CN108948182A - The application of the anti-alloimmunity rejection of TIGIT-ECD recombinant protein - Google Patents

Abstract

The present invention provides the applications of the anti-alloimmunity rejection of TIGIT-ECD recombinant protein.The amino acid sequence of recombinant protein TIGIT-ECD is as shown in SEQ ID NO:1.The nucleotide sequence of the recombinant protein TIGIT-ECD is encoded as shown in SEQ.ID.NO:2.The recombinant protein can be by activating TIGIT-CD155 signal path, and effective regulation macrophage polarization helps to induce allograft tolerance.Therefore it can be used as the application of graft-rejection inhibitor, can also apply to preparation prevention and treatment graft-rejection drug.

Description

The application of the anti-alloimmunity rejection of TIGIT-ECD recombinant protein

Technical field

The invention belongs to biomedicine technical field, in particular to a kind of recombinant protein TIGIT-ECD and its resist of the same race different The application of body immunological rejection.

Background technique

For the patient of terminal phase organ failure, organ transplant is that surgical intervention is important, even unique Means.Since the 1980s, have benefited from surgical technic and broad immune inhibits the universal of drug, filed of organ transplantation flies Speed development, hematopoietic stem cell transplantation and a variety of solid organ transplantations have become routine operation.90% or more in postoperative First Year Graft can obtain good functional survival, but long-term effect is barely satisfactory always, and the rejection of body is All transplant patients need the problem of facing throughout one's life.Transplant organ receptor's prolonged application immunosuppressive drug, it is easy to directly contribute It suffers from post-transplantation newly to swell the complication such as tumor, diabetes, cardiovascular and cerebrovascular disease, causes additional financial burden.Explore transplanting The amynologic mechanism of rejection regulation, develops new Immunotherapy Strategy to improve transplant organ survival rate, is organ transplant The research emphasis in field.

Immune response and immunological homeostasis are the networks of a finely regulating.In allogene histoorgan migration process, from Tissue damage causes the generation of " danger signal (DAMP) " to graft antigen recognizing, answers from the activation of each subgroup immunocyte Lapsing to for immune response is answered, panimmunity molecule, cell participate in response regulation.Filed of organ transplantation research in recent years compares concern Adjusting molecule have a series of costimulations/inhibition molecule such as CTLA-4, PD-1/PD-L1, ICOS, CD40, TIGIT, CD226, with And regulatory T cells (regulatory T cell, Treg), M2 type polarization macrophage, marrow source property inhibit cell (myeloid-derived suppressor cells, MDSCs), it causes to be resistant to Dendritic Cells etc..Its mechanism of action and clinic Transformation Application has obtained extensive concern, has a good application prospect.

Antigen presenting cell (antigen presenting cell, APC) has central role in immune response, knows Other pathogen associated molecular pattern (PAMP) or plain (Alarmin) the post activation differentiation of alarm are thin to T by specific antigen presentation Born of the same parents start adaptive immune response.And the functional status after APC activation directly determines the direction of T cells differentiation, also The pattern of immune response is regulated and controled.Macrophage is professional antigen presenting cells, to the cells such as M1 type polarized secretion TNF-α because Son promotes inflammation progress, then secretes the cell factors such as IL-10 to the polarization of M2 type, plays and inhibit inflammation, promote the effect repaired. The activation of macrophage, the regulation to polarize by receptors a variety of on cell membrane.CD226/TIGIT is important costimulation/inhibition point Son, can be with the same acceptor molecule CD155 of competitive binding, it is known that function be mainly participate in regulation APC activation and function, The cellular immunologies process such as the differentiation of lymphocyte and killing, cell-cell adhesion.With the deep and experimental technique means of research Development, TIGIT molecule function new on panimmunity cell and regulating effect be constantly found, in clinical application, especially It is to obtain more and more attention in terms of the Mechanism Study of tumour and inflammation related disease.The major ligand of TIGIT is CD155 Molecule, while also there is interaction with two molecules of CD226, CD96 in CD155.In same cell surface, 3 molecule competitiveness In conjunction with same ligand CD155, CD155, CD96 and TIGIT cytoplasmic region contain ITIM motif, can transmit inhibition signal, CD226 Reactivity signal is transmitted, complicated regulated and control network is constituted.TIGIT influences immunological synapse shape by interacting with its ligand At, cell-cell adhesion, regulates and controls the biological processes such as differentiation and the killing of hematopoietic development and immunocyte, take part in multiple types and face The occurrence and development of bed disease, mechanism and application study in clinical practice are also increasingly goed deep into.

