CN110124056A - It is a kind of for the liposome and preparation method of anti-organ transplant rejection and application - Google Patents

It is a kind of for the liposome and preparation method of anti-organ transplant rejection and application Download PDF

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CN110124056A
CN110124056A CN201910406036.2A CN201910406036A CN110124056A CN 110124056 A CN110124056 A CN 110124056A CN 201910406036 A CN201910406036 A CN 201910406036A CN 110124056 A CN110124056 A CN 110124056A
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liposome
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CN110124056B (en
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郭猛
刘艳芳
刘芳
张璐定
王全兴
丁国善
曹雪涛
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Second Military Medical University SMMU
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

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Abstract

The invention discloses a kind of liposomes for anti-organ transplant rejection, be to be prepared from the following materials: the GM2 single-chain antibody of Streptavidin coupling is supported in artificial liposome, it is 0.1~1ng/ μ L that the GM2 single-chain antibody of Streptavidin coupling, which is supported on concentration in artificial liposome, biotinylated Qa-1 or HLA-E and biotinylation PD-L1 is equimolar ratio, and the molar ratio for the GM2 single-chain antibody load artificial liposome that biotinylated Qa-1 or HLA-E, the total amount of biotinylation PD-L1 and above-mentioned Streptavidin are coupled is 100:1~200:1.Anti- rejection effect can be played in vivo provided by the present invention for the liposome of anti-organ transplant rejection, be a kind of novel lipide drug, the anti-rejection effect postoperative for a variety of organ transplants.

Description

It is a kind of for the liposome and preparation method of anti-organ transplant rejection and application
Technical field
The invention belongs to immunization therapies and liposome medicament targeted therapy technical field, relate in particular to one kind and are mounted with The liposome and preparation method and application for anti-organ transplant rejection of immunologic test point NKG2A/PD1 ligand fragment.
Background technique
Organ transplant has become the unique effective treatment means for treating various internal organs whole Terminal Diseases, and many patients pass through Organ transplant extends life.In recent years, the clinical application of FK506, rapamycin neotype immunosuppressant, although reducing shifting The incidence of rejection is planted, but still there are two large problems: first, immunosuppressor can not be fully solved rejection Problem prompts traditional to inhibit IL-2 gene expression to realize the limitation of immune inhibition strategies；Second, the immune suppression of prolonged application There are many adverse reactions for preparation, infection and nascent tumor etc. caused by toxicity, extra-inhibitory immunity of organisms including drug. Therefore, it by excavating activated immune cell and its regulatory mechanism, therefrom finds regulatory molecule existing for body and is applied to anti-row The treatment of different reaction is the effective way for further increasing the anti-rejection effect of post-transplantation.
The immune function of human body can activate when being excited, but this activation is generally confined within a controllable range It is interior, overactivity will not occur and cause body injury, in important mechanisms be " immunologic test point molecule " regulation.It is this kind of Molecule can be such that the activation of immune system is maintained in time " brake " similar to the brake system of automobile in immune system activation Within the scope of normal.Constantly there are new costimulatory molecules discovery in past more than ten years, and are proved maintaining immunologic balance i.e. Crucial work is played in immune response for pathogen antigen and the immune tolerance for autoantigen or homograft antigen With.Currently, the method by antagonism immunologic test point signal pathway activating immune cell for oncotherapy has been applied to clinic, And achieve good reactivity.But organ post-transplantation could be inhibited immune thin by activated immune checkpoint signals approach Born of the same parents' activation, so that playing anti-rejection still lacks research.
NKG2A and PD1 is negative regulator important in immunocyte, and ligand is respectively non-classical MHC I class molecule HLA-E (homologue is Qa-1 in mouse) and PDL1.NKG2A/CD94 dimer can interact with HLA-E (Qa-1), pass through Intracellular section of ITIM motif activates SHP molecule, and then inhibits the transcription factors such as Syk, Vav1, and NK cell is inhibited to kill with CD8+T cell Wound activity.PD-L1 can promote SHP-1 phosphorylation in conjunction with the PD1 on activating T cell surface, inhibit the activation of downstream Syk, resistance Disconnected TCR signal is gone to, and keeps T cell secrete cytokines, proliferation and killing ability impaired.In tumour immunity, pass through blocking NKG2A or PD1 signal can effectively inhibit growth, the transfer of tumour, increase the killing of tumour immunity microenvironment medium size lymphocyte Effect, and preferable reactivity is achieved in clinical trial.But it can inhibit to arrange by activating NKG2A, PD1 signal Reprimand reaction there is no report.Wherein important reason is that the ligand Extracellular domain protein molecular weight of NKG2A and PD1 is small, is metabolized pole in vivo Fastly, it is difficult to effective blood concentration is locally maintained in graft by the method for direct injection, limit its test in vivo and Direct application in clinical research.
Liposome is the duplicature lipid vesicle that amphiphilic rouge molecule is self-assembly of in water phase, and liposome not only may be used Predetermined substance (such as drug and genetic fragment) is embedded in liposome interior, specific transportation system is formed, it can also will be special Albumen carry is determined in surface of liposome.There are several researchs by the way that surface of liposome is loaded bioactive molecule recently, in animal Effective treatment is realized in model.Such as: IL-10 molecule carry is used to treat obesity mice, control in surface of liposome by Toita Make its internal inflammation；Francesca etc. in surface of liposome, successfully constructs MHC- peptide complexes and adhesion molecule carry The artificial antigen presenting cells (aAPC) of activating T cell；Nano antibody is coupled at surface of liposome by Razazan etc., is used to spy Opposite sex delivery tumour antigen.The studies above prompt, can extend it in surface of liposome for NKG2A and PD1 ligand peptide fragment carry Half-life in vivo interacts with immunocyte in a long time, activates NKG2A and PD1 downstream signal, is expected to overcome at present Bottleneck of the immunologic test point signal pathway molecule in resisting transplant rejection application study.
Summary of the invention
The object of the present invention is to provide a kind of liposome for anti-organ transplant rejection, by by immunologic test point Molecule NKG2A and PD1 ligand peptide fragment is loaded to artificial liposome surface, thus provide a kind of targeting, can be with activated immune The liposome of checkpoint signals can be applied to the treatment of anti-rejection after organ transplant.
A second object of the present invention is to provide the preparations for the liposome that anti-organ transplant rejection is used for described in one kind Method.
Third object of the present invention is to provide the purposes for the liposome that anti-organ transplant rejection is used for described in one kind.
To achieve the goals above, The technical solution adopted by the invention is as follows:
The first aspect of the invention provides a kind of liposome for anti-organ transplant rejection, is by following original Material is prepared:
The GM2 single-chain antibody of Streptavidin coupling is supported in artificial liposome, and the GM2 of Streptavidin coupling is single-stranded It is 0.1~1ng/ μ L that antibody, which is supported on concentration in artificial liposome,
Biotinylated Qa-1 or HLA-E and biotinylation PD-L1 is equimolar ratio,
The GM2 single-chain antibody of biotinylated Qa-1, the total amount of biotinylation PD-L1 and the coupling of above-mentioned Streptavidin are negative The molar ratio for carrying artificial liposome is 100:1~200:1；
Or, the total amount of biotinylated HLA-E, biotinylation PD-L1 and the GM2 of above-mentioned Streptavidin coupling are single-stranded anti- The molar ratio that body loads artificial liposome is 100:1~200:1.
The artificial liposome is prepared by following raw material:
Phosphatidyl choline 10mg~100mg, cholesterol 1mg~10mg, 0.1~1mg Ganglioside GM2, pH=6.5's PBS；Artificial liposome concentration is 105~106A/mL.
The GM2 single-chain antibody of the Streptavidin coupling presses VL- (G4S)3-VH-G3The mode of S-SA-GFP synthesizes entirely It is long；
In the biotinylated Qa-1, biotinylation PD-L1 and biotinylated HLA-E Qa-1, PD-L1, HLA-E with The molar ratio of Sulfo-NHS-Biotin is 1:3~1:5.
