CN106008667B - Small peptide, its application and vaccine as vaccine adjuvant - Google Patents
Small peptide, its application and vaccine as vaccine adjuvant Download PDFInfo
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- CN106008667B CN106008667B CN201610396553.2A CN201610396553A CN106008667B CN 106008667 B CN106008667 B CN 106008667B CN 201610396553 A CN201610396553 A CN 201610396553A CN 106008667 B CN106008667 B CN 106008667B
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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Abstract
Small peptide, its application and vaccine as vaccine adjuvant.Be used as the vaccine of vaccine adjuvant as the application of vaccine adjuvant and using above-mentioned small peptide the present invention provides a kind of small peptide, the small peptide, small peptide preparation is simple, easily with after antigen physical mixed can effective enhancement antigen immune response ability.The short peptide sequence is X-GFFYK, and wherein X is end-capping group.Preferably, the small peptide is D configuration.Above-mentioned small peptide can be used as the immunogenicity that immunologic adjuvant carrys out enhancement antigen, so that the cellular immunity and humoral immune response of strong antigentic specificity occur for main body, and the small peptide can be suitably used for all kinds of antigens；The small peptide is easily prepared, composition is single controllable.
Description
Technical field
The present invention relates to a kind of small peptides, its application and vaccine as vaccine adjuvant.
Background technique
With the hair at full speed of the immunologic research that deepens continuously, increasingly mature technique for gene engineering and synthetic technology
Exhibition, the mankind have been developed that the new generation vaccines such as a variety of DNA recombinant vaccines, small peptide vaccine.Although these emerging vaccines have many excellent
Point, for example, be readily synthesized, purify, antigentic specificity it is strong, but its weaker immunogenicity becomes its fatal disadvantage, leads to it
Strong immune response cannot be induced, its use clinically is limited.In practical applications, it often needs with immune assistant
Agent come enhance its immunogenicity or enhance host versus original specific response.Up to the present, aluminium adjuvant is unique logical for cut-off
The adjuvant that can be used for people of FDA approval is crossed, however it can only excite corresponding humoral immunity, not can induce body and generate accordingly
Cellular immunity, and cellular immunity be in terms of the immunization therapy of many diseases (virus infection, tumour etc.) it is essential, because
For this aluminium adjuvant to antigens such as many viruses, diseases without adjuvanticity effect, this, which allows for developing, novel can be used in human body
Therapeutic immunological adjuvants seem extremely urgent.
In addition to the aluminium adjuvant applied to human body of FDA approval, now under study for action or the adjuvant master of animal can be applied to
There are Freund's adjuvant, liposome, monophosphoryl lipid A, cell factor, CpG immune modulatory sequence etc..However, these immune assistants
Agent is not that safety is poor, is exactly that cost is too high, be not easy to synthesizing and purifying or the ability of enhancement antigen immune response is poor, still not
Them are able to satisfy in the demand of clinical use.
Summary of the invention
Goal of the invention
It is an object of the present invention to provide a kind of small peptides, prepare simply, easily and after antigen physical mixed
The ability of effective enhancement antigen immune response.
Application it is a further object to provide the small peptide as vaccine adjuvant, small peptide preparation are simple, simple
Just with after antigen physical mixed can effective enhancement antigen immune response ability.
It is a further object to provide more than one to state vaccine of the small peptide as vaccine adjuvant.
Summary of the invention
Inventors have found that a series of small peptides comprising sequence FFY easily can effectively enhance with after antigen physical mixed
The ability of antigen immune response, such as sequence are the small peptide of X-GFFY, X-GFFYK, and the effect of D configuration small peptide is more excellent.
According to the first aspect of the invention, the present invention provides a kind of small peptides, and sequence X-GFFYK, wherein X is sealing end
Group.End-capping group containing aromatic rings commonly used in the art, such as Nap may be selected in the group X.
" small peptide " is term commonly used in the art, refers to the short-chain peptide being made of 3-9 amino acid residue.
Heretofore described amino acid sequence if not otherwise specified, then to its configuration without limitation.
Preferably, the small peptide is D configuration, sequence X-GDFDFDYDK。
Preferably, X Nap, the structural formula of the small peptide is as follows at this time:
Well known FMOC- solid phase synthesis process synthesis can be used in the small peptide.
According to the second aspect of the invention, application of the small peptide as vaccine adjuvant is additionally provided.
The vaccine can be a variety of vaccines, for example, protein vaccine or small peptide vaccine.
A variety of physical forms can be selected when applying as vaccine adjuvant in the small peptide, enhance it ability of immune response not
Substantial effect can be generated, such as vaccine can be mixed to get with antigen after mixing with water, alternatively, since the small peptide has
There is good gel-forming property, can also be applied in the form of small peptide hydrogel, specifically, the aqueous mixtures of the small peptide pass through
The cooling method of heating forms small peptide hydrogel, then mixes with antigen, and the hydrogel formed after standing is used as vaccine.As this
The aqueous mixtures of field common sense, the small peptide should have good biocompatibility, and therefore, it is necessary to by small peptide and with good raw
The aqueous solution of object compatibility is mixed to get the aqueous mixtures of the small peptide.Common, the water with good biocompatibility
Solution can be physiological saline or PBS solution.This vaccine preparation method is simple and practical, ingredient is controllable.The formation hydrogel
Heating cooling means be known method, specifically: it is mixed using the water of the heating devices heats small peptide such as hair dryer or oil bath pan
It closes object to small peptide to be completely dissolved and (generally require and be heated to 90 DEG C or more), forms hydrogel after cooling and (be generally cooled to 20-
40 DEG C).Pass through the method judgement for being inverted bottle whether plastic, being retained in bottle bottom is hydrogel, if it is flowing
It is then liquid.
