CN110393797A - The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant - Google Patents

The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant Download PDF

Info

Publication number
CN110393797A
CN110393797A CN201910444050.1A CN201910444050A CN110393797A CN 110393797 A CN110393797 A CN 110393797A CN 201910444050 A CN201910444050 A CN 201910444050A CN 110393797 A CN110393797 A CN 110393797A
Authority
CN
China
Prior art keywords
vaccine
muc1
galcer
aunp
antigen
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
CN201910444050.1A
Other languages
Chinese (zh)
Inventor
赵炜
刘永辉
于凡
王昭予
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nankai University
Original Assignee
Nankai University
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 Nankai University filed Critical Nankai University
Priority to CN201910444050.1A priority Critical patent/CN110393797A/en
Publication of CN110393797A publication Critical patent/CN110393797A/en
Pending legal-status Critical Current

Links

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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • 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
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma

Abstract

The present invention discloses construction method and its application of a kind of MUC1 polypeptide vaccine, and vaccine of the present invention is using the tandem repetitive sequence of MUC1 glycopeptide as antigen, using glycolipid α-GalCer as adjuvant, with gold nano grain (AuNP) for multivalent carrier.The MUC1 glycopeptide sequence is synthesized by Solid-phase synthesis peptides strategy, and glycolipid α-GalCer passes through chemical reactive synthesis.Using AuNP as multivalent carrier, MUC1 and α-GalCer is connected on AuNP jointly by S-Au key high forces, the MUC1 glycopeptide vaccine that AuNP of the invention is multivalent carrier can be obtained.MUC1 glycopeptide vaccine of the invention includes the galactoside rouge α-GalCer that can be combined with CD1d albumen strength, after being offered by CD1d molecule, it can be in conjunction with the TCR of NKT cell surface, inducing cytotoxic simultaneously secretes a large amount of Th1 types and the Th2 type immunological effect factor, then the proliferation of effective stimulus NKT and NK cell, there is stronger lethal effect to tumour cell, can be used for the prevention and treatment of tumour.