The amino acid sequence of the GM2 single-chain antibody of the Streptavidin coupling is as shown in SeqNo.1.
The amino acid sequence of described Qa-1, PD-L1 and HLA-E are respectively such as Seq No.2, Seq No.3 and SeqNo.4 institute Show, respectively with Qa1-G4S-FLAG, that is, Seq No.5, PDL1-G4S-FLAG, that is, Seq No.6, HLAE-G4S-FLAG, that is, Seq The form of No.7 fusion protein is expressed.
The second aspect of the invention provides a kind of preparation method of liposome for anti-organ transplant rejection, The following steps are included:
The first step carries the liposome preparation in carry site
The preparation of artificial liposome: artificial liposome is prepared using ultrasonic membrane method
Phosphatidyl choline 10mg~100mg, cholesterol 1mg~10mg, 0.1~1mg neuromere are proportionally weighed respectively Glycosides rouge GM2, room temperature concussion dissolution overnight in chloroform/methanol solution, drains organic solvent using 37 DEG C of Rotary Evaporators, is allowed to Uniform liposome membrane is formed on eggplant type flask wall, and dry gas 5min is filled into bottle, residual organic solvents is made sufficiently to volatilize； PBS (0.5M) 5mL of pH=6.5 is added, concussion is sufficiently dissolved to film, Ultrasound Instrument 1~2min of ultrasound, until liposome solutions disperse For uniform and stable transparency liquid, artificial liposome is obtained；Later every 20 μ L artificial liposome be added DSPE-PEG100~ 800mg, 50 DEG C of water-bath 10min are modified, its stability is enhanced；
Second step, single-chain antibody-Streptavidin expressing fusion protein purifying of Ganglioside GM2
The GM2 single-chain antibody of Streptavidin coupling presses VL- (G4S)3-VH-G3The mode of S-SA-GFP synthesizes overall length, egg The expression of white matter uses ExpiCHO system, and the single-chain antibody of GM2-Streptavidin fusion protein is cloned into pcDNA3.4 and is carried After body, transfection CHO cell；37 DEG C, 8%CO2Under environment after shaken cultivation 8 days, cell suspension is collected；5000rpm~6000rpm After being centrifuged 10~15min removal cell, diluted using isometric 2 × PBS；The GM2 single-chain antibody of Streptavidin coupling uses The purifying of iminobiotin sepharose 4B；Wherein single-chain antibody scFv (VL+VH) sequence derives from patent US5830470；
Third step, the expression and purification of carry element
Carry element includes Qa-1 extracellular fragment, PD-L1 extracellular fragment and HLA-E extracellular fragment, and 3 carry elements are respectively with Qa1- G4S-FLAG, that is, Seq No.5, PDL1-G4S-FLAG, that is, Seq No.6 and HLAE-G4S-FLAG, that is, Seq No.7 fusion protein Form is expressed, after three above fusion protein is cloned into pcDNA3.4 carrier, transfection CHO cell, and 37 DEG C, 8%CO2Ring Under border after shaken cultivation 8 days, collects cell suspension and used after 5000rpm~6000rpm is centrifuged 10~15min removal cell 2 × PBS of volume dilution, carry element connect FLAG label and human IgG1 Fc sections；All albumen use OptiCHOTM The expression of Express system secretion, carry element carry out first round purifying using ProteinA column, complete to use Tris- after purification In HCl and pH value is to 8.0, cuts off the end Fc using enterokinase, stays overnight digestion by 0.2U enterokinase/25 DEG C of 1mg total protein, digestion produces Object carries out secondary affinity purification using FLAG affinity column；Purified product is neutralized to pH=7.5 using Tris-HCl, uses 35kDa Super filter tube protein concentrate；
4th step, the biotinylation of carry element
The biotinylation of carry element is reacted using Sulfo-NHS-Biotin
After quantitative to the carry element of third step preparation, in molar ratio with Sulfo-NHS-Biotin by carry element peptide fragment 1:3~1:5 reacts at room temperature 30min in pH=8.5PBS, and unbonded Sulfo-NHS- is removed using 10kDa bag filter Biotinylated carry element is concentrated by ultrafiltration in Biotin, obtains biotinylated carry element；
5th step, liposome medicament load
The GM2 single-chain antibody of the Streptavidin coupling of second step preparation is added in the artificial liposome of first step preparation, Concentration is 0.1~1ng/ μ L, and 4 DEG C are mixed by inversion overnight；
After the biotinylated Qa-1 or HLA-E of third step preparation are mixed with PD-L1 equimolar, by 100:1~200:1 Molar ratio mixed with above-mentioned liposome, room temperature is mixed by inversion, and dialysis removes unbonded albumen, completes liposome preparation.
Membrane process is preferentially used in preparation method of the present invention and prepares liposome, and in addition to this, reverse evaporation, gradient are anti- The liposome prepared to other schemes such as stowage, double emulsifications can also be used for this system.
In addition, in addition in semicrystalline material load peptide fragment, for inhibiting activated immune cell, prepared by liposome itself In journey can also package immunomodulator further increase immunosuppressive effect, or play in specific organ transplant type specific Effect.Such as, during liposome preparation package immune suppressive cyclosporin A, FK506, rapamycin, mycophenolate mofetil or The mixture of several drugs, may be implemented medicament slow release, and help steadily improves immunosuppressor concentration after transplanting.
Further, in addition in semicrystalline material load peptide fragment, for inhibiting activated immune cell, prepared by liposome itself In the process can also package drug specifically transplanting type in play other effects.It can package in the liposome as used in pancreatic islets transplantation Promote revascularization drug, such as VEGF, promotes graft angiogenesis.
The third aspect of the invention provides a kind of liposome for anti-organ transplant rejection and is used to prepare suppression Application in activated immune cell drug processed.
The fourth aspect of the invention provides a kind of liposome for anti-organ transplant rejection and is used to prepare suppression Application in lymphocyte activation drug processed.
The fifth aspect of the invention provides a kind of liposome for anti-organ transplant rejection and is used to prepare pancreas Application in the post-transplantation drug of island.
Liposome prepared by the present invention for anti-organ transplant rejection, can in vivo effective activation NKG2A and PD1 dual signal access plays the anti-rejection effect of post-transplantation.
Liposome prepared by the present invention for anti-organ transplant rejection passes through Qa-1/PD-L1 rouge after detection MLR Influence of the plastid to associated signal paths, the results showed that the liposome can remarkably promote the activation of SHP1 Yu SHP2 molecule, from And inhibit Syk protein tyrosine kinase and the downstream activation of ERK1/2.
Liposome prepared by the present invention for anti-organ transplant rejection, Western-blot detect SHP1/2, Syk With the Activation of ERK1/2, the results showed that Qa-1 molecule/PD-L1 liposome can activate liver lymphocyte SHP1/2, thus Inhibit the activation of Syk and ERK1/2.
Due to the adoption of the above technical scheme, the present invention has the following advantages and beneficial effects:
Provided by the present invention for the preparation method of the liposome of anti-organ transplant rejection, the liposome of preparation can be Anti- rejection effect is played in vivo, is a kind of novel lipide drug, the anti-rejection effect postoperative for a variety of organ transplants.
Provided by the present invention for the preparation method of the liposome of anti-organ transplant rejection, the liposome of preparation is one Kind has the liposome of targeting for immunologic test point molecule, has the feature that 1. liposome preparation passes through in the process and mixes Enter gangliosides GM2Form carry site；2. forming carry center in surface of liposome by single-chain antibody-Streptavidin； 3. biotinylated Qa-1 segment, HLA-E segment, PD-L1 segment hang over liposome by biotin-Streptavidin reaction Surface.The present invention can act in effective activation NKG2A and PD1 dual signal access, the anti-rejection for playing post-transplantation in vivo.