In the third aspect of the present invention, more than one vaccines for stating small peptide as vaccine adjuvant are additionally provided.The small peptide
Vaccine is obtained with antigen physical mixed with can be convenient.It is preferred that the method for the heated cooling of the aqueous mixtures of the small peptide formed it is short
Peptide hydrogel, then mixes with antigen, and the hydrogel formed after standing is used as vaccine.The vaccine can be a variety of vaccines, example
For example protein vaccine or small peptide vaccine.The dosage of the small peptide can determine by those skilled in the art through simple experiment, generally
When for protein vaccine and small peptide vaccine, the mass ratio of the small peptide and antigen is 5:1-30:1, more preferably 5:1-20:1.
Inventors have found that above-mentioned small peptide can be used as the immunogenicity that immunologic adjuvant carrys out enhancement antigen, so that main body occurs
The cellular immunity and humoral immune response of strong antigentic specificity, and the small peptide can be suitably used for all kinds of antigens；The small peptide
Easily prepared, composition is single controllable.
Invention increases the types of immunologic adjuvant, provide for the immunologic adjuvant that exploitation can apply to human body valuable
Information.
Detailed description of the invention
Fig. 1: the antibody titer that protein vaccine vac-1, vac-2, OVA, Alum-OVA are triggered an immune response；
Fig. 2: the antibody titer that protein vaccine vac-3, OVA, Alum-OVA are triggered an immune response；
Fig. 3: the antibody titer that small peptide vaccine vac-4, epitope, Alum-epitope are triggered an immune response；
Fig. 4: cell vaccine vac-5, vac-6, XTC, L-gel, D-gel stimulate CD8+IFN- γ+T cell proliferation effect
(Fig. 4 a)) and their tumor inhibitory effect (Fig. 4 b)).
Specific embodiment
The present invention is further described with embodiment below, but the embodiment is only used for the present invention rather than limits this hair
It is bright.
In following embodiment, whether examining the formation of hydrogel by the method for inversion bottle commonly used in the art.
Involved preparation source is as follows in following embodiment:
Culture medium, RMPI 1640 is scientific and technological (ThermoFisher Scientific) purchased from the silent winged generation that of match, sterile；
Fetal calf serum, it is scientific and technological (ThermoFisher Scientific) purchased from the silent winged generation that of match, it is sterile；
2-cl-Trt resin is purchased from Tianjin Nankai Hecheng S&T Co., Ltd., active 1.2mmol/mL；
N,N-diisopropylethylamine (is indicated with DIEPA) below, is purchased from sigma aldrich company (Sigma-
Aldrich), purity 99%；
Benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (being indicated below with HBTU), it is biochemical purchased from gill
(Shanghai) Co., Ltd., purity 98%；
Trifluoroacetic acid (is indicated with TFA) below, is purchased from sigma aldrich company (Sigma-Aldrich), purity
99%；
Tri isopropyl silane (is indicated with TIS) below, is purchased from sigma aldrich company (Sigma-Aldrich), pure
Degree 99%；
L-configuration and D amino acids are purchased from gill biochemistry (Shanghai) Co., Ltd., purity 98%；
The chicken egg white (hereinafter referred to as OVA albumen) of endotoxin-free is purchased from InvivoGen company, purity 99%；
Aluminium adjuvant is scientific and technological (ThermoFisher Scientific) purchased from the silent winged generation that of match, purity 99%；
Methyl α-naphthyl acetate, fluoro phenthazine, biotin are purchased from sigma aldrich company (Sigma-Aldrich, purity
99%；
Small peptide B+T epitope is purchased from gill biochemistry (Shanghai) Co., Ltd., purity 98%；
Cancer cell EG7 sings all biologies purchased from Shanghai, and cultivates according to the following steps:
1) water-bath is warming up to 37 DEG C in advance, culture medium, serum etc. is put into water-bath preheating, and at the same time opening super
Net platform ultraviolet light irradiation half an hour；
2) the cancer cell EG7 frozen is taken out from liquid nitrogen container, being placed on rapidly in 37 DEG C of water-bath makes cell thaw, it
After be quickly transferred to proceed as follows in super-clean bench: celliferous solution is carefully transferred to pipettor containing culture medium
Centrifuge tube in, be centrifuged 5 minutes, remove supernatant, with containing 10% fetal calf serum culture medium be resuspended, be transferred to containing 10% tire ox
In the culture dish of the culture medium of serum, it is then placed in 37 DEG C of incubators and cultivates；
3) second day observation cell state passes a generation and carries out following experiment later after cell state is good；
4) cell culture fluid is collected, is drawn into centrifuge tube, 4min is centrifuged under 1000rpm revolving speed, discards solution later, is added
Enter fresh culture medium to be blown and beaten uniformly with rifle, is counted as 5 × 10 with cell counting board7It is every milliliter a.
