.2A CN201710455438A CN107007831A CN 107007831 A CN107007831 A CN 107007831A CN 201710455438 A CN201710455438 A CN 201710455438A CN 107007831 A CN107007831 A CN 107007831A
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- Prior art keywords
- enzymolysis
- hepatitis
- enzymolysis polypeptide
- adjuvant
- dirty
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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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- A61K2039/55516—Proteins; Peptides
-
- 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
Abstract
The invention discloses the immunologic adjuvant of hepatitis B DNA vaccine, the immunologic adjuvant is a kind of enzymolysis polypeptide, is prepared via a method which to form：Digested clam meat is dirty through alkali protease, with the 3000u of ultrafiltration retaining molecular weight 5000 component, freeze-drying produces the enzymolysis polypeptide.The enzymolysis polypeptide that the present invention is provided can significantly increase the immune response efficiency of hepatitis B DNA vaccine, can both improve humoral immune response, and cellullar immunologic response can be improved again, can be used as DNA vaccination adjuvant.
Description
Technical field
The invention belongs to vaccines arts, it is related to vaccine adjuvant, and in particular to the immunologic adjuvant of hepatitis B DNA vaccine.
Background technology
Hepatitis B is a kind of influence infectious disease as caused by hepatitis type B virus.HB vaccination is current control second
The maximally effective measure of type virus infection, but traditional hepatitis B subunit vaccine is resisted by the surface of hepatitis type B virus
Former and aluminium adjuvant composition, aluminium adjuvant vaccine is only capable of causing good humoral immune reaction and effective cellular immunity can not being caused anti-
Should.
DNA vaccination is also known as gene vaccine or nucleic acid vaccine, and its principle is that encoding gene is inserted into the matter with promoter
Grain carrier, after be conducted into internal cell with the method for physics, also exposed DNA is injected after body, target gene exists
Destination protein is given expression in vivo, so as to induce the immune response of body.After the albumen of expression is processed through APC, pass through MHC I and MHC
II two passes offer, to immune system, to stimulate body to produce specificity humoral and cell immune response.DNA vaccination has following
Advantage：Can be while inducing cell and humoral immunity；Different immunization routes can induce immune response；With appropriate configuration and group
Into allogenic gene can be expressed in human body, and induce neutrality antibody；Easy to maintain, transport, production.But it exempts from induction
It is relatively inefficient in epidemic disease response, especially in larger animal and the mankind, it have impact on its practical application.
Adjuvant refers to apply simultaneously or in advance with antigen, can strengthen the immune response ability that body is directed to antigen, or change
The material of immune response type.Immunologic adjuvant can strengthen antibody response；Strengthen the mucous membrane transmission of vaccine, promote immune contact；
Strengthen cellular immunity；Strengthen the immunogenicity of weak immunogene, such as highly purified antigen or recombinant antigen；Reduce antigen inoculation agent
Amount and inoculation times；Promote immune effect of the vaccine in the weak crowd of immune response ability；Accelerate immune response speed and
Extend the duration；Change the configuration of antigen；Change the compatibility of species, IgG subclass and the antibody of humoral antibody.
The application of adjuvant is most important for the efficiency for improving DNA vaccination induction immune response.
The content of the invention
The present invention is intended to provide a kind of immunologic adjuvant of hepatitis B DNA vaccine, the immunologic adjuvant is a kind of enzymolysis polypeptide,
To strengthen the immune response efficiency of hepatitis B DNA vaccine.
The present invention is achieved by following technical solution：
A kind of enzymolysis polypeptide, is prepared via a method which to form：Digested clam meat is dirty through alkali protease, use milipore filter
Molecular cut off 5000-3000u component, freeze-drying produces the enzymolysis polypeptide.