Liposome prepared by the present invention for anti-organ transplant rejection passes through Qa-1/PD-L1 rouge after detection MLR Influence of the plastid to associated signal paths, the results showed that the liposome can remarkably promote the activation of SHP1 Yu SHP2 molecule, from And inhibit Syk protein tyrosine kinase and the downstream activation of ERK1/2.
Detailed description of the invention
Fig. 1 is the preparation flow schematic diagram of the artificial liposome of the embodiment of the present invention.
Fig. 2 is the carry ideograph of various elements in Qa-1 molecule/PD-L1 artificial liposome in the embodiment of the present invention.
Fig. 3 be in the embodiment of the present invention using red fluorescent protein be Quality Control Molecular Detection carry efficiency (Scale bar: 10μm)。
Fig. 4 is Qa-1 molecule/influence of the PD-L1 liposome to lymphopoiesis and activation, in which: A, B are in figure The influence that CFSE method detection Qa-1 molecule/PD-L1 liposome is proliferated MLR medium size lymphocyte, result are 3 independent experiment results (n=3)；C, D is FACS detection CD107a positive cell ratio (n=3)；E is IFN-γ expression in ELISA detection culture supernatant It measures (n=3)；F be after Western-blot detection MLR 1 hour with the Activation of 2 hours SHP1/2, Syk and ERK1/2；**p < 0.01, p < 0.001.
Fig. 5 is Qa-1 molecule/PD-L1 liposome to transplantation site lymphocyte activation effect；Wherein: A is FACS detection CD107a positive cell ratio (n=3) in liver lymphocyte；B is Western blot detection SHP1/2, Syk and ERK1/2 Activation (n=3).
Fig. 6 be Qa-1 molecule/PD-L1 liposome in congenic mouse pancreatic islet transplant model to the protective effect of graft, Wherein: A is mouse blood sugar variation (n=10) after transplanting；B is mouse C peptide variation (n=10) after transplanting；C is exempted within 14 days after transplanting The survival condition (Scale bar:50 μm) of Islet allografts in epidemic disease Fluorometric assay liver.
Specific embodiment
In order to illustrate more clearly of the present invention, below with reference to preferred embodiment, the present invention is described further.Ability Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this The protection scope of invention.
Phosphatidyl choline (article No. V900485) used in invented liposomes building process be purchased from Sigma, purity >= 99%；Cholesterol (article No. C8667) is purchased from Sigma, purity >=99%；Ganglioside GM2 (article No. G4651) is purchased from Sigma, Purity ≈ 95%.The expression of protein (including single-chain antibody-SA fusion protein and three carry elements) is using Gibco company ExpiCHO (A29133) eukaryotic protein expression system.
Unless otherwise described, implementation of the invention will be using molecular biology, microbiology, recombinant DNA and immunologic Routine techniques, these are known to those skilled in the art.These technologies have complete description in the following documents: for example, Sambrook " Molecular Cloning:A Laboratory guide " second edition (1989)；" DNA clone " I and II volumes (D.N.Glover edits 1985)； " oligonucleotide synthesis " (M.J.Gait is edited, 1984)；" nucleic acid hybridization " (B.D.Hames and S.J.Higgins are edited .1984)；" protein purification: principle and practice " second edition (Springer-Verlag, N.Y.), and " experiment immunization learns to do Volume " I-IV volumes (D.C.Weir and C.C.Blackwell edit 1986).Alternatively, can be according to explanation provided by reagent manufacturer Book carries out.
Unless otherwise stated, otherwise percentage and number are calculated by weight.Unless otherwise defined, as used herein all Professional and scientific terms have the same meanings as commonly understood by one of ordinary skill in the art.In addition, any similar or equal to described content Deng method and material all can be applied in the present invention.The preferred methods and materials described herein are for illustrative purposes only.
The design and preparation of embodiment 1:Qa-1/PD-L1 liposome
Experimental method:
The first step carries the liposome preparation in carry site
The preparation of artificial liposome: artificial liposome is prepared using ultrasonic membrane method
Phosphatidyl choline 20mg, cholesterol 5mg, 0.25mg Ganglioside GM2 are proportionally weighed respectively, and room temperature is in chlorine Concussion dissolution overnight, drains organic solvent using 37 DEG C of Rotary Evaporators, is allowed in imitative/methanol (volume ratio 5:1) 15mL solution Uniform liposome membrane is formed on eggplant type flask wall, and dry gas 5min is filled into bottle, residual organic solvents is made sufficiently to volatilize. PBS (0.5M) 5mL of pH=6.5 is added, concussion is sufficiently dissolved to film, Ultrasound Instrument 1~2min of ultrasound, until liposome solutions disperse For uniform and stable transparency liquid, artificial liposome is obtained, artificial liposome concentration is 105~106A/mL.Every 20 μ L later DSPE-PEG200mg is added in artificial liposome, and 50 DEG C of water-bath 10min are modified, and enhances its stability.
If you need to package immunomodulator (glucocorticoid, FK506, cyclosporine etc.), promotion revascularization in liposome Drug (vegf protein and derived peptide segment, hepatocyte growth factor, the platelet proliferation factor etc.), operate it is as follows: respectively according to than Example weighs phosphatidyl choline 20mg, cholesterol 5mg, 0.25mg Ganglioside GM2, and room temperature is at chloroform/methanol (volume ratio 5:1) Concussion dissolution overnight, drains organic solvent using 37 DEG C of Rotary Evaporators, is allowed to be formed on eggplant type flask wall in 15mL solution Uniform liposome membrane fills dry gas 5min into bottle, residual organic solvents is made sufficiently to volatilize.Use the PBS of pH=6.5 (glucocorticoid, FK506, cyclosporine etc. are the immunosuppressor of representative or vegf protein and spread out (0.5M) 2mL dissolution drug Raw peptide fragment, hepatocyte growth factor, platelet proliferation factor etc. are the promotion revascularization drug of representative), reach drug concentration To 100~1000ng/mL.Detect hydrating fluid concentration using pH meter, using the citrate buffer of 10 × pH=3.0 or 10 × The Tris salt buffer of pH=8.5 readjusts liquid to pH=6.5.Eggplant type flask is added in liquid, is shaken sufficiently molten to film Solution, Ultrasound Instrument 1~2min of ultrasound obtain artificial liposome, people until liposome solutions are separated into uniform and stable transparency liquid Work concentration of liposomes is 105~106A/mL.DSPE-PEG200mg, 50 DEG C of water-baths are added in every 20 μ L artificial liposome later 10min is modified, its stability is enhanced.
Second step, single-chain antibody-Streptavidin expressing fusion protein purifying of Ganglioside GM2
The GM2 single-chain antibody of Streptavidin coupling presses VL- (G4S)3-VH-G3The mode of S-SA-GFP synthesizes overall length, For amino acid sequence as shown in Seq No.1, the expression of protein uses ExpiCHO (Gibco, A29133) system, and GM2 is single-stranded After antibody-SA fusion protein is cloned into pcDNA3.4 carrier, transfection CHO cell.37 DEG C, 8%CO2Shaken cultivation 8 days under environment Afterwards, cell suspension is collected.After 5000rpm~6000rpm is centrifuged 10~15min removal cell, diluted using isometric 2 × PBS. The GM2 single-chain antibody of Streptavidin coupling uses iminobiotin sepharose 4B purifying (PurKine, KTP20306).Its Middle single-chain antibody scFv (VL+VH) sequence derives from patent US5830470.