The OVA protein solution is self-control, and OVA albumen is dissolved in PBS solution (pH=7.0) and obtains the OVA egg of 5mg/mL
White solution；
Described 5 × 107Cancer cell after a every milliliter of X-ray irradiation is self-control: taking 1.5 milliliter 5 × 107A every milliliter
Cancer cell EG7 is irradiated 8.5 minutes in irradiation instrument with the electric current of 320 volts of voltages, 12.5 peaces, and irradiation is twice.
Remaining reagent is ommercially available AR.
Prepare embodiment 1
The preparation of the vaccine vac-1 of small peptide hydrogel load OVA albumen at pH 7.0 and 20 DEG C of room temperature
(1) L-configuration small peptide Nap-GFFY is synthesized with FMOC- solid phase synthesis process, structural formula is as follows:
Specific step is as follows:
1) 0.5mmol 2-cl-Trt resin is weighed in synthesis in solid state device, and the anhydrous methylene chloride that 10mL is added is (following
Indicated with DCM), it is placed on shaking table and rocks 5min, be swollen 2-cl-Trt resin sufficiently；
2) with ear washing bulb, DCM, pressure is removed completely from the synthesis in solid state device equipped with 2-cl-Trt resin；
3) the fmoc-protected amino acid of 0.75mmol is dissolved in the anhydrous DCM of 10mL, is added 0.75mmol's
DIEPA is then transferred into above-mentioned solid phase synthesizer, then adds the DIEPA of 0.75mmol, reacts 1h at room temperature；
4) it closes: removing the reaction solution in synthesis in solid state device with ear washing bulb, then washed with anhydrous DCM, each DCM dosage
10mL, washing 1min, are washed 5 times altogether, and it is anhydrous DCM: DIEPA that the volume ratio prepared, which is added: methanol=17: 1: 2 solution 20mL,
10min is reacted at room temperature；
5) reaction solution in synthesis in solid state device is removed with ear washing bulb, is first washed with anhydrous DCM, each DCM dosage 10mL, is washed
Time 1min is washed, is washed altogether 5 times, then is washed with n,N-Dimethylformamide (being indicated below with DMF), each DMF dosage 10mL, is washed
Time 1min is washed, is washed altogether 5 times, the DMF for the piperidines that 10mL is 20% containing percent by volume is added, reacts 25min, then contained with 10mL
The DMF for the piperidines that percent by volume is 20% reacts 5min, is then washed with DMF, each DMF dosage 10mL, wash time
1min is washed 5 times altogether, after carry out next step reaction；
6) second fmoc-protected amino acid 1 mmol, HBTU 1.5mmol, DIEPA 2mmol and 10mlDMF is added,
The solution prepared is added in above-mentioned solid phase synthesizer, 2h is reacted；
7) amino acid or end-capping group that step 5) sequentially adds needs with method 6) are repeated；Then 5 are washed with DMF
Time, methylene chloride is washed 5 times, and the next step is carried out；
8) 95%TFA, 2.5%TIS, 2.5%H are pressed2The solution 10mL of O percent by volume composition is added to above-mentioned solid phase conjunction
In growing up to be a useful person, reaction half an hour (or the volume ratio of TFA and DCM is 1: 99, and it is molten to be configured to the TFA that concentration of volume percent is 1%
Liquid takes each 3mL of the TFA solution to be added in above-mentioned solid phase synthesizer, and altogether plus ten times, each reaction time is 1min), production
Object is cut from 2-cl-Trt resin, and vacuum concentration removes solvent, obtains crude product, uses HPLC separating-purifying later.
Its structural characterization data are as follows:
1H NMR (400MHz, DMSO) δ 8.29 (d, J=7.3Hz, 1H), 8.23 (t, J=5.7Hz, 1H), 8.17 (d, J
=7.9Hz, 1H), 8.02 (d, J=8.6Hz, 1H), 7.89-7.79 (m, 3H), 7.75 (s, 1H), 7.49-7.39 (m, 3H),
7.26-7.11 (m, 12H), 7.08 (d, J=7.9Hz, 2H), 4.60-4.40 (m, 4H), 3.71 (dd, J=16.9,4.9Hz,
2H), 3.57 (dd, J=16.4,5.6Hz, 3H), 2.99-2.85 (m, 5H), 2.81-2.74 (m, 1H), 2.69-2.62 (m,
1H).
(2) it takes 1mg L-configuration small peptide Nap-GFFY to be placed in 1.5 milliliters of vial, 400 microlitres of PBS solution (pH is added
=7.0) its pH value, is adjusted to 7.0 with sodium carbonate liquor, be heated to boiling be completely dissolved compound, be cooled to room temperature it
Afterwards up to small peptide hydrogel.
(3) 20 microlitres of OVA protein solution of 5mg/mL are taken to be added in (2) in hydrogel obtained, with PBS solution (pH
=7.0) constant volume carries out physical mixed in 500 microlitres, stands plastic after ten minutes, obtains protein vaccine vac-1 (final small peptide
Concentration be 2mg/mL, OVA protein concentration be 0.2mg/mL).