Preferably, described enzymolysis polypeptide is prepared via a method which to form：Take the dirty homogenate of clam meat broken, be placed in distillation
In water, alkali protease enzymolysis is added, enzymatic hydrolysis condition is pH value 9.4-9.8, and 42-48 DEG C of hydrolysis temperature, enzymolysis time 6-8 is small
When；After enzymolysis terminates, heating makes basic protein enzyme-deactivating, is cooled to normal temperature, 12000-14000 revs/min of 15-25 points of centrifugation
Clock, takes supernatant, carries out classification separation with the milipore filter that molecular cut off is 10000u, 5000u, 3000u successively, collects retention point
Son amount 5000-3000u component, freeze-drying produces the enzymolysis polypeptide.
Preferably, alkali protease weight is the dirty weight 0.2-0.4% of meat.
Preferably, enzymatic hydrolysis condition is pH value 9.6,45 DEG C of hydrolysis temperature, enzymolysis time 7 hours.
Preferably, heating makes the method for basic protein enzyme-deactivating be：Water-bath 8-12 minutes under the conditions of 80-90 DEG C.
Above-mentioned enzymolysis polypeptide is used as the purposes of hepatitis B DNA vaccine adjuvant.
Advantage of the present invention：
Enzymolysis polypeptide of the present invention can significantly increase the immune response efficiency of hepatitis B DNA vaccine, can be used as DNA vaccination assistant
Agent.
Brief description of the drawings
Fig. 1 is each group Anti-HBsAg antibody IgG concentration；
Fig. 2-4 is each group cytokine-expressing testing result.
Embodiment
In order to preferably explain technical scheme, it is further described with reference to specific embodiment.In embodiment
The experiment material do not emphasized especially is normal experiment material, belongs to the category that those skilled in the art are easily obtained.
Embodiment 1：The preparation of enzymolysis polypeptide
Fresh clam, which is shelled, takes internal organ, and -20 DEG C of freezen protectives are standby；Ultrafiltration cup and milipore filter are purchased from Shanghai and rubbed fast science device
Material Co., Ltd；Alkali protease 37017 (1:2.5U/g) it is purchased from Novozymes Company.
Take the dirty homogenate of clam meat broken, be placed in distilled water, adding alkali protease enzymolysis, (alkali protease adds quality
For the dirty weight of meat 0.3%), enzymatic hydrolysis condition be pH value 9.6,45 DEG C of hydrolysis temperature, enzymolysis time 7 hours；After enzymolysis terminates, 85
Water-bath makes basic protein enzyme-deactivating in 10 minutes under the conditions of DEG C, is cooled to normal temperature, and 13000 revs/min centrifuge 20 minutes, take supernatant,
Classification separation is carried out with the milipore filter that molecular cut off is 10000u, 5000u, 3000u successively, molecular cut off 5000- is collected
3000u component, freeze-drying produces the enzymolysis polypeptide.
Embodiment 2：The immunological enhancement of enzymolysis polypeptide
First, experiment material
Enzymolysis polypeptide is prepared according to the method for embodiment 1.
Rabbit igg, mouse anti-rabbit IgG, tetramethyl azo azoles (MTT), canavaline (ConA), bovine serum albumin(BSA) (BSA),
Phorbol diester (PMA), coban, paraformaldehyde and saponin are Sigma Products；CD3、IL-4、IFN-γ、CD4、
CD8, IgG1 fluorescent monoclonal antibody and Fc gamma antibodies are purchased from BD companies；Hydroxyl fluorescein diacetate succinimide fat
(CFSE) it is MolecularProbes products；Hyclone and RPMI 1640 are GIBCO Products；The pharmacy of HBsAg North China
Group provides；HBsAg specific CTL polypeptide S208-215ILSPFLPL are synthesized by the biochemical Co., Ltd of Shanghai gill；Anti-HBsAg antibody
ELISA detection kit is purchased from Beijing Jin Hao Bioisystech Co., Ltd.
2nd, experimental method
1st, the preparation of hepatitis B DNA
Hepatitis B plasmid, plasmid commission will be obtained in S2 areas before HBV and the gene constructed pcDNA3.0 to carrier for expression of eukaryon in S areas
Guangzhou Rui Bo bio tech ltd is built, and having proven to can high efficient expression.By hepatitis B plasmid and pcDNA3.0 empty plasmids point
Other Transformed E .coli DH5a, extract plasmid, are positive colony through digestion sequencing identification, and positive colony is inoculated in benzyl containing ammonia respectively
In the LB fluid nutrient mediums of penicillin, in 37 DEG C of shaken cultivation 15h, next day presses 1:80 expand the kind that culture 5h prepares fermented and cultured
Son, is finally transferred to fermentation cylinder for fermentation culture, using extensive alkaline lysis method of extracting plasmid, and with physiological saline adjust concentration to
1mg/mL is in -20 DEG C of preservations.