Third step, the expression and purification of carry element
Carry element includes Qa-1 extracellular fragment, PD-L1 extracellular fragment and HLA-E extracellular fragment, and amino acid sequence is respectively such as Seq Shown in No.2, Seq No.3 and Seq No.4,3 carry elements are respectively with Qa1-G4S-FLAG(Seq No.5)、PDL1-G4S- FLAG (Seq No.6) and HLAE-G4The form of S-FLAG (Seq No.7) fusion protein is expressed, and three above is merged After albumen is cloned into pcDNA3.4 carrier, transfection CHO cell.37 DEG C, 8%CO2Under environment after shaken cultivation 8 days, cell is collected Suspension.After 5000rpm~6000rpm is centrifuged 10~15min removal cell, diluted using isometric 2 × PBS.Carry element connects Connect FLAG label and human IgG1 Fc sections；All albumen use OptiCHOTMThe expression of Express system secretion.Carry element makes Carry out first round purifying with ProteinA column (world people and, SA012K), complete after purification using in Tris-HCl and pH value extremely 8.0, the end Fc is cut off using enterokinase (the refined heart in Shanghai, REK08), stays overnight digestion, enzyme by 0.2U enterokinase/25 DEG C of 1mg total protein Cutting product, (people from world carries out secondary affinity purification with SA042001) using FLAG affinity column；Purified product uses Tris-HCl It is neutralized to pH=7.5, uses 35kDa super filter tube protein concentrate.
4th step, the biotinylation of carry element
The biotinylation of carry element is reacted using Sulfo-NHS-Biotin (the raw work in Shanghai, C100213).
Sulfo-NHS-Biotin can efficiently make primary amino group (- NH in ealkaline buffer2) form stable amide Key.Lysine (K) residue side chains and polypeptide N-terminal in protein can react to form stable life with Sulfo-NHS-Biotin The connection of object element.After quantitative to the carry element of third step preparation, by carry element peptide fragment and Sulfo-NHS-Biotin by mole 30min is reacted at room temperature in pH=8.5PBS than 1:3~1:5.Unbonded Sulfo-NHS- is removed using 10kDa bag filter Biotinylated carry element is concentrated by ultrafiltration in Biotin, obtains biotinylated carry element.
5th step, liposome medicament load
The GM2 of the Streptavidin coupling of 100ng second step preparation is added in the 200 μ L of artificial liposome of first step preparation Single-chain antibody, 4 DEG C are mixed by inversion overnight, and fluorescence microscope counts lipid volume density.
By third step prepare biotinylated Qa-1 (or HLA-E) mixed with PD-L1 equimolar after, by 100:1~ The molar ratio of 200:1 is mixed with above-mentioned liposome, and room temperature is mixed by inversion 30min, unbonded using the dialysis removal of 35KD bag filter Albumen completes liposome preparation.Experimental result:
Fig. 1 is the preparation flow schematic diagram of the artificial liposome of the embodiment of the present invention.Fig. 2 is Qa-1 in the embodiment of the present invention The carry ideograph of various elements in molecule/PD-L1 artificial liposome.Qa-1/PD-L1 uses scheme carry as shown in Figure 2 To artificial liposome: the anti-Ganglioside GM2 by expression fusion strepto- affinity prime (Streptavidin) is single-stranded Antibody forms carry bracket in conjunction with the GM2 on film, later incubates Qa-1 the and PD-L1 extracellular fragment of biotin modification altogether therewith It educates, forms Qa-1/PD-L1 artificial liposome.
Embodiment 2
Liposome carry efficiency and specific detection based on GM2 single-chain antibody streptavidin (SA) fusion protein
Experimental method: the efficiency test of liposome carry is carried out using biotinylated red fluorescent protein (dsRed).
The amino acid sequence of red fluorescent protein (dsRed) as shown in Seq No.8, red fluorescent protein (dsRed) with Qa1-G4The form of S-FLAG (Seq No.9) fusion protein is expressed: by Qa1-G4S-FLAG (Seq No.9) fusion protein After being cloned into pcDNA3.4 carrier, transfection CHO cell.37 DEG C, 8%CO2Under environment after shaken cultivation 8 days, cell suspension is collected. After 5000rpm~6000rpm is centrifuged 10~15min removal cell, diluted using isometric 2 × PBS, product is affine using FLAG Column purification, purified product is neutralized to pH=7.5 using 2 × PBS, using 10kDa super filter tube protein concentrate, dsRed after purification It is reacted using Sulfo-NHS-Biotin, after the 5th step carry liposome of method in embodiment 1, fluorescence detection carry Efficiency.After not biotinylated dsRed and liposome are incubated for altogether, the dsRed that dialysis removal is not associated with is special for measuring carry Property.
Experimental result: measuring this using the affine plain fusion protein of scFv and biotinylated dsRed that have GFP label is System carry efficiency, Fig. 3 are to use red fluorescent protein for Quality Control Molecular Detection carry efficiency in the embodiment of the present invention.Wherein GFP Green fluorescence illustrates the liposome after scFv-SA fusion protein combines, and diameter distribution is more uniform, and size is about 50- 500nm (Fig. 3 the picture left above, top right plot, Scale bar are 10 μm).Red fluorescence illustrates biotinylated dsRed in liposome Apparent carry efficiency, the results showed that the system has preferable carry efficiency, and biotinylated dsRed is successfully mounted to all Surface of liposome (lower-left Fig. 3 figure)；The system has good carry specificity simultaneously, and not biotinylated dsRed cannot be hung It is downloaded to surface of liposome (Fig. 3 bottom-right graph.)
Embodiment 3
The influence of Qa-1/PD-L1 artificial liposome prepared by embodiment 1 to mixed lymphocyte reaction (MLP)
Experimental method: C57 Mouse spleen cells break it is red after adjust to 1 × 1062mmolCFSE solution is added in cells/mL, 37 DEG C of incubation 15min are washed 2 times with 10 times of volumes ice cold PBS, and 500 holes μ L/ are inoculated in 24 orifice plates.Balb/c mouse spleen Break it is red after, be added 37 DEG C of complete medium of 10ug/ml mitomycin c and handle 2 hours, wash 6 times with 10 times of volume PBS, tune It is whole to cell concentration to 1 × 106Cells/mL, 500 holes μ L/ and C57 mixing with cells.Continuous culture 3 days, it is thin that FACS detects lymph IFN-γ amount in born of the same parents' proliferative conditions, CD107a positive cell ratio and supernatant.Westernblot detect SHP1, SHP2, Syk with The Activation of ERK1/2.
Experimental result: taking donor mice spleen lymphocyte, is handled using mitomycin c, is allowed to no longer be proliferated.By the two 1:1 is mixed 3 days, and FACS detects lymphopoiesis.CFSE is a kind of dyestuff that fluorescent marker can be carried out to living cells, can With penetrating cell film and intracellular protein covalent bond, and issue green fluorescence.In cell division breeding, fluorescence intensity meeting Weaken with cell division according to generation, the ratio of lymphocyte can be proliferated by this feature detection.The result shows that Qa-1/ PD-L1 liposome can significantly inhibit lymphopoiesis (A, B in Fig. 4).CD107a is NK cell and CD8+T cell activation Important symbol, it is closely related with cytotoxic activity.Have detected CD107a positive cell ratio after MLR, discovery incorporation Qa-1/PD- L1 liposome can be significantly reduced CD107a positive cell ratio (C, D in Fig. 4), prompt liposome that can significantly inhibit lymph thin The cytotoxic activity of born of the same parents.The secretion of IFN-γ is also reflection NK and another important symbol object of T cell activation, has detected culture supernatant The expression of middle IFN-γ, the results showed that Qa-1/PD-L1 liposome can significantly inhibit IFN-γ secretion, further prompt The liposome can inhibit lymphocyte activation (E in Fig. 4).In order to study its related mechanism, have detected after MLR 1 hour with it is 2 small When influence of the Qa-1/PD-L1 liposome to associated signal paths, the results showed that the liposome can remarkably promote SHP1 and SHP2 The activation of molecule, to inhibit Syk protein tyrosine kinase and the downstream activation (F in Fig. 4) of ERK1/2.
