CN110124056B - Liposome for resisting organ transplant rejection, preparation method and application - Google Patents

Liposome for resisting organ transplant rejection, preparation method and application Download PDF

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CN110124056B
CN110124056B CN201910406036.2A CN201910406036A CN110124056B CN 110124056 B CN110124056 B CN 110124056B CN 201910406036 A CN201910406036 A CN 201910406036A CN 110124056 B CN110124056 B CN 110124056B
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CN110124056A (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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/51—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
    • 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
    • A61K47/51—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

Abstract

The invention discloses a liposome for resisting organ transplant rejection, which is prepared from the following raw materials: the streptavidin-coupled GM2 single-chain antibody is loaded in the artificial liposome, the concentration of the streptavidin-coupled GM2 single-chain antibody in the artificial liposome is 0.1-1 ng/mu L, the molar ratio of biotinylated Qa-1 or HLA-E to biotinylated PD-L1 is equal, and the molar ratio of the total amount of biotinylated Qa-1 or HLA-E and biotinylated PD-L1 to the streptavidin-coupled GM2 single-chain antibody loaded artificial liposome is 100: 1-200: 1. The liposome for resisting organ transplantation rejection provided by the invention can play a role in resisting rejection in vivo, is a novel liposome medicament and is used for resisting rejection after various organ transplants.

Description

Liposome for resisting organ transplant rejection, preparation method and application
Technical Field
The invention belongs to the technical field of immunotherapy and liposome drug-targeted therapy, and particularly relates to a liposome loaded with an immune checkpoint NKG2A/PD1 ligand fragment and used for resisting organ transplant rejection, a preparation method and application thereof.
Background
Organ transplantation has become the only effective treatment for the end-stage diseases of various organs, and many patients have prolonged life through organ transplantation. In recent years, clinical application of FK506 and rapamycin novel immunosuppressant reduces incidence rate of transplant rejection, but still has two problems: first, immunosuppressants do not completely solve the problem of rejection, suggesting the limitations of conventional approaches to immunosuppression by inhibiting IL-2 gene expression; secondly, the long-term application of the immunosuppressant has a plurality of adverse reactions, including drug toxicity, infection and new tumors caused by over-inhibition of body immunity, and the like. Therefore, by exploring immune cell activation and regulation mechanisms thereof, regulation molecules existing in the body are discovered and applied to treatment of rejection reaction resistance, and the method is an effective way for further improving the rejection resistance effect after transplantation.
The immune function of human body is activated when being stimulated, but the activation is usually limited in a controllable range, and the body injury caused by over-activation can not happen, and the inherent important mechanism is the regulation and control of 'immune checkpoint molecules'. The molecules are similar to the brake system of an automobile, and can brake in time when the immune system is activated, so that the activation of the immune system is kept within a normal range. Over the past decade, new costimulatory molecules have been discovered and demonstrated to play a key role in maintaining immune balance, i.e., immune response to pathogen antigens and immune tolerance to self-antigens or allograft antigens. Currently, methods for activating immune cells for tumor therapy by antagonizing immune checkpoint signaling pathways have been applied clinically and have achieved good response rates. However, there is no study on whether or not immune cells can be inhibited from being activated after organ transplantation by activating an immune checkpoint signaling pathway, thereby exerting an anti-rejection response.
NKG2A and PD1 are important negative regulatory molecules in immune cells, and their ligands are non-classical MHC class I molecule HLA-E (mouse homolog is Qa-1) and PDL1, respectively. The NKG2A/CD94 dimer can interact with HLA-E (Qa-1), activate SHP molecules through an intracellular ITIM motif, further inhibit transcription factors such as Syk and Vav1 and inhibit killing activity of NK cells and CD8+ T cells. PD-L1 can be combined with PD1 on the surface of an activated T cell to promote SHP-1 phosphorylation, inhibit the activation of downstream Syk, block TCR signal transfer and enable the T cell to secrete cytokines, and have impaired proliferation and killing capacity. In tumor immunity, by blocking NKG2A or PD1 signals, the growth and metastasis of tumors can be effectively inhibited, the killing effect of lymphocytes in a tumor immune microenvironment is increased, and a better response rate is obtained in clinical experiments. However, it has not been reported whether or not the rejection reaction can be inhibited by activating NKG2A and PD1 signals. The important reasons are that the ligand extracellular domain proteins of NKG2A and PD1 have small molecular weight and extremely fast in vivo metabolism, and the effective blood concentration is difficult to maintain in the local part of the graft by a direct injection method, so that the direct application of the protein in vivo experiments and clinical researches is limited.
The liposome is a double-layer membrane lipid vesicle formed by self-assembly of amphiphilic lipid molecules in a water phase, and the liposome can embed specific substances (such as drugs and gene fragments) in the liposome to form a specific transportation system and can also carry specific proteins on the surface of the liposome. Several recent studies have achieved effective treatment in animal models by loading the liposome surface with biologically active molecules. Such as: toita et al, which have IL-10 molecules on the liposome surface, are used for treating obese mice and controlling inflammation in vivo; francesca et al successfully constructed an artificial antigen presenting cell (aAPC) that activated T cells by loading MHC-peptide complexes and adhesion molecules on the surface of liposomes; razazan et al conjugated the nanobody to the surface of liposome for specific delivery of tumor antigen. The research suggests that the NKG2A and PD1 ligand peptide fragments can be carried on the surface of liposome to prolong the half-life period in vivo, interact with immune cells for a long time, activate NKG2A and PD1 downstream signals, and is expected to overcome the bottleneck of the current immune checkpoint signaling pathway molecules in the research of anti-transplant rejection application.
Disclosure of Invention
The invention aims to provide a liposome for resisting organ transplantation rejection, which is used for providing a targeted liposome capable of activating an immune checkpoint signal by loading immune checkpoint molecules NKG2A and PD1 ligand peptide fragments onto the surface of an artificial liposome and can be applied to the treatment of rejection after organ transplantation.
The second purpose of the invention is to provide a preparation method of the liposome for resisting the rejection reaction of organ transplantation.