2nd, animal packet and immunization method
C57BL/6 mouse are randomly divided into 3 groups, and every group 10, packet and processing method are as follows：
Empty plasmid group：Only immune pcDNA3.0 empty plasmids, 100 μ L/ times/only；
Hepatitis B plasmid group：Only immune hepatitis B plasmid, 100 μ L/ times/only；
Adjuvant assisted group：Immune hepatitis B plasmid+enzymolysis polypeptide, 100 μ of hepatitis B plasmid L/ times/, enzymolysis polypeptide 2mg/kg/
Secondary/only.
When immune, muscle multi-point injection is in quadriceps muscle of thigh after mouse two.Respectively the 0th, 2,4 weeks immune mouse, at the 6th week
The dead every amynologic index of mouse detection.
3rd, amynologic index is detected
3.1 Anti-HBsAg antibody IgG Concentration Testings：Anti-HBsAg antibody specific IgG content is anti-by Beijing Jin Hao Bioisystech Co., Ltd
HBs ELISA kits specification is completed.
3.2T lymphopoiesis：2nd week after last time is immune, separating Morr. cell under aseptic condition passes through nylon
Post removes B cell and single cell suspension, adjustment cell concentration to 1 × 10 is made6Individual/mL, viable count is added 90% per hole
On 100 μ L cell suspensions to 96 porocyte culture plates, plus 100 μ LHBsAg antigens to final concentration of 5 μ g/mL be experimental group, plus
100 μ L ConA to final concentration of 5 μ g/mL are positive control, plus 100 μ LBSA to final concentration of 2 μ g/mL are unrelated control, 100
μ L RPMI 1640 are blank control thing, and experimental group and control wells respectively set 3 repeating holes, 37 DEG C, 5%CO248h is cultivated, per hole
Add 20 μ L 5 μ g/mLMTT, 37 DEG C, 5%CO24h is cultivated, supernatant is abandoned in centrifugation, 100 μ LDMSO, 37 DEG C of cultures are added per hole
15min, A570 light absorption values are determined with ELIASA, calculate stimulus index (SI).
3.3 Cytokine Expression Level：2nd week after last time is immune, separating Morr. cell under aseptic condition passes through Buddhist nun
Imperial post removes B cell and single cell suspension, adjustment cell concentration to 5 × 10 is made6Individual/mL, 100 μ L cell suspensions are added per hole and are arrived
In 96 porocyte culture plates, plus HBsAg antigens and anti-CD28 antibody are experimental group per each 50 μ L in hole to final concentration of 5 μ g/mL；Plus
100 μ LPMA to final concentration of 5 μ g/mL are positive controls, plus 100 μ LRPMI 1640 are blank control group, experimental group and right
3 repeating holes are respectively set according to hole.37 DEG C, 5%CO2Cultivate after 48h, cell, anti-Fc gamma antibodies closing, 4% poly first is collected by centrifugation
Aldehyde fixes 8min, and 0.1% saponin rupture of membranes 5min, PBS is washed 2 times, with 10 μ L fluorescent monoclonal antibodies CD4-FITC, CD4-PE,
CD8-PE, IL-4-PE and 4 DEG C of dark place dyeing 30min of IFN-γ-FITC, and anti-corresponding Isotype control is set, take 300 μ L streamings
Cell instrument is detected, compares each group Cytokine Expression Level.