In terms of costimulatory molecules regulation CTL cell and oncotherapy relationship, CTLA-4 and PD-1 are paid close attention to earliest, are resisted The relevant biological therapeutic agent such as body and recombinant protein comes into clinical application.But still there is the trouble of significant proportion in practical application Person does not reach ideal therapeutic effect, prompt there is still a need for carry out deeper into research, seek more costimulatory molecules regulations Mechanism.Johnston et al. after the highly expressed CD155 molecule combination TIGIT of tumor cell surface the study found that inhibit CTL thin The killing ability of born of the same parents then can effectively reverse this phenomenon using the combination of antibody blocking TIGIT.TIGIT antibody and inhibitor A kind of drug has obtained more research in the treatment of malignant tumour.But in contrast, for graft rejection Treatment, then need to intervene that TIGIT/CD226-CD155 is signal-balanced, pass through the signal of enhancing TIGIT, promote CD155 positive Macrophage polarizes to M2 type, inhibits the activation of antigen presenting cell in the Immune discrimination stage.

Summary of the invention

It is arranged the purpose of the present invention is to provide a kind of humanized's recombinant protein TIGIT-ECD and its in anti-alloimmunity Application in reprimand reaction.

The invention discloses a kind of humanized's recombinant protein TIGIT-ECD, the amino acid sequences of recombinant protein TIGIT-ECD Column are as shown in SEQ ID NO:1.

The nucleotide sequence of the recombinant protein TIGIT-ECD is encoded as shown in SEQ.ID.NO:2.

After the invention also discloses above-mentioned recombinant protein TIGIT-ECD as allograft, anti-immunity rejection The application of inhibitor.

The invention also discloses above-mentioned recombinant protein TIGIT-ECD answering in preparation prevention and treatment graft-rejection drug With.

Preferably, the drug is to activate TIGIT-CD155 signal path by stimulation, promotes macrophage to M2 type Polarization, thus the drug for inducing allograft to be resistant to.

Preferably, the drug is the drug for raising macrophage IL-10 and Arg1 (including but not limited to) expression.

Preferably, the drug be lower iNOS, IL-12p40, TNF-α, IFN-γ, MCP-1, IL6 and IL-1 β because The drug of sub (including but not limited to) expression.

Preferably, the drug is the phosphorylation for promoting macrophage ERK molecule intracellular, by increasing STAT3 consideration convey Position, the drug for promoting IL10 to generate.

Preferably, the drug is the drug for promoting Treg differentiation, depression effect T cell proliferation.

Preferably, the drug is that can be improved Treg cell proportion, increases the drug of M2 type macrophage ratio.

Preferably, the drug is the drug that tolerogenic dendritic cells can be promoted to generate.

Compared with prior art, the invention has the following beneficial technical effects:

Recombinant protein TIGIT-ECD disclosed by the invention, can be by activating TIGIT-CD155 signal path, effectively Regulation macrophage polarization, facilitate induce allograft tolerance.The present invention stimulates macrophage using in vitro culture LPS Cell model detects TIGIT-ECD recombinant protein pair by the technological means such as flow cytometry, RT-PCR, Luminex and WB The signal path of the polarized influence of macrophage and IL-10 secretion.As the result is shown:

1, TIGIT-ECD can remarkably promote macrophage and polarize to M2 type, the M2 type characteristic molecular expression such as up-regulation Arg1, Promote IL-10 secretion, lowers the expression of M1 the type characteristic molecule and cell factor such as iNOS, IL-6, IL-1 β；

2, the mechanism of action of TIGIT-ECD is analyzed, discovery TIGIT-ECD promotes macrophage born of the same parents in combination with CD155 molecule The phosphorylation of interior ERK molecule, and then increase STAT3 nuclear translocation, promote IL10 transcription and macrophage to polarize to M2 type；

3, the effect of detection TIGIT-ECD recombinant protein in mixed lymphocyte reaction (MLP) system, inquires into TIGIT-ECD tune Control macrophage polarization and inhibit T cell proliferation effect, discovery TIGIT-ECD can promote macrophage to M2 type polarization, Inhibit it to polarize to M1 type, and inhibits CD4+Th cell-proliferation activity.