Description

The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant
Technical field
Invention belongs to tumor vaccine field, and in particular to the preparation of MUC1 glycopeptide antigen, the synthesis of glycolipid adjuvant and Jenner Rice is building and its Efficacy evaluation of the vaccine of carrier.
Technical background
Tumor is to seriously threaten a kind of disease of human health, recently as the increase and living environment of people's operating pressure Deterioration, tumor patient increases year by year.The immunotherapy of tumour and traditional cancer immunotherapies operative treatment, chemotherapy and Radiotherapy is compared, and is a kind of emerging tumor therapeuticing method.It refers to body and inhibits or kill with the immune system of itself Go out tumour cell, it is considered to be therapeutic field of tumor is most hopeful to cure a kind of method of tumour.Therapeutic tumor vaccine is swollen One important directions of tumor immunotherapy, become the focus of people's research in recent years.
Mucins(mucin) it is a kind of heightOGlycosylated albumen.With more and more glycosylation modified albumen It is found,OGlycosylated importance is increasingly taken seriously.The glycosylation pattern of mucin has a unique feature: GalNAc with Serine/threonine on protein sequence is connected by the oxygen glycosidic bond of α configuration.The identified gene come out of mucin family at present There are 20, comprising: MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19 and MUC20.MUC1 glycoprotein is almost expressed in all epithelial tissues, and Surface is connected with a variety of sugar by oxygen glycosidic bond.It is a transmembrane protein in tumor cell surface overexpression, in the thin of it There are multiple (20 to 125) tandem repetitive sequences in extracellular regions.Each tandem repetitive sequence is made of 20 amino acid: HGVTSAPDTRPAPGSTAPPA has 5 potential glycosylation sites in serine/threonine above.In tumour cell, Since the activity of glycosyl transferase is changed, and new glycosyl transferase is produced, so tumour cell and normal thin Cell phase ratio, the sugar chain on MUC1 shorten, and have new sugar (such as sialic acid) appearance.The biosynthesis of Mucin surface polysaccharide Path is: acetylamino galactosamine is transferred to serine/Soviet Union of albumen under the catalysis of alpha-acetamido galactosyltransferase Tn antigen is obtained on propylhomoserin, and then under the action of beta galactose based transferase, is shifted and is obtained TF on galactolipin to Tn antigen Antigen, this process can carry out in normal cell and tumour cell.In normal cell, and then shifted in gucosamine Acetylglucosamine is shifted under the action of enzyme, obtains acetogenin amino lactose.And in tumour cell, gucosamine transfer Enzyme decline, cannot carry out following glycosyl transfer process as normal cell.Therefore it is anti-to express incomplete Tn and TF for sugar chain It is former to be largely present in tumour cell.In addition, expression of the sialyltransferase in tumour cell increases, and tumour cell is deposited TN and TF antigen carry out sialylated obtaining STn, STF etc..Generally speaking, based on MUC1 glycoprotein in normal cell and The otherness expressed in tumour cell has become tumor vaccine and studies important antigenic targets.
In the less immunogenic of MUC1 glycopeptide antigen itself, in order to enhance its immunogenicity, usually by itself and immune assistant Agent or carrier are connected.Main at present includes following four classes: 1) being two component vaccines of carrier with albumen (BSA, KLH, TTX, DT)； 2) using T epitope or immunologic adjuvant as two component vaccines of carrier, including two component univalent vaccines and two component polyvaccines；3) even Join three components and multicomponent vaccine of T epitope and immunopotentiator；4) multivalent carrier vaccine.But existing adjuvant and carrier Since safety is poor, due to the ability difference of enhancement antigen immune response etc., still reaches to the requirement less than clinical use.
Summary of the invention
The purpose of invention be to provide it is a kind of prepare simple, safety is good, the high tumor vaccine of immunogenicity.This vaccine includes MUC1 glycopeptide antigen, α-Galcer glycolipid adjuvant and AuNP multivalent carrier can induce body and generate humoral immune reaction simultaneously And cell immune response, the prevention and treatment for tumour.
The amino acid sequence of MUC1 glycopeptide antigen provided by the invention is HGVTSAPDT(α-GalNAc) RPAPGSTAPPA.
MUC1 glycopeptide in the present invention is synthesized using Solid-phase synthesis peptides strategy.
Adjuvant provided by the invention is α-Galcer glycolipid.
α-Galcer in the present invention is synthesized using chemical synthesis process.
The carrier of antigen and adjuvant provided by the invention is gold nano grain (AuNP).
MUC1 glycopeptide antigen, α-Galcer glycolipid adjuvant and AuNP multivalent carrier provided by the invention can be used to prepare Dengue Viral vaccine.
Tumor vaccine provided by the invention can be used to prepare the preparation and antibody of MUC1 antigen related neoplasms.
Tumor vaccine provided by the invention can excite the humoral immunity and cellular immunity of strong antigentic specificity in vivo.
Tumor vaccine provided by the invention can inhibit the growth of tumour in animal body, for grinding for mankind's clinical tumor vaccine Hair provides valuable reference.
Detailed description of the invention
Fig. 1 is the synthesis in solid state of MUC1 glycopeptide；
Fig. 2 is the chemical synthesis of α-Galcer glycolipid；
Fig. 3 is using gold nano as carrier, and α-Galcer is the synthesis of the MUC1 glycopeptide vaccine of adjuvant；
Fig. 4 is the antibody titer that vaccine triggers an immune response；
Fig. 5 is the antibody subtype that vaccine triggers an immune response；
Fig. 6 is the CDC activity for the antibody that vaccine generates；
Fig. 7 is vaccine-induced CTL activity；
Fig. 8 is the tumor inhibitory effect of vaccine in vivo.
Specific embodiment
Apply the synthesis of 1 tumor vaccine Antigenic Peptide of example
1. the source of chemical reagent
Fmoc- amino acid, the chloro- trityl chloride resin of 2-, benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (HBTU), it is purchased from gill biochemistry (Shanghai) Co., Ltd..Trifluoroacetic acid (TFA), the chemistry such as n,N-diisopropylethylamine (DIPEA) Reagent is purchased from AlfaAesar (China) Chemical Co., Ltd..Methylene chloride (DCM), n,N-Dimethylformamide (DMF) etc. are changed It learns solvent and is purchased from Tianjin chemical reagent wholesaling firm.