CTL activity in 3.4 bodies：
2nd week after last time is immune, non-immunized blank mouse is put to death, isolated splenocyte is made single
Splenocyte suspension, is divided into two equal portions as target cell, and portion Jia 1 × and 10-6Mol/L HBsAg specific CTL epitope polypeptides
S208-215 is stimulated, and portion is not stimulated, 37 DEG C, 5%CO24h is cultivated, supernatant is abandoned in centrifugation, and PBS is washed 1 time, the target cell not stimulated
10min is dyed with 0.5 μm of ol/L CFSE dark place, the target cell of stimulation dyes 10min with 5 μm of ol/L CFSE dark places, add etc.
Volume calf serum terminates dyeing 2min, and supernatant is abandoned in centrifugation, and PBS is washed 1 time, the finally mixing by two kinds of target cells in equal volume, by 2
×107Individual target cell tail vein injection puts to death small to being immunized in Mice Body after carrying out internal cell toxicant killing reaction, killing 4h
Mouse, the isolated splenocyte in dark place takes 300 μ L flow cytomeries to analyze, and calculates internal CTL killings rate (%).
4th, statistical analysis
T inspections, P are carried out using SPSS20.0 statistics softwares<0.05 thinks significant difference.
3rd, experimental result
1st, Anti-HBsAg antibody IgG Concentration Testings result
2 weeks, ELISA method detection mice serum Anti-HBsAg antibody IgG after last time is immune.As shown in figure 1, Anti-HBsAg antibody IgG
Testing result shows that adjuvant assisted group Anti-HBsAg antibody IgG concentration is 458mIU/mL, and hepatitis B plasmid group Anti-HBsAg antibody IgG concentration is
264mIU/mL, empty plasmid group Anti-HBsAg antibody IgG concentration are 62mIU/mL.As can be seen here, adjuvant assisted group Anti-HBsAg antibody IgG concentration
It is significantly higher than hepatitis B plasmid group Anti-HBsAg antibody IgG concentration and empty plasmid group Anti-HBsAg antibody IgG concentration (P ＜ 0.05).
2nd, T lymphocyte proliferation assays result
2 weeks after last time is immune, after mouse T lymphocyte is stimulated through HBsAg, adjuvant assisted group and hepatitis B plasmid group
T lymphocytes produce proliferated specifically, hence it is evident that higher than empty plasmid group (P ＜ 0.05), and adjuvant assisted group again apparently higher than
Hepatitis B plasmid group (P ＜ 005), each group stimulus index (SI) is as shown in the table.
| Empty plasmid group | Hepatitis B plasmid group | Adjuvant assisted group | |
| Stimulus index (SI) | 1.4±0.3 | 2.2±0.2 | 4.5±0.3 |
3rd, cytokine-expressing testing result
As in Figure 2-4,2 weeks after last time is immune, after mouse T lymphocyte is stimulated through HBsAg, with hepatitis B plasmid
Group is compared, and adjuvant assisted group can significantly raise the expression of CD4+T lymphocytes IL-4 and IFN-γ, while adjuvant assisted group
Significantly increase the expression of CD8+T lymphocyte IFN-γs.
4th, internal CTL activity
2 weeks after last time is immune, internal CTL killings reaction is detected by flow cytometer, is killed in each group body
Hinder rate as shown in the table, adjuvant assisted group is significantly better than hepatitis B plasmid group and empty plasmid group：
| Empty plasmid group | Hepatitis B plasmid group | Adjuvant assisted group | |
| Internal killing rate (%) | 7.9±1.4 | 32.5±2.3 | 73.4±2.9 |
Summary experiment is as can be seen that enzymolysis polypeptide of the present invention can significantly increase the immune of hepatitis B DNA vaccine answers
Efficiency is answered, humoral immune response can be both improved, cellullar immunologic response can be improved again, can be used as DNA vaccination adjuvant.
Above-described embodiment is only used for that technical scheme is explained further, it will be appreciated by those skilled in the art that, appoint
What simple replacement is changed all without departing from the present invention, and protection scope of the present invention is not limited to above-mentioned specific embodiment.
Claims (6)
1. a kind of enzymolysis polypeptide, it is characterised in that be prepared via a method which to form：Clam meat is dirty through alkali protease enzyme
Solution, with ultrafiltration retaining molecular weight 5000-3000u component, freeze-drying produces the enzymolysis polypeptide.