Prepare embodiment 2
Small peptide hydrogel Nap-G at pH 7.0 and 20 DEG C of room temperatureDFDFDY loads the preparation of the vaccine vac-2 of OVA albumen
(1) Nap-G is synthesized with FMOC- solid phase synthesis processDFDFDY, structural formula are as follows:
Specific step is as follows:
1) 0.5mmol 2-cl-Trt resin is weighed in synthesis in solid state device, the DCM of 10mL is added, and is placed on shaking table and is shaken
5min is shaken, is swollen 2-cl-Trt resin sufficiently；
2) with ear washing bulb, DCM, pressure is removed completely from the synthesis in solid state device equipped with 2-cl-Trt resin；
3) the fmoc-protected amino acid of 0.75mmol is dissolved in the anhydrous DCM of 10mL, is added 0.75mmol's
DIEPA is then transferred into above-mentioned solid phase synthesizer, then adds the DIEPA of 0.75mmol, reacts 1h at room temperature；
4) it closes: removing the reaction solution in synthesis in solid state device with ear washing bulb, then washed with anhydrous DCM, each DCM dosage
10mL, washing 1min, are washed 5 times, each 10mL altogether, and it is anhydrous DCM: DIEPA that the volume ratio prepared, which is added: methanol=17: 1: 2
Solution 20mL, reacts 10min at room temperature；
5) reaction solution in synthesis in solid state device is removed with ear washing bulb, is first washed with anhydrous DCM, each DCM dosage 10mL, is washed
Time 1min is washed, is washed altogether 5 times, then washed with DMF, each DMF dosage 10mL, wash time 1min are washed 5 times altogether, and 10mL is added
DMF containing the piperidines that percent by volume is 20% reacts 25min, then the DMF with the 10mL piperidines for being 20% containing percent by volume
React 5min, then washed with DMF, each DMF dosage 10mL, wash time 1min are washed 5 times altogether, after carry out in next step
Reaction；
6) second fmoc-protected amino acid 1 mmol, HBTU 1.5mmol, DIEPA 2mmol and 10mlDMF is added,
The solution prepared is added in above-mentioned solid phase synthesizer, 2h is reacted；
7) amino acid or end-capping group that step 5) sequentially adds needs with method 6) are repeated；Then 5 are washed with DMF
Time, methylene chloride is washed 5 times, and the next step is carried out；
8) 95%TFA, 2.5%TIS, 2.5%H are pressed2The solution 10mL of the volume ratio composition of O is added to above-mentioned solid phase synthesis
In device, reaction half an hour (or the volume ratio of TFA and DCM is 1: 99, and it is molten to be configured to the TFA that concentration of volume percent is 1%
Liquid takes each 3mL of the TFA solution to be added in above-mentioned solid phase synthesizer, and altogether plus ten times, each reaction time is 1min), production
Object is cut from 2-cl-Trt resin, and vacuum concentration removes solvent, obtains crude product, uses HPLC separating-purifying later.
Its structural characterization data are as follows:
1H NMR (400MHz, DMSO) δ 8.29 (d, J=7.3Hz, 1H), 8.23 (t, J=5.7Hz, 1H), 8.17 (d, J
=7.9Hz, 1H), 8.02 (d, J=8.6Hz, 1H), 7.89-7.79 (m, 3H), 7.75 (s, 1H), 7.49-7.39 (m, 3H),
7.26-7.11 (m, 12H), 7.08 (d, J=7.9Hz, 2H), 4.60-4.40 (m, 4H), 3.71 (dd, J=16.9,4.9Hz,
2H), 3.57 (dd, J=16.4,5.6Hz, 3H), 2.99-2.85 (m, 5H), 2.81-2.74 (m, 1H), 2.69-2.62 (m,
1H).
(2) 1mg Nap-G is takenDFDFDY is placed in 1.5 milliliters of vial, and 400 microlitres of PBS solutions (pH=7.0) are added,
Its pH value is adjusted to 7.0 with sodium carbonate liquor, being heated to boiling is completely dissolved compound, is cooled to after room temperature up to short
Peptide hydrogel.
(3) 20 microlitres of OVA protein solution of 5mg/mL are taken to be added in (2) in hydrogel obtained, with PBS solution (pH
=7.0) constant volume is in 500 microlitres, carries out physical mixed, plastic after standing 8 minutes, obtain protein vaccine vac-2 (final small peptide
Concentration is 2mg/mL, and OVA protein concentration is 0.2mg/mL).