The third purpose of the invention is to provide the application of the liposome for resisting the rejection reaction of organ transplantation.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a liposome for resisting organ transplant rejection reaction, which is prepared from the following raw materials:
the streptavidin coupled GM2 single-chain antibody is loaded in the artificial liposome, the concentration of the streptavidin coupled GM2 single-chain antibody loaded in the artificial liposome is 0.1-1 ng/muL,
the molar ratio of biotinylated Qa-1 or HLA-E to biotinylated PD-L1 is equal,
the molar ratio of the total amount of biotinylated Qa-1 and biotinylated PD-L1 to the streptavidin-coupled GM2 single-chain antibody loaded artificial liposome is 100: 1-200: 1;
or the molar ratio of the total amount of biotinylated HLA-E and biotinylated PD-L1 to the streptavidin-coupled GM2 single-chain antibody loaded artificial liposome is 100: 1-200: 1.
The artificial liposome is prepared from the following raw materials:
10 mg-100 mg of phosphatidylcholine, 1 mg-10 mg of cholesterol, 0.1-1 mg of ganglioside GM2, and PBS with pH of 6.5; the artificial liposome has a concentration of 105～106one/mL.
The streptavidin-coupled GM2 single-chain antibody is expressed as VL- (G)4S)3-VH-G3Synthesizing full length by the S-SA-GFP mode;
the molar ratio of Qa-1, PD-L1, HLA-E and Sulfo-NHS-Biotin in the biotinylated Qa-1, biotinylated PD-L1 and biotinylated HLA-E is 1: 3-1: 5.
The amino acid sequence of the streptavidin-conjugated GM2 single-chain antibody is shown in SeqNo. 1.
The amino acid sequences of Qa-1, PD-L1 and HLA-E are respectively shown as Seq No.2, Seq No.3 and Seq No.4, and are respectively Qa1-G4S-FLAG, Seq No.5, PDL1-G4S-FLAG, Seq No.6, HLAE-G4S-FLAG, a form of SeqNo.7 fusion protein, was expressed.
In a second aspect of the present invention, there is provided a method for preparing a liposome for anti-organ transplant rejection, comprising the steps of:
first, preparation of liposome carrying a mounting site
Preparing artificial liposome: the artificial liposome is prepared by adopting an ultrasonic film method
Weighing 10-100 mg of phosphatidylcholine, 1-10 mg of cholesterol and 0.1-1 mg of ganglioside GM2 according to the proportion, shaking and dissolving the phosphatidylcholine, the cholesterol and the ganglioside GM2 in chloroform/methanol solution overnight at room temperature, pumping out the organic solvent by using a rotary evaporator at 37 ℃ to form a uniform liposome membrane on the wall of the eggplant-shaped flask, and filling dry gas into the flask for 5min to fully volatilize the residual organic solvent; adding 5mL of PBS (0.5M) with the pH value of 6.5, shaking until the membrane is fully dissolved, and carrying out ultrasonic treatment for 1-2 min by using an ultrasonic instrument until the liposome solution is dispersed into uniform and stable transparent liquid to obtain the artificial liposome; then adding 100-800 mg of DSPE-PEG into every 20 mu L of artificial liposome, and carrying out modification in water bath at 50 ℃ for 10min to enhance the stability of the artificial liposome;
second, the single chain antibody-streptavidin fusion protein of ganglioside GM2 is expressed and purified
Streptavidin-conjugated GM2 single-chain antibody as VL- (G)4S)3-VH-G3Synthesizing full length by S-SA-GFP, cloning the single chain antibody-streptavidin fusion protein of GM2 to pcDNA3.4 vector by ExpicHO system for protein expression, and transfecting CHO cell; 37 ℃ and 8% CO2Shaking and culturing for 8 days under the environment, collecting cell suspension, centrifuging at 5000-6000 rpm for 10-15min to remove cells, diluting with 2 × PBS (with the same volume), and purifying streptavidin-coupled GM2 single-chain antibody with iminobiotin agarose beads, wherein the scFv (VL + VH) sequence is derived from the patent US 5830470;
thirdly, expression and purification of the mounting element
The mounting elements comprise a Qa-1 extracellular segment, a PD-L1 extracellular segment and an HLA-E extracellular segment, and the 3 mounting elements are respectively expressed as Qa1-G4S-FLAG, Seq No.5, PDL1-G4S-FLAG, Seq No.6 and HLAE-G4Expressing the S-FLAG fusion protein of Seq No.7, cloning the three fusion proteins to pcDNA3.4 vector, transfecting CHO cell at 37 deg.C and 8% CO2Shaking culture under the environment 8Collecting cell suspension, centrifuging at 5000-6000 rpm for 10-15min to remove cells, diluting with equal volume of 2 × PBS, connecting FLAG tag and human IgG1Fc segment with mounting element, wherein all proteins are OptiCHOTMExpress system secretion expression, using a ProteinA column to carry out first round purification by a mounting element, using Tris-HCl to neutralize the pH value to 8.0 after purification is finished, using enterokinase to cut off an Fc end, carrying out overnight enzyme digestion at 25 ℃ according to 0.2U enterokinase/1 mg total protein, and using a FLAG affinity column to carry out secondary affinity purification on the enzyme digestion product; the purified product was neutralized to pH 7.5 using Tris-HCl and the protein was concentrated using a 35kDa ultrafiltration tube;
fourth, biotinylation of the mounted element
Biotinylation of the mounting element Using Sulfo-NHS-Biotin
After quantifying the mounted element prepared in the third step, reacting the peptide fragment of the mounted element with Sulfo-NHS-Biotin at a molar ratio of 1: 3-1: 5 in PBS (pH 8.5) at room temperature for 30min, removing unbound Sulfo-NHS-Biotin by using a 10kDa dialysis bag, and concentrating the biotinylated mounted element by ultrafiltration to obtain a biotinylated mounted element;
fifthly, loading liposome drug
Adding the streptavidin coupled GM2 single-chain antibody prepared in the second step into the artificial liposome prepared in the first step, wherein the concentration is 0.1-1 ng/mu L, and reversing and uniformly mixing the mixture at 4 ℃ overnight;
and (3) mixing the biotinylated Qa-1 or HLA-E prepared in the third step with PD-L1 in an equal molar ratio, mixing the mixture with the liposome according to the molar ratio of 100: 1-200: 1, reversing and uniformly mixing at room temperature, and dialyzing to remove unbound protein to finish the preparation of the liposome.
The preparation method of the invention preferably adopts a film method to prepare the liposome, and in addition, the liposome prepared by other schemes such as a reverse evaporation method, a gradient reverse loading method, double emulsification and the like can also be used in the system.