The Fmoc synthesis in solid state strategy of the preparation polypeptide of 2.MUC1 glycopeptide antigen epitope synthesizes MUC1 glycopeptide, and structural formula is such as Under:
Specifically synthesis step includes:
A) resin 1.0g is weighed in Peptide systhesis pipe, so for solid phase carrier with 2- chlorine trityl resin (0.13mmol/g) DCM is added afterwards to be swollen 30 minutes.Pressurization removes the DCM in synthesis in solid state pipe.
B) it the coupling of amino acid: uses Fmoc-AA-OH (4.0 equiv), HBTU (4.0 equiv), HOBt (4.0 ), equiv DIPEA (8.0 equiv) carries out the coupling of amino acid.
C) closing of amino acid: reacting in acetic anhydride/pyridine=2:1 solution, closes the complete amino of unreacted Acid, to reduce the generation of by-product.
D) the Fmoc protecting group of amino acid the deprotection of Fmoc group: is taken off with 20% piperidines/DMF solution.
E) by b above, the step of c, d, sequentially adds required amino acid, until amino acid is all coupled.Then DMF is used It is successively washed with DCM 3 times.
F) Side chain protective group of amino acid is removed with 90% TFA/5% TIPS/5% H2O, and by amino acid from resin Removing is got off.
G) it is precipitated to obtain polypeptide crude product with ether, then prepares HPLC with half and obtain pure polypeptide solution.Freeze-drying obtains 14 mg of white powder, mass spectrum confirm the molecular weight of product.
Structural characterization data are as follows:
HPLC: Rt (retention time) = 12.3 min (15-40% of acetonitrile and 0.1% trifluoroacetic acid over 25 min on a C-18 column, λ=220 nm)
ESI-MS: m/z for C94H151N27O36 [M+2H]2+ calcd 1118.55, found 1118.45; [M+3H]3+ calcd 746.03, found 745.90;
The synthesis of 2 glycolipid α-Galcer of embodiment
The synthesis of α-Galcer glycolipid is divided into three parts: the synthesis of galactosyl donor, the synthesis of aliphatic side chains, saccharide donor and by The coupling of body.
1. the synthesis of saccharide donor
Saccharide donor synthesis is to be translated into first glycosides in acid condition with methanol using galactolipin as raw material, then use pivaloyl Base carries out selective protection to primary hydroxyl, and the hydroxyl of other of protection is deprotected primary hydroxyl to have obtained the first glycosides of 6 hydroxyls, Then it is connected with the PEG chain of modification, the first methyl is taken off with sour water solution, is made into saccharide donor (Fig. 2).
2. the synthesis of glycosyl acceptor
When synthesizing side chain, using phytosphingosine as raw material, with the cerinic acid and amino of activation at amido bond, protected with silicon-based protecting group Hydroxyl is protected, the protecting group of primary hydroxyl site is selectively taken off, obtains side chain (Fig. 2).
3. the coupling of galactosyl donor and side chain receptor
After galactosyl donor and side chain receptor have synthesized, saccharide donor and side chain receptor are subjected to glycosyl under TMSOTf catalysis Change reaction, be then passed through deprotection, catalytic hydrogenation, obtains 6 galactolipin glycolipids (Fig. 2) of the PEG chain end with amino.
The structural characterization data of product are as follows:
1H NMR (400 MHz, Pyr) δ 8.70 (m, 1H), 5.21 (d, J = 4.7 Hz, 2H), 4.74 – 4.49 (m, 2H), 4.43 – 4.16 (m, 4H), 4.10 – 3.83 (m, 3H), 3.66 (t, J = 7.1 Hz, 2H), 3.63 – 3.51 (m, 4H), 3.51 – 3.35 (m, 1H), 2.98 (q, J = 7.3 Hz, 6H), 2.63 – 2.37 (m, 2H), 2.25 (s, 1H), 2.18 – 1.76 (m, 3H), 1.67 – 1.53 (m, 1H), 1.48 – 1.07 (m, 66H), 0.83 (t, J = 6.6 Hz, 6H).
13C NMR (101 MHz, Pyr) δ 173.16, 149.92, 149.65, 149.38, 135.55, 135.30, 135.05, 123.54, 123.29, 123.04, 101.00, 76.43, 72.08, 71.30, 70.98, 70.82, 70.60, 70.37, 70.23, 69.67, 67.98, 67.39, 51.21, 45.52, 39.62, 36.58, 34.18, 31.90, 30.22, 29.96, 29.80, 29.69, 29.57, 29.40, 29.38, 26.31, 26.23, 22.71, 14.06.
For embodiment 3 using gold nano as carrier, α-Galcer is the synthesis of the MUC1 glycopeptide vaccine of adjuvant
In order to which NH will be had2The MUC1 segment and α-GalCer of linking group are covalently attached to the surface AuNP, we first AuNP is prepared into the form that particle surface is connected with Acibenzolar.Specific method be using triphenyl phosphorus chlorine gold as raw material, first make its with Lauryl mercaptan bonding is then reacted with the sulfydryl of the hendecoic acid end of activation, and the surface AuNP covering carboxyl has been obtained The form of Acibenzolar.Then end is connected with NH by us2MUC1 segment and end be connected with NH2α-GalCer be connected to Gold nano grain surface is constructed using AuNP as the multivalence tumor vaccine (Fig. 3) of carrier.
1. the synthesis of AuNP-a
The round-bottomed flask of reaction is successively used into chloroazotic acid, ultrapure water wash clean, until cleaning solution pH=7.Weigh AuClP (Ph)3 (120 mg) is added and steams benzene (20 mL) again.It is stirred 10 minutes after magneton is added.Lauryl mercaptan (0.13 is added dropwise ML), and reducing agent (CH is added3)3CNH2.BH3(0.21 g).Reaction solution is stirred one hour at 55 DEG C.Then certainly by reaction solution So it is down to room temperature.20 mL dehydrated alcohols are added, then reaction solution in flask is transferred in centrifuge tube.With the speed of 800 r/min Supernatant is poured out in degree centrifugation after ten minutes.It is washed, and is centrifuged three times with dehydrated alcohol repeatedly, centrifugate is collected together, very Sky is spin-dried for solution, obtains solid precipitating.Finally solid is precipitated with 20 mL chloroforms and is redissolved, is placed at 4 DEG C and stores.
2. the synthesis of AuNP-b
MUDHSE is dissolved in DMF(40 mL) in, then it is added slowly with stirring AuNP-a(2.5 mL) solution, at room temperature React 18 h.Reaction terminates to be spin-dried under vacuum with DMF, obtains solid powder.Obtained solid powder is redissolved in DMF In (10 mL), it is placed at 4 DEG C and stores.
3. the synthesis of AuNP-c
MUC1 glycopeptide and glycolipid α-Galcer are weighed in proportion, with being dissolved in the DMF solution dissolved with AuNP-b.Stirring 30 minutes Afterwards, triethylamine is added, then reacts 72 hours at room temperature.It is dialysed three times respectively with DMF and ultrapure water after reaction, and It is saved at 4 DEG C.
Transmission electron microscope analysis is carried out with vaccine of the TEM to building, the partial size of three vaccines is 7 ± 1 nm.
The immunity evaluation of 4 tumor vaccine of embodiment
1. immune grouping