Prepare embodiment 3
Small peptide Nap-G is synthesized with FMOC- solid phase synthesis processDFDFDYDK, the specific steps are as follows:
1) 0.5mmol 2-cl-Trt resin is weighed in synthesis in solid state device, the DCM of 10mL is added, and is placed on shaking table and is shaken
5min is shaken, is swollen 2-cl-Trt resin sufficiently；
2) with ear washing bulb, DCM, pressure is removed completely from the synthesis in solid state device equipped with 2-cl-Trt resin；
3) the fmoc-protected amino acid of 0.75mmol is dissolved in the anhydrous DCM of 10mL, is added 0.75mmol's
DIEPA is then transferred into above-mentioned solid phase synthesizer, then adds the DIEPA of 0.75mmol, reacts 1h at room temperature；
4) it closes: removing the reaction solution in synthesis in solid state device with ear washing bulb, then washed with anhydrous DCM, each DCM dosage
10mL, wash time 1min are washed 5 times altogether, and it is anhydrous DCM: DIEPA that the volume ratio prepared, which is added: methanol=17: 1: 2 solution
20mL reacts 10min at room temperature；
5) reaction solution in synthesis in solid state device is removed with ear washing bulb, is first washed with anhydrous DCM, each DCM dosage 10mL, is washed
Time 1min is washed, is washed altogether 5 times, then washed with DMF, each DMF dosage 10mL, wash time 1min are washed 5 times altogether, and 10mL is added
DMF containing the piperidines that percent by volume is 20% reacts 25min, then the DMF with the 10mL piperidines for being 20% containing percent by volume
React 5min, then washed with DMF, each DMF dosage 10mL, wash time 1min are washed 5 times altogether, after carry out in next step
Reaction；
6) second fmoc-protected amino acid 1 mmol, HBTU 1.5mmol, DIEPA 2mmol and 10mlDMF is added,
The solution prepared is added in above-mentioned solid phase synthesizer, 2h is reacted；
7) amino acid or end-capping group that step 5) sequentially adds needs with method 6) are repeated；Then 5 are washed with DMF
Time, methylene chloride is washed 5 times, and the next step is carried out；
8) 95%TFA, 2.5%TIS, 2.5%H are pressed2The solution 10mL of the volume ratio composition of O is added to above-mentioned solid phase synthesis
In device, reaction half an hour (or the volume ratio of TFA and DCM is 1: 99, and it is molten to be configured to the TFA that concentration of volume percent is 1%
Liquid takes each 3mL of the TFA solution to be added in above-mentioned solid phase synthesizer, and altogether plus ten times, each reaction time is 1min), production
Object is cut from 2-cl-Trt resin, and vacuum concentration removes solvent, obtains crude product, uses HPLC separating-purifying later.
Its structural characterization data are as follows:
1H NMR (400MHz, DMSO) δ 8.28 (d, J=6.3Hz, 2H), 8.18 (d, J=8.3Hz, 1H), 8.08 (dd, J
=8.0,4.0Hz, 2H), 7.89-7.72 (m, 7H), 7.51-7.39 (m, 3H), 7.24-7.05 (m, 13H), 6.65 (d, J=
8.4Hz, 2H), 4.49 (dt, J=21.4,10.8Hz, 3H), 3.72 (dd, J=16.8,5.8Hz, 1H), 3.65-3.53 (m,
3H),3.01–2.88(m,3H),2.70(m,6H),1.66–1.49(m,3H),1.41–1.28(m,3H).
Prepare embodiment 4
Nap-G at pH 7.0 and 20 DEG C of room temperatureDFDFDYDK small peptide hydrogel loads the preparation of the vaccine vac-3 of OVA albumen
(1) Nap-G for taking 1mg preparation embodiment 1 to obtainDFDFDYDK is placed in 1.5 milliliters of vial, and it is micro- to be added 400
It rises PBS solution (pH=7.0), its pH value is adjusted to 7.0 with sodium carbonate liquor, being heated to boiling is completely dissolved compound,
It is cooled to after room temperature up to small peptide hydrogel.
(2) 20 microlitres of OVA protein solution of 5mg/mL are taken to be added in hydrogel made from (1), with PBS solution (pH=
7.0) constant volume is in 500 microlitres, carries out physical mixed, plastic after standing 16 minutes, obtain protein vaccine vac-3 (final small peptide
Concentration is 2mg/mL, and OVA protein concentration is 0.2mg/mL).
Prepare embodiment 5
Nap-G at pH 7.0 and 20 DEG C of room temperatureDFDFDYDThe vaccine vac- of K small peptide hydrogel load B+T epitope small peptide
4 preparation
(1) Nap-G for taking 1mg preparation embodiment 1 to prepareDFDFDYDK is placed in 1.5 milliliters of vial, and it is micro- to be added 400
It rises PBS solution (pH=7.0), its pH value is adjusted to 7.0 with sodium carbonate liquor, being heated to boiling is completely dissolved compound,
It is cooled to after room temperature up to small peptide hydrogel.
(2) 25 microlitres 20mg/mL B+T epitope small peptide (500 microgram) are taken to be added in hydrogel made from (1),
Physical mixed is carried out, with PBS solution (pH=7.0) constant volume in 500 microlitres, plastic after standing 14 minutes obtains small peptide vaccine
Vac-4 (concentration of final small peptide is 2mg/mL, and the concentration of B+T epitope small peptide is 1mg/mL).