In addition, besides loading peptide fragments on the surface of liposome membrane for inhibiting immune cell activation, the liposome can also encapsulate immunomodulators in the preparation process to further improve the immunosuppressive effect or play a specific role in a specific organ transplantation type. For example, the immunosuppressive agent cyclosporin A, FK506, rapamycin, mycophenolate mofetil or a mixture of several drugs is encapsulated in the liposome preparation process, so that the drug can be slowly released, and the concentration of the immunosuppressive agent can be smoothly increased after transplantation.
Further, in addition to loading peptide fragments on the surface of liposome membrane for inhibiting immune cell activation, the liposome itself can also encapsulate drugs to play other roles in specific transplantation types during the preparation process. For example, the liposome used for islet transplantation can be encapsulated with angiogenesis promoting drugs, such as VEGF, etc., so as to promote the angiogenesis of the transplant.
The third aspect of the invention provides an application of liposome for resisting organ transplant rejection in preparing a medicament for inhibiting immune cell activation.
The fourth aspect of the invention provides an application of liposome for resisting organ transplant rejection in preparing a medicament for inhibiting lymphocyte activation.
The fifth aspect of the invention provides an application of liposome for resisting organ transplant rejection reaction in preparing a post-islet transplantation medicament.
The liposome for resisting organ transplant rejection reaction prepared by the invention can effectively activate double signal paths of NKG2A and PD1 in vivo and play a role in resisting rejection after transplantation.
The liposome for resisting organ transplant rejection reaction prepared by the invention detects the influence of Qa-1/PD-L1 liposome on related signal paths after MLR, and the result shows that the liposome can remarkably promote the activation of SHP1 and SHP2 molecules, thereby inhibiting the activation of Syk protein tyrosine kinase and downstream ERK1/2 thereof.
Western-blot detection on the activation conditions of SHP1/2, Syk and ERK1/2 shows that the Qa-1 molecule/PD-L1 liposome can activate liver lymphocyte SHP1/2, so that activation of Syk and ERK1/2 is inhibited.
Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:
the liposome prepared by the method for preparing the liposome for resisting the organ transplantation rejection can play a role in resisting rejection in vivo, is a novel liposome medicament, and is used for resisting rejection after various organ transplants.
The invention provides a preparation method of liposome for resisting organ transplant rejection, the prepared liposome is a liposome with targeting aiming at immune checkpoint molecule, and has the following characteristics that ① ganglioside G is doped in the preparation process of liposomeM2The invention can effectively activate NKG2A and PD1 dual-signal channels in vivo and play the role of anti-rejection after transplantation.
The liposome for resisting organ transplant rejection reaction prepared by the invention detects the influence of Qa-1/PD-L1 liposome on related signal paths after MLR, and the result shows that the liposome can remarkably promote the activation of SHP1 and SHP2 molecules, thereby inhibiting the activation of Syk protein tyrosine kinase and downstream ERK1/2 thereof.
Drawings
FIG. 1 is a schematic diagram of a process for preparing an artificial liposome according to an embodiment of the present invention.
FIG. 2 is a diagram showing the mode of carrying the various elements of the Qa-1 molecule/PD-L1 artificial liposome in the example of the present invention.
FIG. 3 shows the efficiency of the detection of the carrier by using red fluorescent protein as the quality control molecule (Scale bar: 10 μm) in the examples of the present invention.
FIG. 4 is a graph of the effect of Qa-1 molecule/PD-L1 liposomes on lymphocyte proliferation and activation, wherein: a, B shows the effect of Qa-1 molecule/PD-L1 liposome on the proliferation of lymphocytes in MLR measured by CFSE method, which is the result of 3 independent experiments (n-3); C. d is the proportion of CD107a positive cells detected by FACS (n-3); e is IFN-gamma expression level (n-3) in ELISA detection culture supernatant; f is the activation conditions of SHP1/2, Syk and ERK1/2 after detecting MLR by Western-blot at 1 hour and 2 hours; p <0.01, p < 0.001.
FIG. 5 shows the effect of Qa-1 molecule/PD-L1 liposomes on lymphocyte activation at the site of transplantation; wherein: a is the proportion of CD107a positive cells in hepatic lymphocytes detected by FACS (n-3); b is the activation of SHP1/2, Syk and ERK1/2 detected by Western blot (n is 3).
FIG. 6 is the protective effect of Qa-1 molecule/PD-L1 liposome on the graft in the same mouse islet transplantation model, wherein: a is post-transplant mouse blood glucose change (n-10); b is the mouse C-peptide change after transplantation (n-10); c is the survival of islet grafts in the liver as detected by immunofluorescence 14 days after transplantation (Scale bar: 50 μm).
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The phosphatidylcholine (cargo number V900485) used in the construction process of the liposome is purchased from Sigma, and the purity is more than or equal to 99 percent; cholesterol (cat # C8667) was purchased from Sigma and has a purity of 99% or more; ganglioside GM2 (cat # G4651) was purchased from Sigma with a purity of 95%. Expression of proteins (including single chain antibody-SA fusion protein and three mounting elements) the ExpicHO (A29133) eukaryotic protein expression system from Gibco was used.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are fully described in the following documents: for example, Sambrook molecular cloning, A laboratory Manual, 2 nd edition (1989); DNA cloning, volumes I and II (D.N. Glover editor 1985); oligonucleotide synthesis (edited by m.j. gait, 1984); nucleic acid hybridization (edited by b.d. hames and s.j. higgins, 1984); protein purification: principles and practices, 2 nd edition (Springer-Verlag, n.y.), and experimental immunology handbook, volumes I-IV (d.c. well and c.c. blackwell, editors 1986). Alternatively, the procedure may be followed according to the instructions provided by the reagent manufacturer.
Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1: design and preparation of Qa-1/PD-L1 liposome
The experimental method comprises the following steps:
first, preparation of liposome carrying a mounting site
Preparing artificial liposome: the artificial liposome is prepared by adopting an ultrasonic film method
Weighing phosphatidylcholine 20mg, cholesterol 5mg and ganglioside GM2 0.25mg according to the proportion, dissolving in chloroform/methanol (volume ratio 5: 1)15mL solution overnight by shaking at room temperature, pumping out the organic solvent at 37 ℃ by using a rotary evaporator to form a uniform liposome membrane on the wall of the eggplant-shaped flask, and filling dry gas in the flask for 5min to fully volatilize the residual organic solvent. Adding 5mL of PBS (0.5M) with pH of 6.5, shaking until the membrane is fully dissolved, performing ultrasonic treatment for 1-2 min by using an ultrasonic instrument until the liposome solution is dispersed into uniform and stable transparent liquid to obtain the artificial liposome, wherein the concentration of the artificial liposome is 105～106one/mL. Then adding DSPE-PEG200mg into each 20 μ L of artificial liposome, and performing modification in 50 deg.C water bath for 10min to enhance its stability.