Detailed description of the invention

Fig. 1 is TIGIT amino acid sequence hydrophobicity analysis result figure；

Fig. 2 is the clone of TIGIT after birth outskirt genetic fragment and the experimental result picture of vector construction；

Fig. 3 is the experimental result picture of SDS-PAGE protein electrophoresis after TIGIT-ECD recombinant protein purification；

Fig. 4 is that TIGIT-ECD promotes macrophage system THP-1 to M2 polarization result figure；

Fig. 5 is that TIGIT-ECD promotes macrophage ERK phosphorylation level experimental result picture；

Fig. 6 is that TIGIT-ECD processing promotes STAT3 nuclear translocation experimental result picture；

Fig. 7 is the experimental result picture that TIGIT-ECD inhibits T cell proliferation in heart xenotransplantaion；Wherein, (a) is Wherein primary representative proliferation results, (b) statistical analysis to test three times.

Specific embodiment

The invention firstly uses bioinformatic analysis humanized's TIGIT sequences, clone the outer region sequence of after birth, are inserted into table Up to carrier pSectag2-Fc, eukaryon expression plasmid is constructed.Through conventional gene sequencing, determine that the gene order is correct；Transfection Cells and supernatant is collected after HEK293T cell, is purified into TIGIT-ECD recombinant protein using anti-Fc affinity chromatography technology；? Detection TIGIT-ECD recombinant protein is on the polarized influence of macrophage on the model of macrophages in vitro culture and LPS stimulation；Inspection Survey influence of the TIGIT-ECD for T cell Proliferation, Differentiation in mixed lymphocyte reaction (MLP).

The present invention is done specifically below with reference to preparation method and the biological activity detection of TIGIT-ECD recombinant protein Bright, the explanation of the invention is not limited.

1, the design and preparation of TIGIT-ECD recombinant protein

1.1 TIGIT-ECD gene orders: bioinformatic analysis is utilized, by TIGIT molecule after birth outskirt (Extracellular domain, ECD) sequence is inserted into carrier for expression of eukaryon pSectag2-Fc, and gene sequencing determines the fusion Gene order.Specifically, (carrier has public affairs to the expression vector pSectag2-Fc containing Fc sections of human IgG1 source fusion Open document record: " research of CD226 and its ligand CD112/CD155 interaction of molecules mechanism " (The Fourth Military Medical University doctor Academic dissertation), the public, which can contact author and ask for or deliver document according to this, to be voluntarily synthetically prepared)；Detect human IgG and soluble type Fc sections of monoclonal antibodies (mAb) of sandwich ELISA lcits, mouse anti human IgG molecule of Fc fusion protein are the quotient being commercially available Product product.Coli strain DN5 α, human embryonic kidney epithelial cells system HEK293T (are to buy quotient by biotech company Product product) it is incubated in 10%NCS RPMI 1640 or DMEM culture medium (Hyclone Products).CNBr- Activated Sepharose 4B chromatographs column packing and is purchased from Pharmacia company, transfection reagent liposome Lipofectamin2000 (LF2000) is purchased from Invitrogen company.RNA extracts reagent Trizol and is purchased from Gibco company.With In AMV reverse transcriptase, the reverse transcriptase primer oligo (dT) of reverse transcription synthesis cDNA, rTaq archaeal dna polymerase, Pyrobest high are protected True polymerization enzyme, dNTPs, pMD18-T carrier and connection kit (DNA Ligation Kit Ver.2.0) etc. are purchased from precious biology Engineering (Dalian) Co., Ltd.Sequence specific primers are synthesized by Beijing AudioCodes company.Tryptone, yeast extract and eutectic Point agarose is purchased from Britain OXOID company.DNA rapidly purifies kit, plastic recovery kit, small amount plasmid extraction kit purchase From Shanghai Tiangeng Bioisystech Co., Ltd.