B+T epitope small peptide structural formula is as follows:
Prepare embodiment 6
The vaccine vac- of cancer cell at pH 7.0 and 20 DEG C of room temperature after L-configuration Nap-GFFY small peptide hydrogel load irradiation
5 preparation
(1) it takes the L-configuration Nap-GFFY prepared in 1mg preparation embodiment 1 to be placed in 1.5 milliliters of vial, is added 400
Microlitre PBS solution (pH=7.0), is adjusted to 7.0 for its pH value with sodium carbonate liquor, and being heated to boiling keeps compound completely molten
Solution is cooled to after room temperature up to small peptide hydrogel.
(2) 100 microlitre 5 × 10 is taken7Cancer cell after a every milliliter of X-ray irradiation is added to hydrogel obtained in (1)
In, physical mixed is carried out, plastic after standing 13 minutes, obtaining cell vaccine vac-5, (concentration of final small peptide is 2mg/mL, spoke
The concentration of cancer cell according to after is 1 × 107It is every milliliter a).
Prepare embodiment 7
Nap-G at pH 7.0 and 20 DEG C of room temperatureDFDFDThe vaccine vac-6's of cancer cell after the load irradiation of Y small peptide hydrogel
Preparation
(1) Nap-G prepared in 1mg preparation embodiment 2 is takenDFDFDY is placed in 1.5 milliliters of vial, and it is micro- to be added 400
It rises PBS solution (pH=7.0), its pH value is adjusted to 7.0 with sodium carbonate liquor, being heated to boiling is completely dissolved compound,
It is cooled to after room temperature up to small peptide hydrogel.
(2) 100 microlitre 5 × 10 is taken7Cancer cell after a every milliliter of X-ray irradiation is added to hydrogel obtained in (1)
In, physical mixed is carried out, plastic after ten minutes is stood, obtaining cell vaccine vac-6, (concentration of final small peptide is 2mg/mL, spoke
The concentration of cancer cell according to after is 1 × 107It is every milliliter a).
Compare preparation example 1
20 microlitres of OVA protein solution of 5mg/mL are taken, 500 microlitres is settled to PBS solution (pH=7.0), is free of
The protein vaccine OVA of adjuvant (final OVA protein concentration is 0.2mg/mL).
Compare preparation example 2
(1) 62.5 microlitres of aluminium adjuvant of 40mg/mL are taken, are settled to 250 microlitres with PBS solution (pH=7.0), obtain aluminium assistant
Agent dispersing liquid.
(2) it takes 20 microlitres of OVA protein solution of 5mg/mL to be added to aluminium adjuvant dispersion liquid obtained in (1), uses PBS solution
(pH=7.0) constant volume carries out physical mixed in 500 microlitres, and obtained mixture is the protein vaccine Alum-OVA containing aluminium adjuvant.
(concentration of final aluminium adjuvant is 5mg/mL, and OVA protein concentration is 0.2mg/mL)
Compare preparation example 3
Take 100 microlitre 5 × 107Cancer cell after a every milliliter of X-ray irradiation, is settled to PBS solution (pH=7.0)
500 microlitres, obtaining the cell vaccine XTC without adjuvant, (concentration of the cancer cell after final irradiation is 1 × 107It is every milliliter a).
Compare preparation example 4
The preparation of small peptide hydrogel L-gel at pH 7.0 and 20 DEG C of room temperature
The L-configuration small peptide Nap-GFFY for taking 1mg preparation embodiment 1 to prepare is placed in 1.5 milliliters of vial, is added 400
Microlitre PBS solution (pH=7.0), is adjusted to 7.0 for its pH value with sodium carbonate liquor, and being heated to boiling keeps compound completely molten
Solution is cooled to room temperature and forms hydrogel later, with PBS solution (pH=7.0) constant volume in 500 microlitres, stands plastic after ten minutes,
As hydrogel L-gel (concentration of final small peptide is 2mg/mL).
Compare preparation example 5
The preparation of small peptide hydrogel D-gel at pH 7.0 and 20 DEG C of room temperature
The Nap-G for taking 1mg preparation embodiment 2 to prepareDFDFDY is placed in 1.5 milliliters of vial, and 400 microlitres of PBS are added
Its pH value is adjusted to 7.0 with sodium carbonate liquor by solution (pH=7.0), and being heated to boiling is completely dissolved compound, is cooled to
Hydrogel is formed after room temperature, with PBS solution (pH=7.0) constant volume in 500 microlitres, plastic, as hydrogel after standing 8 minutes
D-gel (concentration of final small peptide is 2mg/mL).
Compare preparation example 6
25 microlitres of 20mg/mL B+T epitope small peptides (500 microgram) are taken, are settled to 500 with PBS solution (pH=7.0)
Microlitre, obtain the small peptide vaccine epitope without adjuvant (concentration of final B+T epitope small peptide is 1mg/mL).
Compare preparation example 7
(1) 62.5 microlitres of aluminium adjuvant of 40mg/mL are taken, are settled to 250 microlitres with PBS solution (pH=7.0), obtain aluminium assistant
Agent dispersing liquid.
(2) 25 microlitres of 20mg/mL small peptide B+T epitope (500 microgram) is taken to be added to aluminium adjuvant dispersion obtained in (1)
Liquid carries out physical mixed with PBS solution (pH=7.0) constant volume in 500 microlitres, and obtained mixture is the small peptide containing aluminium adjuvant
Vaccine Alum-epitope.