If immune regulators (glucocorticoid, FK506, cyclosporine and the like) and angiogenesis promoting drugs (VEGF protein and derived peptide fragments, hepatocyte growth factor, platelet proliferation factor and the like) need to be encapsulated in the liposome, the operation is as follows: weighing phosphatidylcholine 20mg, cholesterol 5mg and ganglioside GM2 0.25mg according to the proportion, dissolving in chloroform/methanol (volume ratio 5: 1)15mL solution overnight by shaking at room temperature, pumping out the organic solvent at 37 ℃ by using a rotary evaporator to form a uniform liposome membrane on the wall of the eggplant-shaped flask, and filling dry gas in the flask for 5min to fully volatilize the residual organic solvent. 2mL of PBS (0.5M) at pH 6.5 was used to dissolve drugs (immunosuppressants typified by glucocorticoids, FK506, cyclosporin, etc., or blood-promoting agents typified by VEGF protein and derived peptide fragments, hepatocyte growth factor, platelet growth factor, etc.)Tube regeneration medicine) to enable the medicine concentration to reach 100-1000 ng/mL, detecting the concentration of a hydration solution by using a pH meter, readjusting the liquid to be pH 6.5 by using a citrate buffer solution with pH being 10 × and 3.0 or a Tris buffer solution with pH being 10 × and 8.5, adding the liquid into an eggplant-type flask, shaking until a membrane is fully dissolved, carrying out ultrasonic treatment for 1-2 min by using an ultrasonic instrument until a liposome solution is dispersed into a uniform and stable transparent liquid to obtain an artificial liposome, wherein the concentration of the artificial liposome is 105～106one/mL. Then adding DSPE-PEG200mg into each 20 μ L of artificial liposome, and performing modification in 50 deg.C water bath for 10min to enhance its stability.
Second, the single chain antibody-streptavidin fusion protein of ganglioside GM2 is expressed and purified
Streptavidin-conjugated GM2 single-chain antibody as VL- (G)4S)3-VH-G3S-SA-GFP synthesis of full length, the amino acid sequence is shown in Seq No.1, expression of the protein using ExpicHO (Gibco, A29133) system, GM2 single chain antibody-SA fusion protein was cloned into pcDNA3.4 vector, transfection of CHO cells. 37 ℃ and 8% CO2After shaking culture for 8 days under the environment, collecting cell suspension, centrifuging at 5000-6000 rpm for 10-15min to remove cells, diluting with equal volume of 2 × PBS, and purifying the streptavidin-coupled GM2 single-chain antibody by using iminobiotin agarose beads (PurKine, KTP20306), wherein the sequence of the single-chain antibody scFv (VL + VH) is derived from the patent US 5830470.
Thirdly, expression and purification of the mounting element
The mounting elements comprise a Qa-1 extracellular segment, a PD-L1 extracellular segment and an HLA-E extracellular segment, the amino acid sequences of the mounting elements are respectively shown as Seq No.2, Seq No.3 and Seq No.4, and 3 mounting elements are respectively shown as Qa1-G4S-FLAG(Seq No.5)、PDL1-G4S-FLAG (Seq No.6) and HLAE-G4The S-FLAG (Seq No.7) fusion protein is expressed, and after the three fusion proteins are cloned to pcDNA3.4 vector, CHO cell is transfected. 37 ℃ and 8% CO2After shaking culture for 8 days under the environment, collecting cell suspension, centrifuging at 5000-6000 rpm for 10-15min to remove cells, diluting with equal volume of 2 × PBS, connecting the FLAG tag with human IgG1Fc segment by a mounting element, and using OptiCHO for all proteinsTMExpress system secretory expression. The carrier element is subjected to first round purification by a ProteinA column (Tiandi human and SA012K), the pH value is neutralized to 8.0 by Tris-HCl after the purification is finished, an Fc end is cut by enterokinase (Shanghai ya Xin, REK08), the enzyme is cut overnight at 25 ℃ according to 0.2U enterokinase/1 mg total protein, and the enzyme cut product is subjected to second affinity purification by a FLAG affinity column (Tiandi human and SA 042001); the purified product was neutralized to pH 7.5 using Tris-HCl and the protein was concentrated using a 35kDa ultrafiltration tube.
Fourth, biotinylation of the mounted element
Biotinylation of the mounting element was performed using Sulfo-NHS-Biotin (Shanghai Biotech, C100213).
Sulfo-NHS-Biotin can efficiently make primary amino (-NH) in alkaline buffer2) A stable amide bond is formed. The side chain of lysine (K) residue in protein and the N-terminal of polypeptide can react with Sulfo-NHS-Biotin to form stable Biotin connection. After quantifying the mounted element prepared in the third step, reacting the mounted element peptide segment with Sulfo-NHS-Biotin at a molar ratio of 1: 3-1: 5 in PBS at room temperature for 30min at the pH of 8.5. Unbound sulfos-NHS-Biotin was removed using a 10kDa dialysis bag and the biotinylated load element was concentrated by ultrafiltration to obtain biotinylated load elements.
Fifthly, loading liposome drug
100ng of the streptavidin-conjugated GM2 single-chain antibody prepared in the second step was added to 200. mu.L of the artificial liposome prepared in the first step, the mixture was inverted and mixed overnight at 4 ℃, and the liposome density was counted by a fluorescence microscope.
And (3) mixing the biotinylated Qa-1 (or HLA-E) prepared in the third step with PD-L1 in an equal molar ratio, mixing the mixture with the liposome according to a molar ratio of 100: 1-200: 1, reversing and uniformly mixing the mixture at room temperature for 30min, and dialyzing by using a 35KD dialysis bag to remove unbound protein to complete preparation of the liposome. The experimental results are as follows:
FIG. 1 is a schematic diagram of a process for preparing an artificial liposome according to an embodiment of the present invention. FIG. 2 is a diagram showing the mode of carrying the various elements of the Qa-1 molecule/PD-L1 artificial liposome in the example of the present invention. Qa-1/PD-L1 was loaded onto the artificial liposomes using the protocol shown in figure 2: the anti-ganglioside GM2 single-chain antibody fused with Streptavidin (Streptavidin) is expressed to combine with GM2 on a membrane to form a hanging scaffold, and then biotin-modified Qa-1 and PD-L1 extracellular segments are incubated with the hanging scaffold to form the Qa-1/PD-L1 artificial liposome.