People's TIGIT cloning vector is purchased from Beijing Yi Qiao Divine Land biotech company (article No. HG10917-M), according to The TIGIT gene order of GenBank record and bioinformatic analysis TIGIT signal peptide, after birth outskirt, transmembrane region and born of the same parents The start-stop site in matter area is as shown in Figure 1, design expands the upstream and downstream primer of after birth outskirt gene are as follows:

TIGIT-ECD-F:GGGGTACCatgatgacaggcacaata (adds Kpn I restriction enzyme site)

TIGIT-ECD-B:CGGGATCCtggaatctggaacctggc (adds BamH I restriction enzyme site)

Using people TIGIT cloning vector as template, carried out after high-fidelity DNA polymerase Pyrobest and primer mixing is added PCR.Reaction condition are as follows: 94 DEG C of denaturation 10min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s prolong after 35 circulations then at 72 DEG C totally Stretch 10min.It expands obtained DNA fragmentation and adds rTaq enzyme, dNTP, PCR buffer, carry out the second wheel reaction.Parameter: 94 DEG C It is denaturalized 10min, 72 DEG C of 40min.Reaction product confirms length through 1% agarose gel electrophoresis, as shown in Figure 2.Glue reclaim reagent Box colloidal sol recycles target fragment, and Kpn I and BamH I double digestion handles PCR product and pSectag2-Fc carrier, recycles digestion Segment and digestion carrier are attached, and convert bacillus coli DH 5 alpha by product.Parameter: 16 DEG C of connection 2h, with competence DH5 α Ice bath 10min after bacterium softly mixes, 42 DEG C of heat shock 90s are coated on Amp resistance (120 μ g/ml of Amp concentration) LB agar plate, It is incubated overnight rear picking positive transformant clone and carries out PCR identification, positive colony is transferred to liquid LB training with ampicillin again Support base (120 μ g/ml of Amp concentration) expansion culture, a small amount of extraction Plasmid DNA carry out Kpn I and BamH I double digestion and identify, picking Positive colony carries out the correctness that DNA sequencing determines fusion gene sequence.Clone containing correct sequence is expanded into culture, is extracted Plasmid.

The amino acid sequence of recombinant protein TIGIT-ECD are as follows:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSL TVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP.(as shown in SEQ ID NO:1)

The gene order for the TIGIT-ECD recombinant protein for designing and correctly expressing is as follows:

(shown in SEQ.ID.NO:2)

The expression of 1.2 TIGIT-ECD plasmid transfections and albumen:

HEK293 cell is inoculated in 24 porocyte culture plates, cell reaches about 90% and converges rate after overnight incubation.It will PSectag2-TIGIT-ECD plasmid is transfected according to calcium phosphate method.Cells and supernatant is collected after 5 days, with detection soluble type people Fc The sandwich ELISA lcits of section fusion protein detect recombinant protein expression.

2, a large amount of preparation and purification of TIGIT-ECD recombinant protein:

It is thin in 15cm Tissue Culture Dish large-scale culture HEK293 with 1640 culture medium of RPMI containing 10% newborn bovine serum Born of the same parents, cell reaches about 90% and converges rate after overnight incubation.PSectag2-TIGIT-ECD plasmid is transfected according to calcium phosphate method.It is flat Each 15cm Tissue Culture Dish can harvest supernatant 25ml.Fc fusion protein in supernatant is measured by sampling during this at any time to contain Amount.Every batch of harvest supernatant is frozen immediately in -30 DEG C.

By anti-human Fc monoclonal antibody (mAb, this teaching and research room preparation, clone WT6) crosslinking CNBr-activated Sepharose 4B Ago-Gel, prepares affinity column.In short, CNBr-activated Sepharose 4B agar Sugar is first through 1mmol/L hydrochloric acid activation, and room temperature softly mixes 2h in pH8.3 cross-linking buffer with the mAb of purifying immediately, and ratio is Every ml swell gel adds 8mg to purify mAb, then switches into pH8.3 glycine solution closing residual activity site, pH4.0 and Alternately washing 3 times of pH8.3 buffer, PBS are washed 2 times, and final concentration of 0.01%NaN is added₃Plastics chromatographic column is filled afterwards, is placed in 4 DEG C refrigerator saves.