Immune embodiment 1
(1) mouse is immune for the first time
6-8 weeks mouse is taken, is denoted as 0 day with injection time first time, preparation embodiment 1, preparation embodiment 2, comparison are taken
Hydrogel is dispersed as after viscous solution respectively with every by protein vaccine obtained in preparation example 1, comparison preparation example 2 with vortex instrument
The dosage that 100 microlitres of mouse is subcutaneously injected at mouse groin.
(2) it is immunized for second of mouse
The 14th day time point, takes preparation embodiment 1, preparation embodiment 2, comparison preparation example 1, compares in preparation example 2
Hydrogel is dispersed as after viscous solution respectively with every 100 microlitres of mouse of dosage small by protein vaccine obtained with vortex instrument
It is subcutaneously injected at mouse groin.
(3) measurement of antibody titer
Mice serum is taken when the 21st day, using BioTek microplate reader, carries out corresponding antibodies drop with the method for Elisa
The measurement of degree.As a result as shown in Figure 1.
Find out from the result in Fig. 1: wherein IgG represents total antibody titer；IgG1 is one of phenotype, indicates body fluid
Immune response；IgG2a and IgG2b is also one of phenotype, indicates cellular immune level.With the OVA protein groups for being free of adjuvant
IgG antibody can be improved using the group (preparation embodiment 1) of L-configuration polypeptide hydrogel and OVA albumen in (comparison preparation example 1) comparison
It 60 times of titre, can be improved 580 times using the group (preparation embodiment 2) of D configuration polypeptide hydrogel and OVA albumen, and helped using aluminium
The group (comparison preparation example 2) of agent and OVA albumen only can be improved 165 times；With the OVA protein groups (comparison preparation example 1) without adjuvant
Comparison can be improved 167 times of IgG1 antibody titer using the group (preparation embodiment 1) of L-configuration polypeptide hydrogel and OVA albumen, make
It 2) only can be improved 135 times with D configuration polypeptide hydrogel and the preparation of OVA egg；With OVA protein groups (the comparison preparation example without adjuvant
1) it compares, can be improved 10 times of IgG2a antibody titer using the group (preparation embodiment 1) of L-configuration polypeptide hydrogel and OVA albumen,
It can be improved 60 times using the group (preparation embodiment 2) of D configuration polypeptide hydrogel and OVA albumen, and use aluminium adjuvant and OVA albumen
Group (comparison preparation example 2) only can be improved 12 times；With OVA protein groups (comparison preparation example 1) comparison without adjuvant, L structure is used
The group (preparation embodiment 1) of type polypeptide hydrogel and OVA albumen can be improved 30 times of IgG2b antibody titer, use D configuration polypeptide water
The group (preparation embodiment 2) of gel and OVA albumen can be improved 90 times, and use group (the comparison preparation example of aluminium adjuvant and OVA albumen
2) 60 times only be can be improved.
Immune embodiment 2
(1) mouse is immune for the first time
6-8 weeks mouse is taken, is denoted as 0 day with injection time first time, preparation embodiment 4, comparison preparation example 1, comparison are taken
Hydrogel is dispersed as after viscous solution respectively with every 100 microlitres of mouse by protein vaccine obtained in preparation example 2 with vortex instrument
Dosage be subcutaneously injected at mouse groin.
(2) it is immunized for second of mouse
The 14th day time point, takes preparation embodiment 4, comparison preparation example 1, compares albumen epidemic disease obtained in preparation example 2
Seedling with vortex instrument by hydrogel be dispersed as after viscous solution respectively with every 100 microlitres of mouse of dosage at mouse groin into
Row subcutaneous injection.
(3) measurement of antibody titer
Mice serum is taken when the 21st day, using BioTek microplate reader, carries out corresponding antibodies drop with the method for Elisa
The measurement of degree.As a result as shown in Figure 2.
Find out from the result in Fig. 2: short using D configuration with OVA protein groups (comparison preparation example 1) comparison without adjuvant
The group (preparation embodiment 4) of peptide hydrogel and OVA albumen can be improved 233 times of IgG antibody titre, and use aluminium adjuvant and OVA egg
White group (comparison preparation example 2) only can be improved 64 times.
Immune embodiment 3
(1) mouse is immune for the first time
6-8 weeks mouse is taken, is denoted as 0 day with injection time first time, preparation embodiment 5, comparison preparation example 6, comparison are taken
Hydrogel is dispersed as after viscous solution respectively with every 100 microlitres of mouse by small peptide vaccine obtained in preparation example 7 with vortex instrument
Dosage be subcutaneously injected at mouse groin.
(2) it is immunized for second of mouse
The 14th day time point, takes preparation embodiment 5, comparison preparation example 6, compares small peptide epidemic disease obtained in preparation example 7
Seedling with vortex instrument by hydrogel be dispersed as after viscous solution respectively with every 100 microlitres of mouse of dosage at mouse groin into
Row subcutaneous injection.
(3) measurement of antibody titer
Mice serum is taken when the 21st day, using BioTek microplate reader, carries out corresponding antibodies drop with the method for Elisa
The measurement of degree.As a result as shown in Figure 3.