As shown in figure 4, Fig. 4 is Qa-1 molecule/influence of the PD-L1 liposome to lymphopoiesis and activation, in which: figure Middle A, B are the influences that CFSE method detection Qa-1 molecule/PD-L1 liposome is proliferated MLR medium size lymphocyte, and result is 3 independences Experimental result (n=3)；C, D is FACS detection CD107a positive cell ratio (n=3)；E is in ELISA detection culture supernatant IFN-γ expression quantity (n=3)；F be after Western-blot detection MLR 1 hour with the work of 2 hours SHP1/2, Syk and ERK1/2 Change situation；* p < 0.01, p < 0.001.
Embodiment 4
Inhibit the effect disquisition of rejection in Qa-1 molecule/PD-L1 Via Liposomes prepared by embodiment 1
Experimental method:
Isolated pancreatic islet and transplanting: Liberase TL is diluted to the work of 200 μ g/mL with Perfusion solution Concentration after Balb/c mice pancreatic sufficiently is perfused along choledochus, is open using blood vessel clip closing ductus pancreaticus, wins entire pancreas.37 DEG C water-bath digests 10-15min, is softly mixed using Pasteur's dropper within every 2 minutes.Total volume 10% is added immediately after the completion of digestion FBS terminates digestion, after Optiprep density gradient purification pancreas islet, draws enrichment pancreas islet using wide-mouth suction nozzle under microscope, It is resuspended in CMRL1066 liquid.The pancreatic islets transplantation that every 2-3 is separated only for body mouse is to 1 Recipient mice.C57 Recipient mice uses After the anesthesia of 2mg/kg chloraldurate intraperitoneal administration, opens abdomen exposure portal vein and be inserted into remaining needle and establish transplanting channel, will be resuspended in Pancreas islet (heparin sodium 100U is added) in 250 μ L CMRL1066 is slowly injected into portal vein, and injection terminates to use tissue glue's water seal needle Simultaneously clog gelfoam in hole.
Administration mode: Qa-1 molecule/PD-L1 liposome prepared by embodiment 1 injects portal vein with graft for the first time together； 10 μ L Qa-1 molecules/PD-L1 liposome was diluted to 100 μ L, tail vein administration in every 3 days later.Control administration unloaded liposome Group, every group of 10 mouse.
Liver endolymph cell Activation: 48 hours execution mouse after pancreatic islets transplantation take mouse liver, use 100 mesh steel After net grinding, it is resuspended in 5mL physiological saline；Configuring discontinuous Optiprep density gradient, (upper layer 24mL density is 1.056g/mL, lower layer's 12mL density are 1.090g/mL), cell suspension is slowly added to density fluid upper layer, 500g centrifugation 20min, drawing intermediate tunica albuginea layer is the liver lymphocyte purified.FACS detects CD107a positive cell ratio, The Activation of Westernblot detection SHP1, SHP2, Syk and ERK1/2.
Experimental result: 48 hours execution mouse after pancreatic islets transplantation take mouse liver and separate liver lymphocyte.FACS detection CD107a positive cell ratio in lymphocyte, the results showed that compared with empty liposome processing group, Qa-1 molecule/PD-L1 lipid After body processing, CD107a positive cell ratio significantly lowers (20.93 ± 1.94%vs.13.40 ± 1.89%) in liver, prompts rouge Plastid can inhibit liver endolymph cell to activate (A in Fig. 5).The activation of Western-blot detection SHP1/2, Syk and ERK1/2 Situation, the results showed that Qa-1 molecule/PD-L1 liposome can activate liver lymphocyte SHP1/2, to inhibit Syk and ERK1/2 Activation (B in Fig. 5).As shown in figure 5, Fig. 5 is Qa-1 molecule/PD-L1 liposome to transplantation site lymphocyte activation effect； Wherein: A is CD107a positive cell ratio (n=3) in FACS detection liver lymphocyte；B is Westernblot detection SHP1/ 2, the Activation (n=3) of Syk and ERK1/2.
Embodiment 5
The research of Qa-1 molecule/PD-L1 liposome prepared by embodiment 1 to transplantation effect in islet object
Experimental method: the foundation of diabetic mouse model: the fasting of C57 mouse after rs, is infused for 24 hours by 100mg/kg dosage abdominal cavity The STZ solution for penetrating 10mg/mL, 7 days detection mouse tail vein blood glucose and C peptide concentration after injection.Blood sugar concentration > 16.7mmol/L, C peptide concentration is less than 100pmol/L, it is believed that models successfully.If single injection does not reach modeling standard, by 50% first administration agent Amount injects STZ again, until modeling successfully.
Isolated pancreatic islet and transplanting: Liberase TL is diluted to the work of 200 μ g/mL with Perfusion solution Concentration after Balb/c mice pancreatic sufficiently is perfused along choledochus, is open using blood vessel clip closing ductus pancreaticus, wins entire pancreas.37 DEG C water-bath digests 10-15min, is softly mixed using Pasteur's dropper within every 2 minutes.Total volume 10% is added immediately after the completion of digestion FBS terminates digestion, after Optiprep density gradient purification pancreas islet, draws enrichment pancreas islet using wide-mouth suction nozzle under microscope, It is resuspended in CMRL1066 liquid.Every 3 only for body mouse separation pancreatic islets transplantation give 1 Recipient mice.C57 Recipient mice uses After the anesthesia of 2mg/kg chloraldurate intraperitoneal administration, opens abdomen exposure portal vein and be inserted into remaining needle and establish transplanting channel, will be resuspended in Pancreas islet (heparin sodium 100U is added) in 250 μ L CMRL1066 is slowly injected into portal vein, and injection terminates to use tissue glue's water seal needle Simultaneously clog gelfoam in hole.
Administration mode: Qa-1 molecule/PD-L1 liposome prepared by embodiment 1 injects portal vein with graft for the first time together； 10 μ L liposomes were diluted to 100 μ L, tail vein administration in every 3 days later.300 μ g/kgday gastric infusion of rapamycin.Root According to administration mode difference, mouse is divided into 4 groups: unloaded liposome group, unloaded liposome+low dosage rapamycin group, Qa-1/PD- L1 liposome group and Qa-1/PD-L1 liposome+low dosage rapamycin group, every group of 10 mouse.
Islet function inspection: 20~30 μ L of blood is taken from vena orbitalis posterior clump using test tube of hepari glass capillary within every 24 hours, taken Chlormycetin eye ointment water sterilization is used after blood, detects C peptide, blood glucose.
Survival condition in immuno-fluorescence assay graft liver: after mouse liver is fixed using 4% paraformaldehyde, paraffin packet It buries and is prepared into paraffin section.Slice through dewaxing, antigen retrieval, closing and etc. after, be added have fluorescent marker insulin (1:400) and glucagon antibody (1:400), 4 DEG C are protected from light incubation 1 hour, and DAPI (1 μ g/mL) is after antibody incubation 45min It is added.Mounting is washed after the completion of dyeing, in fluorescence microscopy microscopic observation and the typical visual field is taken to take pictures.