Example 2
Detection of liposome mounting efficiency and specificity based on GM2 single-chain antibody Streptavidin (SA) fusion protein
The experimental method comprises the following steps: the liposome mounting efficiency assay was performed using biotinylated red fluorescent protein (dsRed).
The amino acid sequence of the red fluorescent protein (dsRed) is shown as Seq No.8, and the amino acid sequence of the red fluorescent protein (dsRed) is Qa1-G4Expression of the S-FLAG (Seq No.9) fusion protein: general formula Qa1-G4The S-FLAG (Seq No.9) fusion protein was cloned into pcDNA3.4 vector and transfected into CHO cells. 37 ℃ and 8% CO2After shaking culture for 8 days under the environment, collecting cell suspension, centrifuging at 5000-6000 rpm for 10-15min to remove cells, diluting with equal volume of 2 × PBS, purifying the product with a FLAG affinity column, neutralizing the purified product with 2 × PBS to pH 7.5, concentrating protein with a 10kDa ultrafiltration tube, reacting the purified dsRed with Sulfo-NHS-Biotin, carrying out liposome loading according to the fifth step of the method in example 1, detecting the loading efficiency by fluorescence, carrying out co-incubation on the non-biotinylated dsRed and the liposome, dialyzing to remove the non-bound dsRed, and determining the loading specificity.
The experimental results are as follows: the mounting efficiency of the system is determined by using scFv avidin fusion protein with GFP label and biotinylated dsRed, and FIG. 3 shows that red fluorescent protein is used as quality control molecule to detect the mounting efficiency in the embodiment of the invention. The GFP green fluorescence shows that the diameter distribution of the liposome after the scFv-SA fusion protein is combined is relatively uniform, and the size of the liposome is about 50-500nm (the upper left graph and the upper right graph in FIG. 3, and the Scale bar is 10 μm). Red fluorescence shows the apparent efficiency of loading of biotinylated dsRed on liposomes, and the results show that the system has better efficiency of loading, and the biotinylated dsRed is successfully loaded on all liposome surfaces (figure 3, lower left panel); meanwhile, the system has good mounting specificity, and non-biotinylated dsRed can not be mounted on the surface of the liposome (the lower right picture of the figure 3.)
Example 3
Effect of Qa-1/PD-L1 Artificial liposomes prepared in example 1 on Mixed lymphocyte reaction
Experimental method C57 mice spleen cells are adjusted to 1 × 10 after red blood breaking6cells/mL, 2mmol CFSE solution, 37 degrees C were incubated for 15min, 10 volume ice-cold PBS washing 2 times, 500 u L/hole inoculated in 24-well plate Balb/c mice spleen broken red, added 10ug/mL mitomycin C complete medium 37 degrees C treatment for 2 hours, 10 volume PBS washing 6 times, adjusted to cell concentration to 1 × 106cells/mL, 500. mu.L/well were mixed with C57 cells. After 3 days of continuous culture, the proliferation of lymphocytes, the proportion of CD107a positive cells and the amount of IFN-gamma in the supernatant were measured by FACS. Westernblot detects the activation of SHP1, SHP2, Syk and ERK 1/2.
The experimental results are as follows: donor mouse spleen lymphocytes were removed and treated with mitomycin c to prevent further proliferation. And (3) mixing the two components: 1 mixed culture for 3 days, and detecting lymphocyte proliferation by FACS. CFSE is a fluorescent dye that labels living cells, penetrates the cell membrane, covalently binds to intracellular proteins, and fluoresces green. In the process of cell division and proliferation, the fluorescence intensity can be weakened along with cell division, and the proportion of the proliferated lymphocytes can be detected by the characteristic. The results show that Qa-1/PD-L1 liposome can obviously inhibit lymphocyte proliferation (A, B in figure 4). CD107a is an important marker for activation of NK cells and CD8+ T cells, and is closely related to cytotoxic activity. The proportion of CD107a positive cells after MLR is detected, and the Qa-1/PD-L1 liposome is doped to obviously reduce the proportion of CD107a positive cells (C, D in figure 4), which indicates that the liposome can obviously inhibit the cytotoxic activity of lymphocytes. IFN-gamma secretion is another important marker reflecting NK and T cell activation, the expression condition of IFN-gamma in culture supernatant is detected, and the result shows that Qa-1/PD-L1 liposome can obviously inhibit IFN-gamma secretion, and further indicates that the liposome can inhibit lymphocyte activation (E in figure 4). In order to research the related mechanism, the influence of Qa-1/PD-L1 liposome on the related signal path at 1 hour and 2 hours after MLR is detected, and the result shows that the liposome can remarkably promote the activation of SHP1 and SHP2 molecules, thereby inhibiting the activation of Syk protein tyrosine kinase and downstream ERK1/2 thereof (F in figure 4).
As shown in fig. 4, fig. 4 is the effect of Qa-1 molecule/PD-L1 liposome on lymphocyte proliferation and activation, wherein: a, B shows the effect of Qa-1 molecule/PD-L1 liposome on the proliferation of lymphocytes in MLR measured by CFSE method, which is the result of 3 independent experiments (n-3); C. d is the proportion of CD107a positive cells detected by FACS (n-3); e is IFN-gamma expression level (n-3) in ELISA detection culture supernatant; f is the activation conditions of SHP1/2, Syk and ERK1/2 after detecting MLR by Western-blot at 1 hour and 2 hours; p <0.01, p < 0.001.
Example 4
Effect of Qa-1 molecule/PD-L1 liposomes prepared in example 1 on in vivo rejection inhibition
The experimental method comprises the following steps:
islet isolation and transplantation: liberase TL is diluted to a working concentration of 200 mug/mL by using Perfusion solution, after Balb/c mouse pancreas is fully perfused along the common bile duct, the opening of the pancreatic duct is closed by using a vascular clamp, and the whole pancreas is extracted. Digesting in water bath at 37 deg.C for 10-15min, and mixing gently with Pasteur dropper every 2 min. Immediately after digestion was complete, digestion was stopped by addition of 10% total volume of FBS, and after purification of islets using Optiprep density gradient, the enriched islets were aspirated under microscope using a wide mouth tip and resuspended in CMRL1066 liquid. Islets isolated from 2-3 donor mice were transplanted into 1 recipient mouse. C57 recipient mice were anesthetized with 2mg/kg chloral hydrate by intraperitoneal administration, the portal vein was exposed by opening the abdomen and an indwelling needle was inserted to establish a graft channel, islets of langerhans (100U heparin sodium was added) resuspended in 250 μ L CMRL1066 were slowly injected into the portal vein, and the needle hole was closed with tissue glue and filled with gelatin sponge at the end of injection.