2. enzymolysis polypeptide according to claim 1, it is characterised in that be prepared via a method which to form：Take clam meat dirty
Homogenate is broken, is placed in distilled water, adds alkali protease enzymolysis, and enzymatic hydrolysis condition is pH value 9.4-9.8, hydrolysis temperature 42-48
DEG C, enzymolysis time 6-8 hours；After enzymolysis terminates, heating makes basic protein enzyme-deactivating, is cooled to normal temperature, and 12000-14000 turns/
Minute centrifugation 15-25 minutes, takes supernatant, is classified successively with the milipore filter that molecular cut off is 10000u, 5000u, 3000u
Separation, collects molecular cut off 5000-3000u component, and freeze-drying produces the enzymolysis polypeptide.
3. enzymolysis polypeptide according to claim 2, it is characterised in that：Alkali protease weight is the dirty weight 0.2- of meat
0.4%.
4. enzymolysis polypeptide according to claim 2, it is characterised in that：Enzymatic hydrolysis condition be pH value 9.6,45 DEG C of hydrolysis temperature,
Enzymolysis time 7 hours.
5. enzymolysis polypeptide according to claim 2, it is characterised in that heating makes the method for basic protein enzyme-deactivating be：
Water-bath 8-12 minutes under the conditions of 80-90 DEG C.
6. any enzymolysis polypeptides of claim 1-5 are used as the purposes of hepatitis B DNA vaccine adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710455438.2A CN107007831A (en) | 2017-06-16 | 2017-06-16 | The immunologic adjuvant of hepatitis B DNA vaccine |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710455438.2A CN107007831A (en) | 2017-06-16 | 2017-06-16 | The immunologic adjuvant of hepatitis B DNA vaccine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097425A (en) * | 2018-08-28 | 2018-12-28 | 青海七彩花生物科技有限公司 | A kind of polypeptide and the purposes as influenza vaccines immunologic adjuvant |
| CN112229696A (en) * | 2020-10-23 | 2021-01-15 | 杭州联科生物技术股份有限公司 | Fixed film breaking agent and preparation method thereof |
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| CN1424110A (en) * | 2002-12-30 | 2003-06-18 | 北京市希波医学技术开发公司 | Vaccine adjuvant against hepatitis B |
| CN104789626A (en) * | 2015-03-06 | 2015-07-22 | 浙江海洋学院 | Preparation method and application of Cyclina sinensis enzymolysis polypeptide |
| CN105732780A (en) * | 2014-12-12 | 2016-07-06 | 郭文江 | Kucaoying peptide and application thereof |
| CN106434811A (en) * | 2016-11-21 | 2017-02-22 | 浙江海洋大学 | Preparation method and application of cyclina sinensis immune-enhancing peptide |
-
2017
- 2017-06-16 CN CN201710455438.2A patent/CN107007831A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1424110A (en) * | 2002-12-30 | 2003-06-18 | 北京市希波医学技术开发公司 | Vaccine adjuvant against hepatitis B |
| CN105732780A (en) * | 2014-12-12 | 2016-07-06 | 郭文江 | Kucaoying peptide and application thereof |
| CN104789626A (en) * | 2015-03-06 | 2015-07-22 | 浙江海洋学院 | Preparation method and application of Cyclina sinensis enzymolysis polypeptide |
| CN106434811A (en) * | 2016-11-21 | 2017-02-22 | 浙江海洋大学 | Preparation method and application of cyclina sinensis immune-enhancing peptide |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097425A (en) * | 2018-08-28 | 2018-12-28 | 青海七彩花生物科技有限公司 | A kind of polypeptide and the purposes as influenza vaccines immunologic adjuvant |
| CN109097425B (en) * | 2018-08-28 | 2022-06-07 | 浙江康嘉基因技术有限公司 | Polypeptide and application of polypeptide as influenza vaccine immunologic adjuvant |
| CN112229696A (en) * | 2020-10-23 | 2021-01-15 | 杭州联科生物技术股份有限公司 | Fixed film breaking agent and preparation method thereof |
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