Experimental result: after separating mouse pancreas islet, transplant congenic mouse (Balb/c → C57), respectively using unloaded liposome, Unloaded liposome+low dosage rapamycin (300 μ g/kgday), Qa-1/PD-L1 liposome and Qa-1/PD-L1 liposome+ Mouse (every group 10) after the transplanting of low dosage rapamycin treatment, monitoring mouse blood sugar variation in every 24 hours and C peptide level (Fig. 6 Middle A, B).As shown in fig. 6, Fig. 6 be Qa-1 molecule/PD-L1 liposome in congenic mouse pancreatic islet transplant model to graft Protective effect, in which: A is mouse blood sugar variation (n=10) after transplanting；B is mouse C peptide variation (n=10) after transplanting；C is to move After plant in 14 days immuno-fluorescence assay livers Islet allografts survival condition (Scale bar:50 μm).The result shows that pancreas Third day starts to restore island function after the transfer, the blood glucose of four groups of mouse respectively from 18.51 ± 1.53,18.29 before transplanting ± 1.86,18.16 ± 0.69,17.84 ± 0.51mmol/L drop to 9.94 ± 0.65,10.43 ± 0.89,9.04 ± 0.75, 7.95±0.76mmol/L；C peptide from 52.52 ± 21.97,40.85 ± 17.99,68.30 ± 21.58,61.38 before transplanting ± 23.97pmol/L rises to 440.99 ± 73.57,347.25 ± 104.82,747.08 ± 79.31,600.40 ± 119.96pmol/L；But unloaded liposome group C peptide compared to Qa-1/PD-L1 liposome group restore amplitude it is smaller (440.99 ± 73.57pmol/L vs.747.08 ± 79.31pmol/L), and islet function is gradually lost after the 7th day, C peptide was at the 8th day Drop to 186.94 ± 81.52pmol/L, blood glucose rise to 17.62 ± 0.89mmol/L.Compared to unloaded liposome group, give Low dosage rapamycin treatment group mouse C peptide level rises more obvious, and blood glucose level maintains 8 for 4~9 days after the transfer~ In the range of 11mmol/L, in the range of C peptide maintains 403.5~1288.8pmol/L, but since after transplanting the 10th day, C peptide gradually drops to 223.1~390.6pmol/L, and blood glucose gradually rises to 13.2~15.8mmol/L, although still there is part pancreas Island function retains, but is not enough to maintain normal blood glucose level；Compared to above two groups, Qa-1/PD-L1 liposome therapeutic group with Qa-1/PD-L1 liposome joint low dosage rapamycin treatment group achieves preferable anti-repulsion effect, and after the transfer the 4th Its blood glucose is remarkably decreased (9.04 ± 0.75mmol/L, 7.95 ± 0.76mmol/L), C stabilized peptide in 1000pmol/L or more, with Normal mouse is without significant difference；Wherein it is worth noting that: although Qa-1/PD-L1 liposome be administered alone effect slightly below join Administering effect is closed, but curative effect difference less, group blood glucose level was only administered alone at the 14th day and is slightly above administered in combination between two groups Group (8.9mmol/L vs.6.92mmol/L, p < 0.001), C peptide are slightly below administering drug combinations group (1070.21pmol/L Vs.1461.52pmol/L, p < 0.001).It is worth noting that, 12-16 hours after the transfer, there is C peptide in all mouse Fulminant of short duration raising (B in Fig. 6), C peptide at this time mostly come from transplanting Deaths islet cells content, and with Prognosis mala is related.Compared with unloaded liposome group, after Qa-1/PD-L1 liposome-treated, early stage C peptide burst size is transplanted Appearance is remarkably decreased (2444.59 ± 349.11pmol/L vs.957.40 ± 241.06pmol/L, p < 0.001)；It is mould with thunder pa Plain group is compared, Qa-1/PD-L1 liposome for transplant early stage pancreas islet loss protective effect it is more significant (1417.02 ± 401.30pmol/L vs.957.40 ± 241.06pmol/L, p < 0.01).Empty liposome is taken to handle respectively within the 14th day after transplanting Group and Qa-1/PD-L1 liposome therapeutic group mouse 1, using insulin and glucagon antibody test after taking liver fixed Islet survival situation in liver.The result shows that most islet cells apoptosis in sinus hepaticus in empty liposome processing group, only The survival of minute quantity islet cells, and Islet allografts form is complete in Qa-1/PD-L1 liposome therapeutic group sinus hepaticus, survival is good (C in Fig. 6).
SeqNo.1:
MHFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWI YSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPYTFGGGTKLEIKRGGGGSGGGGSGGGGSMG WSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGYIYPNNGGTGY NQKFKSKATLTVDKSSSTAYMELHSLTSEDSAVYYCATYGHYYGYMFAYWGQGTLVTVSAGGGGSMRCTIVLGIRAA SPIKEALARPAPRPGRLPSIHRSGRRNMQRLEHVLRRVKAGTGAPIDFSGTWKNELGSTMRIEQSGDSVSGTYESAV SENGGATSGQLSGYVDGDLIAFVVHWDQFQAITAWVGQGGPGASSDRINTLWQMTQQVEAGEEWASINAGADIFVKT VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYKSGAAAAAAAAAAEFPGLEKLGSTGSR
Seq No.2:
PHSLRYFTTAVSRPGLGEPRFIIVGYVDDTQFVRFDSDA61ENPRMEPRARWIEQEGPEYWERETWKA RDMGRNFRVNLRTLLGYYNQSNDESHTLQWMYG121CDVGPDGRLLRGYCQEAYDGQDYISLNEDLRSWTANDIAS QISKHKSEAVDEAHQQRAYLQGPCVEWLHRYLRLGNETL
Seq No.3:
NKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTT NEIFYCTFRRLDPEENH
Seq No.4:
SHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDT AQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKS NDASEAEHQRAYLEDTCVEWLHKYLEKGKETL
Seq No.5:
PHSLRYFTTAVSRPGLGEPRFIIVGYVDDTQFVRFDSDA61ENPRMEPRARWIEQEGPEYWERETWKA RDMGRNFRVNLRTLLGYYNQSNDESHTLQWMYG121CDVGPDGRLLRGYCQEAYDGQDYISLNEDLRSWTANDIAS QISKHKSEAVDEAHQQRAYLQGPCVEWLHRYLRLGNETLGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq No.6:
NKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTT NEIFYCTFRRLDPEENHGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
Seq No.7:
SHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDT AQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKS NDASEAEHQRAYLEDTCVEWLHKYLEKGKETLGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq No.8:
MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGS KVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEAS TERLYPRDGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHL FLSEQ
Seq No.9:
MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGS KVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEAS TERLYPRDGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHL FLSEQDYKGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWY VDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK
The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this patent Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little change or be modified to The equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, it is right according to the technical essence of the invention Any simple modification, equivalent change and modification made by above embodiments, in the range of still falling within the present invention program.