The administration mode comprises the following steps: the Qa-1 molecule/PD-L1 liposome prepared in example 1 was first injected into the portal vein along with the graft; then 10. mu.L of Qa-1 molecule/PD-L1 liposomes were diluted to 100. mu.L every 3 days and administered into the tail vein. Controls were administered to groups of empty liposomes, 10 mice per group.
Activation of intrahepatic lymphocytes: killing the mice 48 hours after the islet transplantation, taking the livers of the mice, grinding the livers by using a 100-mesh steel mesh, and then suspending the livers in 5mL of physiological saline; preparing discontinuous Optiprep density gradient liquid (the density of the upper layer is 24mL and is 1.056g/mL, the density of the lower layer is 12mL and is 1.090g/mL), slowly adding the cell suspension to the upper layer of the density liquid, centrifuging for 20min at 500g, and sucking the middle leucocyte layer, namely the purified hepatic lymphocyte. FACS measures the proportion of CD107a positive cells, and Westernblot measures the activation of SHP1, SHP2, Syk and ERK 1/2.
The experimental results are as follows: mice were sacrificed 48 hours after islet transplantation, and mouse livers were removed and hepatic lymphocytes were isolated. FACS measures the proportion of CD107a positive cells in lymphocytes, and the results show that the proportion of intrahepatic CD107a positive cells is significantly down-regulated (20.93 ± 1.94% vs.13.40 ± 1.89%) after Qa-1 molecule/PD-L1 liposome treatment compared to the empty liposome-treated group, suggesting that liposomes can inhibit intrahepatic lymphocyte activation (a in fig. 5). Western-blot detection of activation of SHP1/2, Syk and ERK1/2 shows that Qa-1 molecule/PD-L1 liposome can activate hepatic lymphocyte SHP1/2, thereby inhibiting activation of Syk and ERK1/2 (B in FIG. 5). As shown in FIG. 5, FIG. 5 shows the effect of Qa-1 molecule/PD-L1 liposome on lymphocyte activation at the site of transplantation; wherein: a is the proportion of CD107a positive cells in hepatic lymphocytes detected by FACS (n-3); b is activation of Westernblot detection SHP1/2, Syk and ERK1/2 (n is 3).
Example 5
Study on the Effect of Qa-1 molecule/PD-L1 liposomes prepared in example 1 on in vivo transplantation of allograft
The experimental method comprises the following steps: establishing a diabetes mouse model: after fasting for 24hrs, C57 mice were intraperitoneally injected with 10mg/mL STZ solution at a dose of 100mg/kg, and the blood glucose and C peptide concentration in the tail vein of the mice were measured 7 days after the injection. The blood glucose concentration was > 16.7mmol/L and the C-peptide concentration was less than 100pmol/L, which was considered successful in modeling. If a single injection fails to meet the modeling criteria, the STZ is re-injected at 50% of the first dose until the modeling is successful.
Islet isolation and transplantation: liberase TL is diluted to a working concentration of 200 mug/mL by using Perfusion solution, after Balb/c mouse pancreas is fully perfused along the common bile duct, the opening of the pancreatic duct is closed by using a vascular clamp, and the whole pancreas is extracted. Digesting in water bath at 37 deg.C for 10-15min, and mixing gently with Pasteur dropper every 2 min. Immediately after digestion was complete, digestion was stopped by addition of 10% total volume of FBS, and after purification of islets using Optiprep density gradient, the enriched islets were aspirated under microscope using a wide mouth tip and resuspended in CMRL1066 liquid. Islets isolated from 3 donor mice were transplanted into 1 recipient mouse. C57 recipient mice were anesthetized with 2mg/kg chloral hydrate by intraperitoneal administration, the portal vein was exposed by opening the abdomen and an indwelling needle was inserted to establish a graft channel, islets of langerhans (100U heparin sodium was added) resuspended in 250 μ L CMRL1066 were slowly injected into the portal vein, and the needle hole was closed with tissue glue and filled with gelatin sponge at the end of injection.
The administration mode comprises the following steps: the Qa-1 molecule/PD-L1 liposome prepared in example 1 was first injected into the portal vein along with the graft; then 10. mu.L of liposomes were diluted to 100. mu.L every 3 days and administered into the tail vein. Rapamycin was administered at 300. mu.g/kg day by intragastric administration. Depending on the mode of administration, mice were divided into 4 groups: the group of unloaded liposomes, the group of unloaded liposomes + low dose rapamycin, the group of Qa-1/PD-L1 liposomes and the group of Qa-1/PD-L1 liposomes + low dose rapamycin, each group consisting of 10 mice.
Pancreatic islet function test: and (3) taking 20-30 mu L of blood from the retroorbital venous plexus by using a heparinized capillary glass tube every 24 hours, disinfecting the blood by using chloramphenicol eye drops, and detecting C peptide and blood sugar.
Detecting the survival condition in the liver of the transplant by an immunofluorescence method: mouse livers were fixed with 4% paraformaldehyde, embedded in paraffin and prepared into paraffin sections. The section is dewaxed, antigen repaired, sealed and the like, then insulin (1:400) with fluorescent label and glucagon antibody (1:400) are added, incubated for 1 hour at 4 ℃ in dark place, DAPI (1 mu g/mL) is added after the antibody incubation for 45 min. After staining was complete, the slides were washed, observed under a fluorescent microscope and photographed in a typical field of view.