Find out from the result in Fig. 3: being compared with the small peptide vaccine group (comparison preparation example 6) without adjuvant, with D configuration polypeptide
Hydrogel Nap-GDFDFDYDK load small peptide B+T epitope group (preparation embodiment 5) can be improved 26 times of IgG antibody titre, and
Small peptide vaccine group (comparison preparation example 7) containing aluminium adjuvant does not improve.
Immune embodiment 4
(1) mouse is immune for the first time
6-8 weeks mouse is taken, is denoted as 0 day with injection time first time, endotoxin-free, sterile PBS solution and system are taken
Hydrogel is dispersed as after viscous solution respectively with vortex instrument for cell vaccine obtained in embodiment 6-7, comparison preparation example 3-5
It is subcutaneously injected with every 100 microlitres of mouse of dosage in back of mice.
(2) it is immunized for second of mouse
The 14th day time point, endotoxin-free, sterile PBS solution and preparation embodiment 6-7, comparison preparation example are taken
Hydrogel is dispersed as after viscous solution respectively with every 100 microlitres of mouse of agent by cell vaccine obtained in 3-5 with vortex instrument
Amount is subcutaneously injected in back of mice.
(3) mouse third time is immune
The 21st day time point, endotoxin-free, sterile PBS solution and preparation embodiment 6-7, comparison preparation example are taken
Hydrogel is dispersed as after viscous solution respectively with every 100 microlitres of mouse of agent by cell vaccine obtained in 3-5 with vortex instrument
Amount is subcutaneously injected in back of mice.
(4) mouse stimulation CD8+IFN- γ+T cell proliferation test
Mouse boosting cell was taken at the 28th day, flow cytometer detection is carried out by using BD FACS Calibur flow cytometer.Knot
Fruit such as Fig. 4 a) shown in.
(5) mouse tumor Inhibition test
At the 28th day with non-irradiated cancer cell inoculation back of mice, inoculum concentration 1 × 106A cancer cell, as tumour is raw
It is long, there is macroscopic tumour in back of mice, routine test tumor size evaluates tumor inhibitory effect with this.Test side
Method is as follows: with the length (longest diameter) and width (perpendicular to the width in longest diameter direction) of vernier caliper measurement mouse tumor, tumour body
Product is calculated according to formula [long × wide × (long+wide)/2].As a result as shown in Fig. 4 b).
Cellullar immunologic response is quite crucial for the treatment of tumour.Experimental result is shown, by Nap-GDFDFDY hydrogel is negative
It carries the mouse that cancer cell (vac-6) is immune after irradiating and illustrates stronger CD8+IFN- γ+enhancing compared to other experimental groups
Effect (such as Fig. 4 a)), this shows that it can improve the expression of cytotoxic T lymphocyte (CTL), and CTL is conducive to tumor suppression.Such as
Fig. 4 b), in PBS group and hydrogel group D-gel, L-gel, tumor growth curve does not have significant difference, but in inoculating cell epidemic disease
Seedling XTC (in figure filled square indicate), vac-5 (solid diamond indicates in figure), vac-6 (hollow triangle table shows in figure)
Experimental group tumour growth is inhibited and extends the life cycle of mouse.The wherein cancer after D configuration hydrogel load irradiation
The immune effect of cell (vac-6) is the most obvious.These results are consistent with the experimental result of protein vaccine before.
Claims (9)
1. a kind of application of small peptide as vaccine adjuvant, which is characterized in that the short peptide sequence as vaccine adjuvant is X-
GFFYK, wherein X is end-capping group, and the vaccine is small peptide vaccine.
2. application as described in claim 1, which is characterized in that the small peptide is D configuration, sequence X-GDFDFDYDK。
3. application as claimed in claim 1 or 2, which is characterized in that X Nap.
4. application as claimed in claim 1 or 2, which is characterized in that the method for the heated cooling of the aqueous mixtures of the small peptide
Small peptide hydrogel is formed, is then mixed with antigen, the hydrogel formed after standing is used as vaccine.
5. the small peptide vaccine using a kind of small peptide as vaccine adjuvant, which is characterized in that the short peptide sequence as vaccine adjuvant
For X-GFFYK, wherein X is end-capping group.
6. small peptide vaccine as claimed in claim 5, which is characterized in that the small peptide is D configuration, sequence X-GDFDFDYDK。
7. such as small peptide vaccine described in claim 5 or 6, which is characterized in that X Nap.
8. such as small peptide vaccine described in claim 5 or 6, which is characterized in that the small peptide as vaccine adjuvant and antigen object
Reason is mixed to get the small peptide vaccine, and the antigen is short peptide antigens.
9. vaccine as claimed in claim 8, which is characterized in that the aqueous mixtures of the small peptide as vaccine adjuvant are heated
Cooling method forms small peptide hydrogel, then mixes with antigen, and the hydrogel formed after standing is used as small peptide vaccine, described anti-
It originally is short peptide antigens.
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| CN113527417B (en) * | 2021-07-05 | 2022-09-27 | 中国医学科学院放射医学研究所 | Self-assembly polypeptide nano adjuvant containing fluorine group and preparation method and application thereof |
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