Sequence table
<110>Second Military Medical University, PLA
<120>a kind of liposome and preparation method and application for anti-organ transplant rejection
<130>specification, claims
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 718
<212> PRT
<213>artificial sequence (Artificial)
<400> 1
Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser
50 55 60
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg
100 105 110
Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
145 150 155 160
Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
165 170 175
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
180 185 190
Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu
195 200 205
Glu Trp Ile Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Tyr Asn
210 215 220
Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
225 230 235 240
Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val
245 250 255
Tyr Tyr Cys Ala Thr Tyr Gly His Tyr Tyr Gly Tyr Met Phe Ala Tyr
260 265 270
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser
275 280 285
Met Arg Cys Thr Ile Val Leu Gly Ile Arg Ala Ala Ser Pro Ile Lys
290 295 300
Glu Ala Leu Ala Arg Pro Ala Pro Arg Pro Gly Arg Leu Pro Ser Ile
305 310 315 320
His Arg Ser Gly Arg Arg Asn Met Gln Arg Leu Glu His Val Leu Arg
325 330 335
Arg Val Lys Ala Gly Thr Gly Ala Pro Ile Asp Phe Ser Gly Thr Trp
340 345 350
Lys Asn Glu Leu Gly Ser Thr Met Arg Ile Glu Gln Ser Gly Asp Ser
355 360 365
Val Ser Gly Thr Tyr Glu Ser Ala Val Ser Glu Asn Gly Gly Ala Thr
370 375 380
Ser Gly Gln Leu Ser Gly Tyr Val Asp Gly Asp Leu Ile Ala Phe Val
385 390 395 400
Val His Trp Asp Gln Phe Gln Ala Ile Thr Ala Trp Val Gly Gln Gly
405 410 415
Gly Pro Gly Ala Ser Ser Asp Arg Ile Asn Thr Leu Trp Gln Met Thr
420 425 430
Gln Gln Val Glu Ala Gly Glu Glu Trp Ala Ser Ile Asn Ala Gly Ala
435 440 445
Asp Ile Phe Val Lys Thr Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
450 455 460
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
465 470 475 480
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
485 490 495
Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
500 505 510
Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr
515 520 525
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
530 535 540
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
545 550 555 560
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
565 570 575
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
580 585 590
His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala
595 600 605
Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn
610 615 620
Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
625 630 635 640
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
645 650 655
Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
660 665 670
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
675 680 685
Glu Leu Tyr Lys Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
690 695 700
Glu Phe Pro Gly Leu Glu Lys Leu Gly Ser Thr Gly Ser Arg
705 710 715
<210> 2
<211> 178
<212> PRT
<213>artificial sequence (Artificial)
<400> 2
Pro His Ser Leu Arg Tyr Phe Thr Thr Ala Val Ser Arg Pro Gly Leu
1 5 10 15
Gly Glu Pro Arg Phe Ile Ile Val Gly Tyr Val Asp Asp Thr Gln Phe
20 25 30
Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Met Glu Pro Arg Ala
35 40 45
Arg Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu Thr Trp
50 55 60
Lys Ala Arg Asp Met Gly Arg Asn Phe Arg Val Asn Leu Arg Thr Leu
65 70 75 80
Leu Gly Tyr Tyr Asn Gln Ser Asn Asp Glu Ser His Thr Leu Gln Trp
85 90 95
Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly Tyr
100 105 110
Cys Gln Glu Ala Tyr Asp Gly Gln Asp Tyr Ile Ser Leu Asn Glu Asp
115 120 125
Leu Arg Ser Trp Thr Ala Asn Asp Ile Ala Ser Gln Ile Ser Lys His
130 135 140
Lys Ser Glu Ala Val Asp Glu Ala His Gln Gln Arg Ala Tyr Leu Gln
145 150 155 160
Gly Pro Cys Val Glu Trp Leu His Arg Tyr Leu Arg Leu Gly Asn Glu
165 170 175
Thr Leu
<210> 3
<211> 86
<212> PRT
<213>artificial sequence (Artificial)
<400> 3
Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser Glu
1 5 10 15
His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val Ile
20 25 30
Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr Thr
35 40 45
Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu Arg
50 55 60
Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg Leu
65 70 75 80
Asp Pro Glu Glu Asn His
85
<210> 4
<211> 178
<212> PRT
<213>artificial sequence (Artificial)
<400> 4
Ser His Ser Leu Lys Tyr Phe His Thr Ser Val Ser Arg Pro Gly Arg
1 5 10 15
Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln Phe
20 25 30
Val Arg Phe Asp Asn Asp Ala Ala Ser Pro Arg Met Val Pro Arg Ala
35 40 45
Pro Trp Met Glu Gln Glu Gly Ser Glu Tyr Trp Asp Arg Glu Thr Arg
50 55 60
Ser Ala Arg Asp Thr Ala Gln Ile Phe Arg Val Asn Leu Arg Thr Leu
65 70 75 80
Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln Trp
85 90 95
Met His Gly Cys Glu Leu Gly Pro Asp Gly Arg Phe Leu Arg Gly Tyr
100 105 110
Glu Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Leu Thr Leu Asn Glu Asp
115 120 125
Leu Arg Ser Trp Thr Ala Val Asp Thr Ala Ala Gln Ile Ser Glu Gln
130 135 140
Lys Ser Asn Asp Ala Ser Glu Ala Glu His Gln Arg Ala Tyr Leu Glu
145 150 155 160
Asp Thr Cys Val Glu Trp Leu His Lys Tyr Leu Glu Lys Gly Lys Glu
165 170 175
Thr Leu
<210> 5
<211> 416
<212> PRT
<213>artificial sequence (Artificial)
<400> 5
Pro His Ser Leu Arg Tyr Phe Thr Thr Ala Val Ser Arg Pro Gly Leu
1 5 10 15
Gly Glu Pro Arg Phe Ile Ile Val Gly Tyr Val Asp Asp Thr Gln Phe
20 25 30
Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Met Glu Pro Arg Ala
35 40 45
Arg Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu Thr Trp
50 55 60
Lys Ala Arg Asp Met Gly Arg Asn Phe Arg Val Asn Leu Arg Thr Leu
65 70 75 80
Leu Gly Tyr Tyr Asn Gln Ser Asn Asp Glu Ser His Thr Leu Gln Trp
85 90 95
Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly Tyr
100 105 110
Cys Gln Glu Ala Tyr Asp Gly Gln Asp Tyr Ile Ser Leu Asn Glu Asp
115 120 125
Leu Arg Ser Trp Thr Ala Asn Asp Ile Ala Ser Gln Ile Ser Lys His
130 135 140
Lys Ser Glu Ala Val Asp Glu Ala His Gln Gln Arg Ala Tyr Leu Gln
145 150 155 160
Gly Pro Cys Val Glu Trp Leu His Arg Tyr Leu Arg Leu Gly Asn Glu
165 170 175
Thr Leu Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Thr
180 185 190
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
195 200 205
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
210 215 220
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
225 230 235 240
Gln Val Lys Phe Asn Trp Tyr Val Asp Gly Val Gln Val His Asn Ala
245 250 255
Lys Thr Lys Pro Arg Glu Gln Gln Tyr Asn Ser Thr Tyr Arg Val Val
260 265 270
Ser Val Leu Thr Val Leu His Gln Asn Trp Leu Asp Gly Lys Glu Tyr
275 280 285
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
290 295 300
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
305 310 315 320
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
325 330 335
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
340 345 350
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
355 360 365
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
370 375 380
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
385 390 395 400
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410 415
<210> 6
<211> 324
<212> PRT
<213>artificial sequence (Artificial)
<400> 6
Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser Glu
1 5 10 15
His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val Ile
20 25 30
Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr Thr
35 40 45
Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu Arg
50 55 60
Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg Leu
65 70 75 80
Asp Pro Glu Glu Asn His Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp
85 90 95
Asp Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
100 105 110
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
115 120 125
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
130 135 140
His Glu Asp Pro Gln Val Lys Phe Asn Trp Tyr Val Asp Gly Val Gln
145 150 155 160
Val His Asn Ala Lys Thr Lys Pro Arg Glu Gln Gln Tyr Asn Ser Thr
165 170 175
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asn Trp Leu Asp
180 185 190
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
210 215 220
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
225 230 235 240
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
245 250 255
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
260 265 270
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
275 280 285
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
290 295 300
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
305 310 315 320
Ser Pro Gly Lys
<210> 7
<211> 416
<212> PRT
<213>artificial sequence (Artificial)
<400> 7
Ser His Ser Leu Lys Tyr Phe His Thr Ser Val Ser Arg Pro Gly Arg
1 5 10 15
Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln Phe
20 25 30
Val Arg Phe Asp Asn Asp Ala Ala Ser Pro Arg Met Val Pro Arg Ala
35 40 45
Pro Trp Met Glu Gln Glu Gly Ser Glu Tyr Trp Asp Arg Glu Thr Arg
50 55 60
Ser Ala Arg Asp Thr Ala Gln Ile Phe Arg Val Asn Leu Arg Thr Leu
65 70 75 80
Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln Trp
85 90 95
Met His Gly Cys Glu Leu Gly Pro Asp Gly Arg Phe Leu Arg Gly Tyr
100 105 110
Glu Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Leu Thr Leu Asn Glu Asp
115 120 125
Leu Arg Ser Trp Thr Ala Val Asp Thr Ala Ala Gln Ile Ser Glu Gln
130 135 140
Lys Ser Asn Asp Ala Ser Glu Ala Glu His Gln Arg Ala Tyr Leu Glu
145 150 155 160
Asp Thr Cys Val Glu Trp Leu His Lys Tyr Leu Glu Lys Gly Lys Glu
165 170 175
Thr Leu Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Thr
180 185 190
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
195 200 205