The experimental results are as follows: after isolation of mouse islets, syngeneic mice were transplanted (Balb/C → C57), and the transplanted mice (10 per group) were treated with empty liposomes, empty liposomes + low dose rapamycin (300. mu.g/kg. day), Qa-1/PD-L1 liposomes and Qa-1/PD-L1 liposomes + low dose rapamycin, respectively, and the blood glucose changes and C-peptide levels of the mice were monitored every 24 hours (A, B in FIG. 6). As shown in fig. 6, fig. 6 is the protective effect of Qa-1 molecule/PD-L1 liposome on the graft in the allogeneic mouse islet transplantation model, wherein: a is post-transplant mouse blood glucose change (n-10); b is the mouse C-peptide change after transplantation (n-10); c is the survival of islet grafts in the liver as detected by immunofluorescence 14 days after transplantation (Scale bar: 50 μm). The results show that the islet function is recovered at the third day after transplantation, and the blood sugar of the four groups of mice is respectively reduced to 9.94 +/-0.65, 10.43 +/-0.89, 9.04 +/-0.75 and 7.95 +/-0.76 mmol/L from 18.51 +/-1.53, 18.29 +/-1.86, 18.16 +/-0.69 and 17.84 +/-0.51 mmol/L before transplantation; c peptide rises from 52.52 + -21.97, 40.85 + -17.99, 68.30 + -21.58, 61.38 + -23.97 pmol/L to 440.99 + -73.57, 347.25 + -104.82, 747.08 + -79.31, 600.40 + -119.96 pmol/L before transplantation; however, the recovery amplitude of the unloaded liposome group C peptide is smaller (440.99 +/-73.57 pmol/L vs.747.08 +/-79.31 pmol/L) compared with that of the Qa-1/PD-L1 liposome group, the islet function is gradually lost after the 7 th day, the C peptide is reduced to 186.94 +/-81.52 pmol/L on the 8 th day, and the blood sugar is increased to 17.62 +/-0.89 mmol/L. Compared with the no-load liposome group, the mice given the low-dose rapamycin treatment group have obviously raised C peptide level, the blood sugar level is maintained in the range of 8-11 mmol/L4-9 days after transplantation, the C peptide is maintained in the range of 403.5-1288.8 pmol/L, but from the 10 th day after transplantation, the C peptide gradually decreases to 223.1-390.6 pmol/L, the blood sugar gradually increases to 13.2-15.8 mmol/L, and although partial pancreatic islet function is remained, the normal blood sugar level is not sufficiently maintained; compared with the two groups, the Qa-1/PD-L1 liposome treatment group and the Qa-1/PD-L1 liposome combined low-dose rapamycin treatment group both achieve better anti-rejection effect, the blood sugar is obviously reduced (9.04 +/-0.75 mmol/L and 7.95 +/-0.76 mmol/L) on the 4 th day after transplantation, the C peptide is stabilized above 1000pmol/L, and the C peptide has no obvious difference with a normal mouse; among them is noteworthy: although Qa-1/PD-L1 liposome was slightly less effective when administered alone than when administered in combination, the therapeutic effect was not significantly different between the two groups, and only on day 14, the blood glucose level was slightly higher in the group administered alone than in the group administered in combination (8.9mmol/L vs.6.92mmol/L, p <0.001), and the C peptide was slightly lower in the group administered in combination (1070.21pmol/Lvs.1461.52pmol/L, p < 0.001). It is noteworthy that all mice exhibited a transient increase in C peptide burst (B in FIG. 6) 12-16 hours after transplantation, when C peptide was mainly derived from islet cell content dead early in the transplantation and was associated with a poor prognosis. Compared with the no-load liposome group, the release amount of C peptide in the early stage of transplantation is obviously reduced after the Qa-1/PD-L1 liposome treatment (2444.59 +/-349.11 pmol/L vs.957.40 +/-241.06 pmol/L, p is less than 0.001); compared with the rapamycin group, the Qa-1/PD-L1 liposome has more remarkable protection effect on the loss of pancreatic islets in the early stage of transplantation (1417.02 +/-401.30 pmol/L vs.957.40 +/-241.06 pmol/L, p is less than 0.01). On 14 days after transplantation, 1 mouse in the empty liposome treatment group and the Qa-1/PD-L1 liposome treatment group was taken, and the survival condition of the islets in the liver was detected by using insulin and glucagon antibodies after the liver was fixed. The results showed that most islet cells in the antrum of the empty liposome-treated group were apoptotic and only a very small number of islet cells survived, whereas the islet graft in the antrum of the Qa-1/PD-L1 liposome-treated group was morphologically intact and survived well (C in fig. 6).
SeqNo.1:
MHFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPYTFGGGTKLEIKRGGGGSGGGGSGGGGSMGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKSKATLTVDKSSSTAYMELHSLTSEDSAVYYCATYGHYYGYMFAYWGQGTLVTVSAGGGGSMRCTIVLGIRAASPIKEALARPAPRPGRLPSIHRSGRRNMQRLEHVLRRVKAGTGAPIDFSGTWKNELGSTMRIEQSGDSVSGTYESAVSENGGATSGQLSGYVDGDLIAFVVHWDQFQAITAWVGQGGPGASSDRINTLWQMTQQVEAGEEWASINAGADIFVKTVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGAAAAAAAAAAEFPGLEKLGSTGSR
Seq No.2:
PHSLRYFTTAVSRPGLGEPRFIIVGYVDDTQFVRFDSDA61ENPRMEPRARWIEQEGPEYWERETWKARDMGRNFRVNLRTLLGYYNQSNDESHTLQWMYG121CDVGPDGRLLRGYCQEAYDGQDYISLNEDLRSWTANDIASQISKHKSEAVDEAHQQRAYLQGPCVEWLHRYLRLGNETL
Seq No.3:
NKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENH
Seq No.4:
SHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETL
Seq No.5:
PHSLRYFTTAVSRPGLGEPRFIIVGYVDDTQFVRFDSDA61ENPRMEPRARWIEQEGPEYWERETWKARDMGRNFRVNLRTLLGYYNQSNDESHTLQWMYG121CDVGPDGRLLRGYCQEAYDGQDYISLNEDLRSWTANDIASQISKHKSEAVDEAHQQRAYLQGPCVEWLHRYLRLGNETLGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq No.6:
NKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq No.7:
SHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Seq No.8:
MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHLFLSEQ
Seq No.9:
MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHLFLSEQDYKGGGGSDYKDDDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> second military medical university of China people liberation army
<120> liposome for resisting organ transplant rejection, preparation method and application
<130> description, 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
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Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser
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Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser
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Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro
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Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
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Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg
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Ser Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
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Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
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Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
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Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
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Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu
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Glu Trp Ile Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Tyr Asn
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Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
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Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val
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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
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Arg Val Lys Ala Gly Thr Gly Ala Pro Ile Asp Phe Ser Gly Thr Trp
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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
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Ser Gly Gln Leu Ser Gly Tyr Val Asp Gly Asp Leu Ile Ala Phe Val
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Val His Trp Asp Gln Phe Gln Ala Ile Thr Ala Trp Val Gly Gln Gly
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Gly Pro Gly Ala Ser Ser Asp Arg Ile Asn Thr Leu Trp Gln Met Thr
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Gln Gln Val Glu Ala Gly Glu Glu Trp Ala Ser Ile Asn Ala Gly Ala
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Asp Ile Phe Val Lys Thr Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
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Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
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Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
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Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
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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 555560
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 135140
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
130135 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
290295 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
145150 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
5055 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 215220
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 375380
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 (7)

1. A liposome for resisting organ transplant rejection is characterized by being prepared from the following raw materials:
the streptavidin coupled GM2 single-chain antibody is loaded in the artificial liposome, the concentration of the streptavidin coupled GM2 single-chain antibody loaded in the artificial liposome is 0.1-1 ng/muL,
the molar ratio of biotinylated Qa-1 or HLA-E to biotinylated PD-L1 is equal,
the molar ratio of the total amount of biotinylated Qa-1 and biotinylated PD-L1 to the streptavidin-coupled GM2 single-chain antibody loaded artificial liposome is 100: 1-200: 1;
or the molar ratio of the total amount of biotinylated HLA-E and biotinylated PD-L1 to the streptavidin-coupled GM2 single-chain antibody loaded artificial liposome is 100: 1-200: 1;
the artificial liposome is prepared from the following raw materials: 10 mg-100 mg of phosphatidylcholine, 1 mg-10 mg of cholesterol, 0.1-1 mg of ganglioside GM2, and PBS with pH of 6.5; the artificial liposome has a concentration of 105～106Per mL;
the amino acid sequence of the streptavidin-coupled GM2 single-chain antibody is shown in Seq No. 1;
the amino acid sequences of Qa-1, PD-L1 and HLA-E are respectively shown as Seq No.2, Seq No.3 and Seq No.4, and are respectively represented as Qa1-G4S-FLAG, Seq No.5, PDL1-G4S-FLAG, Seq No.6, HLAE-G4S-FLAG, i.e., the form of Seq No.7 fusion protein, was expressed.