In order to improve the immunogenicity of polypeptide antigen, it is contemplated that being mixed with nanofiber polypeptide adjuvant and Antigenic Peptide, exempt from jointly Epidemic disease mouse.Polypeptide vaccine is prepared by following scheme respectively, 100 μ L are administered in every mouse every time, and every 6 mouse are one group, every The dosage of mouse is as follows:
1) PBS group: 100 μ L PBS
2) antigen+adjuvant group: (MUC1+ α-GalCer)/100 μ L PBS
3) gold nano group AuNP-1:(antigen/adjuvant=2:1)/100 μ L PBS
4) gold nano group AuNP-2:(antigen/adjuvant=1:1)/100 μ L PBS
5) gold nano group AuNP-3:(antigen/adjuvant=1:2)/100 μ L PBS
2. immunization protocol
The Balb/c mouse for selecting 6 ~ 8 week old, is randomly divided into four groups, subcutaneous administrations.Epidemic disease was injected respectively at the 0th, 2,4,6 week Seedling, immune tail after a week takes blood every time, and centrifuging and taking supernatant does immunity evaluation.By anti-in ELISA method detection mice serum Body titre.
3.ELISA reagent and solution are prepared
A) coating buffer: 0.05mol/L carbonate buffer solution (pH9.6)
0.75g sodium carbonate, 1.46g sodium bicarbonate, adds deionized water to be settled to 500ml.
B) 0.02mol/L phosphate buffer (pH7.4)
0.2g potassium dihydrogen phosphate, 2.90g disodium hydrogen phosphate, 8g sodium chloride add deionized water constant volume to 1000ml.
C) antibody diluent: 0.02mol/L PBS (pH7.4)+0.2%BSA
0.2gBSA adds the 0.02mol/L phosphate buffer prepared dissolution quantitative to 100g.
D) confining liquid: 0.05mol/L carbonate buffer solution (pH9.6)+2.0%BSA
2.0gBSA adds the 0.05mol/L carbonate buffer solution prepared dissolution quantitative to 100g.
E) cleaning solution: 0.02 mol/L PBS (pH7.4)+0.05%Tween-20
50uL Tween-20 is dissolved in 100 mL 0.02mol/L phosphate buffers, concussion mixes.
F) developing solution: TMB solution
TMB solution (3,3',5,5'-tetramethylbenzidine, TMB): it weighs TMB 20mg and is dissolved in 10mL dehydrated alcohol, completely After dissolution, add distilled water to 100mL.
G) terminate liquid: 2 mol/L H2SO4Solution
10 mL, 98% concentrated sulfuric acid is added in 60 mL distilled waters, is settled to 100 mL, room temperature preservation.
ELIAS secondary antibody: the rabbit anti-mouse igg of HRP label dilutes 5000 times with antibody diluent when application.
4. indirect elisa method detects mouse resisting anteserum potency
(1) antigen coat
With coating buffer (carbonate buffer solution of 0.05 mol/L, pH 9.6) antigen diluent be 0.01 mg/mL, will dilute Antigen liquid 100 μ L are added in each ELISA Plate hole.In ELISA Plate merging wet box, stayed overnight in 4 DEG C of coatings.
(2) board-washing
Coating buffer is discarded, fills it up with all enzyme marks hole with cleaning solution, is encased to buckle with gauze and toilet paper and be done.1 time, last is by water button It is dry.
(3) it closes
250 μ L confining liquids (9.6,0.05 mol/L carbonate buffer solution of pH contains 2.0% BSA) is added in every hole on elisa plate, Elisa plate is placed in wet box, 4 DEG C of overnight incubations.It can also 37 DEG C of 2 h of incubation.
(4) confining liquid is discarded, washs 1 time ibid.
(5) add primary antibody (family's rabbit anti-serum)
Immune 10 μ L of mouse resisting anteserum is added to the antiserum that 1:100 is made in the antibody diluent of 990 μ L.(in 1.5 It is operated in the EP pipe of mL).Add the serum of the 1:100 of 100 μ L to No. 1 hole of A row on ELISA Plate.It is arrived to 2 of A row on ELISA Plate 100 μ L of antibody diluent is respectively added in 12 holes, then to A group the second hole be added 100 μ L 1:100 antiserum, pressure-vaccum ten times The serum for drawing 100 μ L afterwards is added to third hole, then pressure-vaccum ten times take 100 μ L to be added to the 4th hole, are sequentially added the 12nd hole, finally 100 μ L are sucked out from the 12nd hole to give up.Hole every in this way is 100 μ L, by 11 doubling dilution, serum dilution 1: 204800.B, C row ibid operates.
The antibody diluent of 100 μ L is only added as blank control to the first two hole of D row, it is dilute that 100 μ L are added in the hole 3-4 100 times of negative blood is released, antigen is not added in the hole 5-6, is only closed with confining liquid, and the antiserum that 100 μ L dilute 100 times is added in every hole It compares.Enzyme connecting plate is placed on 37 DEG C of 1 h of incubation in wet box after adding.
(6) washing is same as above.
(7) add ELIAS secondary antibody
The 100 μ L of ELIAS secondary antibody (rabbit-anti mouse) that extension rate is 1:5000 is added in each hole, is incubated for 1 h in 37 DEG C of wet box.
(8) washing is same as above
(9) it develops the color
Every hole, which adds enzyme connecting plate to be put into wet box after 100 μ L of TMB solution, is protected from light 30 min or so, when the aobvious indigo plant of negative control hole It is terminated when green.The 2 mol/L concentrated sulfuric acids of 50 μ L are added in every hole when termination.
(10) it detects
Rapidly with the value of microplate reader measurement each hole A450 of enzyme connecting plate after termination.
The CTL of 5 tumor vaccine of embodiment is studied
Lactic dehydrogenase (LDH) rich content in endochylema, cannot be by cell membrane, when cell damage wound or death when normal When, releasably arrives extracellular, and LDH activity is directly proportional with the cell number of death in cell culture fluid at this time.By effector cell and target Cell is incubated for jointly, with colorimetric method for determining and compared with target cell control wells LDH activity, is calculated effector cell and is killed to target cell Hurt ability, as cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL) is analyzed.Last time is seven days immune Afterwards, effector cell of the mouse boosting cell as CTL, the cytotoxic T cell that MCF-7 is generated as target cell, MUC1 induction are taken To the lethal effect (Fig. 7) of MCF-7 cell.
The antitumor activity of 6 tumor vaccine of embodiment
In order to probe into prepared vaccine in the intracorporal antitumor activity of mouse, C57BL/6 right side of mice oxter is subcutaneously injected in we Turn people's mucin B16 mouse melanoma cell line-MUC1 (1 × 105).After a week, diameter of tumor reaches 5 mm.7th day, Each group vaccine, every two days measurement tumor sizes is subcutaneously injected at adjacent tumor position within 11 days and the 15th day.Test method are as follows: use vernier calliper Ruler measures the length and width of mouse tumor, gross tumor volume=0.5 × length × wide2The result shows that vaccine obviously inhibits tumour growth (figure 8).