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
210 215 220
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
225 230 235 240
Gln Val Lys Phe Asn Trp Tyr Val Asp Gly Val Gln Val His Asn Ala
245 250 255
Lys Thr Lys Pro Arg Glu Gln Gln Tyr Asn Ser Thr Tyr Arg Val Val
260 265 270
Ser Val Leu Thr Val Leu His Gln Asn Trp Leu Asp Gly Lys Glu Tyr
275 280 285
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
290 295 300
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
305 310 315 320
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
325 330 335
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
340 345 350
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
355 360 365
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
370 375 380
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
385 390 395 400
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410 415
<210> 8
<211> 228
<212> PRT
<213>artificial sequence (Artificial)
<400> 8
Met Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu
20 25 30
Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val
35 40 45
Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln
50 55 60
Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro
65 70 75 80
Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val
85 90 95
Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser
100 105 110
Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn
115 120 125
Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu
130 135 140
Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu
145 150 155 160
Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu
165 170 175
Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr
180 185 190
Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr
195 200 205
Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His Leu Phe
210 215 220
Leu Ser Glu Gln
225
<210> 9
<211> 469
<212> PRT
<213>artificial sequence (Artificial)
<400> 9
Met Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu
20 25 30
Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val
35 40 45
Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln
50 55 60
Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro
65 70 75 80
Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val
85 90 95
Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser
100 105 110
Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn
115 120 125
Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu
130 135 140
Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu
145 150 155 160
Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu
165 170 175
Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr
180 185 190
Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr
195 200 205
Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His Leu Phe
210 215 220
Leu Ser Glu Gln Asp Tyr Lys Gly Gly Gly Gly Ser Asp Tyr Lys Asp
225 230 235 240
Asp Asp Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Gln Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Gln Val His Asn Ala Lys Thr Lys Pro Arg Glu Gln Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asn Trp Leu
325 330 335
Asp Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465

Claims (10)

1. a kind of liposome for anti-organ transplant rejection, which is characterized in that be to be prepared from the following materials:
The GM2 single-chain antibody of Streptavidin coupling is supported in artificial liposome, the GM2 single-chain antibody of Streptavidin coupling Being supported on concentration in artificial liposome is 0.1~1ng/ μ L,
Biotinylated Qa-1 or HLA-E and biotinylation PD-L1 is equimolar ratio,
The GM2 single-chain antibody of biotinylated Qa-1, the total amount of biotinylation PD-L1 and the coupling of above-mentioned Streptavidin load people The molar ratio of work liposome is 100:1~200:1；
Or, the total amount of biotinylated HLA-E, biotinylation PD-L1 and the GM2 single-chain antibody of above-mentioned Streptavidin coupling are negative The molar ratio for carrying artificial liposome is 100:1~200:1.
2. the liposome according to claim 1 for anti-organ transplant rejection, it is characterised in that: the artificial fat Plastid is prepared by following raw material: phosphatidyl choline 10mg~100mg, cholesterol 1mg~10mg, 0.1~1mg gangliosides The PBS of GM2, pH=6.5；Artificial liposome concentration is 105~106A/mL.
3. the liposome according to claim 1 for anti-organ transplant rejection, it is characterised in that: the strepto- parent VL- (G is pressed with the GM2 single-chain antibody of element coupling4S)3-VH-G3The mode of S-SA-GFP synthesizes overall length.
4. the liposome according to claim 1 for anti-organ transplant rejection, it is characterised in that: the biotin Qa-1, PD-L1, HLA-E and Sulfo-NHS-Biotin in Qa-1, the biotinylation PD-L1 and biotinylated HLA-E of change Molar ratio is 1:3~1:5.
5. the liposome according to claim 1 for anti-organ transplant rejection, it is characterised in that: the strepto- parent Amino acid sequence with the GM2 single-chain antibody of element coupling is as shown in SeqNo.1.
6. the liposome according to claim 1 for anti-organ transplant rejection, it is characterised in that: the Qa-1, The amino acid sequence of PD-L1 and HLA-E is respectively as shown in Seq No.2, Seq No.3 and Seq No.4, respectively with Qa1-G4S- FLAG, that is, Seq No.5, PDL1-G4S-FLAG, that is, Seq No.6, HLAE-G4The form of S-FLAG, that is, Seq No.7 fusion protein into Row expression.
7. a kind of preparation method of the liposome as claimed in any one of claims 1 to 6 for anti-organ transplant rejection, It is characterized in that, comprising the following steps:
The first step carries the liposome preparation in carry site
Phosphatidyl choline 10mg~100mg, cholesterol 1mg~10mg, 0.1~1mg gangliosides are proportionally weighed respectively GM2, room temperature concussion dissolution overnight in chloroform/methanol solution, drains organic solvent and is allowed to form uniform liposome membrane, fill dry Pathogenic dryness body makes residual organic solvents sufficiently volatilize；The PBS solution that pH=6.5 is added, which is shaken to film, sufficiently to be dissolved, ultrasound to lipid Liquid solution is separated into uniform and stable transparency liquid, obtains artificial liposome；
Second step, single-chain antibody-Streptavidin expressing fusion protein purifying of Ganglioside GM2
The GM2 single-chain antibody of Streptavidin coupling presses VL- (G4S)3-VH-G3The mode of S-SA-GFP synthesizes overall length, protein Expression use ExpiCHO system, after the single-chain antibody of GM2-Streptavidin fusion protein is cloned into pcDNA3.4 carrier, Transfection CHO cell, shaken cultivation collect cell suspension；It is diluted after centrifugation using isometric PBS；The GM2 of Streptavidin coupling Single-chain antibody is purified using iminobiotin sepharose 4B；
Third step, the expression and purification of carry element
Carry element includes Qa-1 extracellular fragment, PD-L1 extracellular fragment and HLA-E extracellular fragment, and 3 carry elements are respectively with Qa1-G4S- FLAG, that is, Seq No.5, PDL1-G4S-FLAG, that is, Seq No.6 and HLAE-G4The form of S-FLAG, that is, Seq No.7 fusion protein It is expressed, after three above fusion protein is cloned into pcDNA3.4 carrier, transfection CHO cell, shaken cultivation collects cell Suspension is diluted after centrifugation using isometric PBS, and carry element connects FLAG label and human IgG1 Fc sections；All albumen use OptiCHOTMThe expression of Express system secretion, carry element carry out first round purifying using Protein A column, complete after purification Enzyme is stayed overnight by 0.2U enterokinase/25 DEG C of 1mg total protein using the enterokinase excision end Fc with pH value to 8.0 using in Tris-HCl It cuts, digestion products carry out secondary affinity purification using FLAG affinity column；Purified product is neutralized to pH=7.5 using Tris-HCl, Use 35kDa super filter tube protein concentrate；
4th step, the biotinylation of carry element
After quantitative to the carry element of third step preparation, by carry element peptide fragment and Sulfo-NHS-Biotin 1:3 in molar ratio ~1:5 is reacted at room temperature in pH=8.5 PBS, and unbonded Sulfo-NHS-Biotin, ultrafiltration are removed using 10kDa bag filter The carry element of concentrated biological element obtains biotinylated carry element；
5th step, liposome medicament load
The GM2 single-chain antibody of the Streptavidin coupling of second step preparation, concentration are added in the artificial liposome of first step preparation For 0.1~1ng/ μ L, 4 DEG C are mixed by inversion overnight；
After the biotinylated Qa-1 or HLA-E of third step preparation are mixed with PD-L1 equimolar, by rubbing for 100:1~200:1 , than mixing with above-mentioned liposome, room temperature is mixed by inversion for you, and dialysis removes unbonded albumen, completes liposome preparation.
8. the preparation method of the liposome according to claim 7 for anti-organ transplant rejection, it is characterised in that: Further package immunomodulator, promotion revascularization drug in the first step.
9. a kind of liposome as claimed in any one of claims 1 to 6 for anti-organ transplant rejection is used to prepare inhibition Application in activated immune cell drug.
10. a kind of liposome as claimed in any one of claims 1 to 6 for anti-organ transplant rejection is used to prepare inhibition Application or the liposome for anti-organ transplant rejection in lymphocyte activation drug are used to prepare pancreas islet shifting Plant the application in postoperative object.
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