2. The liposome for anti-organ transplant rejection according to claim 1, wherein: the streptavidin-coupled GM2 single-chain antibody is expressed as VL- (G)4S)3-VH-G3Full-length synthesis by way of S-SA-GFP.
3. The liposome for anti-organ transplant rejection according to claim 1, wherein: the molar ratio of Qa-1, PD-L1, HLA-E and Sulfo-NHS-Biotin in the biotinylated Qa-1, biotinylated PD-L1 and biotinylated HLA-E is 1: 3-1: 5.
4. A method for preparing the liposome for anti-organ transplant rejection according to any one of claims 1 to 3, comprising the steps of:
first, preparation of liposome carrying a mounting site
Weighing 10-100 mg of phosphatidylcholine, 1-10 mg of cholesterol and 0.1-1 mg of ganglioside GM2 according to the proportion, shaking and dissolving overnight in a chloroform/methanol solution at room temperature, pumping out an organic solvent to form a uniform liposome membrane, and filling dry gas to fully volatilize the residual organic solvent; adding a PBS solution with the pH value of 6.5, shaking until the membrane is fully dissolved, and carrying out ultrasonic treatment until the liposome solution is dispersed into uniform and stable transparent liquid to obtain the artificial liposome;
second, the single chain antibody-streptavidin fusion protein of ganglioside GM2 is expressed and purified
Streptavidin-conjugated GM2 single-chain antibody as VL- (G)4S)3-VH-G3Synthesizing full length by S-SA-GFP, cloning the single chain antibody-streptavidin fusion protein of GM2 to pcDNA3.4 vector by ExpicHO system, transfecting CHO cell, oscillating and culturing, and collecting cell suspension; after centrifugation, diluting with PBS of equal volume; streptavidin-conjugated GM2 single chain antibody was purified using iminobiotin agarose beads;
thirdly, expression and purification of the mounting element
The mounting elements comprise a Qa-1 extracellular segment, a PD-L1 extracellular segment and an HLA-E extracellular segment, and the 3 mounting elements are respectively expressed as Qa1-G4S-FLAG, Seq No.5, PDL1-G4S-FLAG, Seq No.6 and HLAE-G4Expressing S-FLAG (Seq No.7 fusion protein), cloning the three fusion proteins to a pcDNA3.4 vector, transfecting CHO (Chinese hamster ovary) cells, carrying out shake culture, collecting cell suspension, diluting with PBS (phosphate buffer solution) with the same volume after centrifugation, and connecting a FLAG label and a human IgG1Fc section by using a mounting element; all proteins were used with OptiCHOTMThe Express system secretes and expresses, the mounting element uses Protein A column to carry out the first round of purification, Tris-HCl is used for neutralizing the pH value to 8.0 after the purification is finished, enterokinase is used for cutting off the Fc end, the enzyme digestion is carried out overnight at 25 ℃ according to 0.2U enterokinase/1 mg total Protein, and the enzyme digestion product uses FLAG affinity column to carry out the second affinity purification; the purified product was neutralized to pH 7.5 using Tris-HCl and the protein was concentrated using a 35kDa ultrafiltration tube;
fourth, biotinylation of the mounted element
After quantifying the mounted element prepared in the third step, carrying out room temperature reaction on the peptide fragment of the mounted element and Sulfo-NHS-Biotin in a molar ratio of 1: 3-1: 5 in PBS (pH 8.5), removing unbound Sulfo-NHS-Biotin by using a 10kDa dialysis bag, and carrying out ultrafiltration concentration on the biotinylated mounted element to obtain a biotinylated mounted element;
fifthly, loading liposome drug
Adding the streptavidin coupled GM2 single-chain antibody prepared in the second step into the artificial liposome prepared in the first step, wherein the concentration is 0.1-1 ng/mu L, and reversing and uniformly mixing the mixture at 4 ℃ overnight;
and (3) mixing the biotinylated Qa-1 or HLA-E prepared in the third step with PD-L1 in an equal molar ratio, mixing the mixture with the liposome according to the molar ratio of 100: 1-200: 1, reversing and uniformly mixing at room temperature, and dialyzing to remove unbound protein to finish the preparation of the liposome.
5. The method for preparing a liposome for anti-rejection in organ transplantation according to claim 4, wherein: the first step is further packaged with immune regulator and angiogenesis promoting medicine.
6. Use of the liposome for anti-organ transplant rejection according to any one of claims 1 to 3 for the preparation of a medicament for suppressing activation of immune cells.
7. Use of the liposome for anti-organ transplant rejection according to any one of claims 1 to 3 for the preparation of a medicament for inhibiting lymphocyte activation or for the preparation of a medicament for post-islet transplantation use.
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