Claims (9)

1. a kind of anti-tumor vaccine, which is characterized in that for the vaccine using α-Galcer as adjuvant, MUC1 glycopeptide is antigen, gold nano Particle is multivalent carrier.
2. as claim 1 requires the vaccine, which is characterized in that 6 of α-Galcer adjuvant are attachable amino.
3. vaccine as described in claim 1, which is characterized in that the sequence of MUC1 glycopeptide antigen is HGVTSAPDTRPAPGSTAPPA。
4. vaccine as described in claim 1, which is characterized in that the vaccine further includes gold nano grain carrier.
5. the vaccine as described in claim 2-4, which is characterized in that the adjuvant is by chemical reaction and gold nano with antigen Particle is combined.
6. vaccine as claimed in claim 5, which is characterized in that the adjuvant and antigen are by molar ratio 2:1,1:1 respectively It is coupled with 1:2 and gold nano grain.
7. vaccine as claimed in claim 6, which is characterized in that the gold nano grain conjugate of generation is used as vaccine.
8. vaccine as claimed in claim 7, which is characterized in that the vaccine is glycopeptide vaccine or protein vaccine.
9. vaccine described in claim 1-4 prevents and treats the purposes in tumour medicine in preparation.
CN201910444050.1A 2019-05-27 2019-05-27 The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant Pending CN110393797A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910444050.1A CN110393797A (en) 2019-05-27 2019-05-27 The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910444050.1A CN110393797A (en) 2019-05-27 2019-05-27 The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant

Publications (1)

Publication Number Publication Date
CN110393797A true CN110393797A (en) 2019-11-01

Family

ID=68322985

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910444050.1A Pending CN110393797A (en) 2019-05-27 2019-05-27 The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant

Country Status (1)

Country Link
CN (1) CN110393797A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112603996A (en) * 2020-12-18 2021-04-06 江南大学 Lipoteichoic acid vaccine preparation and application thereof
CN117106024A (en) * 2022-10-21 2023-11-24 南京市妇幼保健院 Human serum polypeptide AGDMP1 and application thereof in improving insulin resistance

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102245204A (en) * 2008-07-11 2011-11-16 中央研究院 Alpha-galactosyl ceramide analogs and their use as immunotherapies, adjuvants, and intiviral, antibacterial, and anticancer agents
CN103933556A (en) * 2004-04-01 2014-07-23 肿瘤防护公司 Mucinous glycoprotein (MUC-1) vaccine
CN107213468A (en) * 2017-04-14 2017-09-29 国家纳米科学中心 A kind of targeted nano pharmaceutical carrier and its preparation method and application
CN107261135A (en) * 2017-07-07 2017-10-20 中国农业科学院兰州兽医研究所 Application of the gold nano grain as adjuvant in virus sample particle vaccines are prepared

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103933556A (en) * 2004-04-01 2014-07-23 肿瘤防护公司 Mucinous glycoprotein (MUC-1) vaccine
CN102245204A (en) * 2008-07-11 2011-11-16 中央研究院 Alpha-galactosyl ceramide analogs and their use as immunotherapies, adjuvants, and intiviral, antibacterial, and anticancer agents
CN107213468A (en) * 2017-04-14 2017-09-29 国家纳米科学中心 A kind of targeted nano pharmaceutical carrier and its preparation method and application
CN107261135A (en) * 2017-07-07 2017-10-20 中国农业科学院兰州兽医研究所 Application of the gold nano grain as adjuvant in virus sample particle vaccines are prepared

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MASAKAZU等: "Structure-Based Discovery of Glycolipids for CD1d-Mediated NKT Cell Activation: Tuning the Adjuvant versus Immunosuppression Activity", 《JACS》 *
MASAKAZU等: "Structure-Based Discovery of Glycolipids for CD1d-Mediated NKT Cell Activation: Tuning the Adjuvant versus Immunosuppression Activity", 《JACS》, vol. 128, 23 June 2006 (2006-06-23), XP002493234, DOI: 10.1021/ja062740z *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112603996A (en) * 2020-12-18 2021-04-06 江南大学 Lipoteichoic acid vaccine preparation and application thereof
CN112603996B (en) * 2020-12-18 2024-05-07 江南大学 Lipoteichoic acid vaccine preparation and application thereof
CN117106024A (en) * 2022-10-21 2023-11-24 南京市妇幼保健院 Human serum polypeptide AGDMP1 and application thereof in improving insulin resistance

Similar Documents

Publication Publication Date Title
Brocke et al. Synthesis of tumor-associated glycopeptide antigens
Compañón et al. Structure-based design of potent tumor-associated antigens: modulation of peptide presentation by single-atom O/S or O/Se substitutions at the glycosidic linkage
Galan et al. Multivalent glyco (cyclo) peptides
Dondoni et al. Recent applications of thiol–ene coupling as a click process for glycoconjugation
US4980462A (en) Antiviral agents
Herzner et al. Synthesis of glycopeptides containing carbohydrate and peptide recognition motifs
Liu et al. Enhancement of the immunogenicity of synthetic carbohydrates by conjugation to virosomes: a leishmaniasis vaccine candidate
WO2018036403A1 (en) Oligosaccharide linker and antibody-drug conjugate with site-specific linkage prepared using the oligosaccharide linker
WO2006017180A2 (en) Glycopeptide dimers and uses thereof
CN103108654A (en) Multivalent glycopeptide constructs and uses thereof
JP2012132025A (en) Novel glycoconjugate, glycoamino acid, intermediates thereto, and uses thereof
US20210085781A1 (en) Neoglycoconjugates as vaccines and therapeutic tools
US10610576B2 (en) Precision glycoconjugates as therapeutic tools
Dziadek et al. Synthesis of tumor‐associated glycopeptide antigens for the development of tumor‐selective vaccines
Gutierrez-Jimenez et al. Bifunctional chiral dehydroalanines for peptide coupling and stereoselective S-Michael addition
WO1995029927A2 (en) Process for preparation of glycosides of tumor-associated carbohydrate antigens
EP0128097B1 (en) Immunostiumulating derivatives, their preparation and their use as medicines
CN110393797A (en) The preparation method and application of glycopeptide vaccine based on glycolipid adjuvant
JP2006514981A (en) GP120-specific antigen, conjugate thereof; method for its preparation and use
Peri et al. Chemoselective ligation in glycochemistry
Goyard et al. Multivalent glycocyclopeptides: conjugation methods and biological applications
Farabi et al. Concise and Reliable Syntheses of Glycodendrimers via Self-Activating Click Chemistry: A Robust Strategy for Mimicking Multivalent Glycan–Pathogen Interactions
Shaik et al. Stereoselective synthesis of natural and non-natural thomsen-nouveau antigens and hydrazide derivatives
CN112604004A (en) Anti-human EGFR antibody drug conjugate and preparation method and application thereof
JP2010540488A (en) Glycoproteins and glycosylated cells and methods for their preparation

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20191101

WD01 Invention patent application deemed withdrawn after publication