HEK293T cells and supernatant is centrifuged through 8500rpm high-speed low temperature, removes cell fragment through 0.45 μm of membrane filtration And particle, using the slow upper prop of medical Transfusion device, flow control is after 1-1.5ml/min, completion of the sample with 10 times of bed volumes PBS rinse affinity column, be added elution buffer elute destination protein, with Eppendorf pipe collect eluent, rapidly plus Enter alkaline neutralizer to neutralize, titrating neutralization ratio in advance is that every 12 drop eluent adds 75 μ l neutralizers.Supernatant before loading passes through liquid The product that keep sample are to be measured.Ultra-filtration centrifuge tube protein concentrate that eluent is 30kDa with molecular cut off simultaneously replaces buffer system extremely 0.15mol/L PBS, ultraviolet spectrophotometry Quantitative Western.SDS-PAGE identifies purity of protein and its molecular weight, such as Fig. 3 institute Show.Supernatant passes through liquid hold-up before detecting the sandwich ELISA lcits detection loading of Fc sections of fusion proteins of soluble type people.

3, recombinant protein TIGIT-ECD inhibits immune response

3.1 detection TIGIT-ECD processing are on the polarized influence of macrophage

Cell culture and LPS stimulating expression of macrophage THP-1 model in vitro pass through the methods of RT-PCR and WB, detection The signal path of the influence polarized on macrophage of TIGIT-ECD recombinant protein and IL-10 secretion.Cultivate humanized's monokaryon macrophage Cell line THP-1, is added LPS stimulation and the processing of TIGIT-ECD recombinant protein, and discovery TIGIT-ECD can remarkably promote macrophage The expression of cell M2 type polar molecule Arg1 and IL-10 inhibit the expression of M1 type polar molecule iNOS, IL-12, as a result referring to figure 4。

3.2TIGIT-ECD can promote the ERK phosphorylation intracellular of LPS stimulating expression of macrophage

Immunoblotting: recording the SDS-PAGE glue of 5%-10%, using cell pyrolysis liquid albumen as antigen, loading (100 μ g/ Road), constant pressure 120V, 180V electrophoresis；On albumen electrotransfer to NC film, skimmed milk power confining liquid room temperature closes 1h；It is pressed with confining liquid The antibody of 1:2000 dilution identification unlike signal protein molecular, is incubated overnight with 4 DEG C of NC film；HRP label goat-anti is added after washing film Mouse secondary antibody, room temperature act on 1h；ECL luminescent solution shines, and Chemiluminescence Apparatus records luminous intensity.Referring to Fig. 5, TIGIT- as the result is shown ECD processing has facilitation for ERK/STAT3 molecule phosphorylation.The endochylema and cytoplasm of different disposal group cell are extracted respectively Ingredient carries out protein electrophoresis and WB detection, as a result referring to Fig. 6, it can be found that the cell of TIGIT-ECD recombinant protein processing, carefully STAT3 content is reduced in cytoplasm, and STAT3 content increases in nucleus, illustrates that albumen processing can promote the consideration convey of STAT3 Position.

The influence that the 3.4 pretreated macrophages of TIGIT-ECD recombinant protein are proliferated T cell

It establishes in the experimental system of external heart xenotransplantaion, is mixed human macrophage system THP-1 and fresh Human peripheral blood single nucleus cell PBMC is separated, and the processing of TIGIT-ECD recombinant protein is added.It was found that TIGIT-ECD can be significant The breeder reaction for inhibiting the Th cell of CD4+, as a result referring to (a) in Fig. 7 and (b), it is shown that utilize CFSE label and immunofluorescence The experimental result of Determination Staining Th ability of cell proliferation, the experiment have carried out biology repetition three times.It (a) is a wherein second generation The proliferation results of table, (b) statistical analysis to test three times.

Sequence table

<110>the Fourth Military Medical University of P.L.A

<120>application of the anti-alloimmunity rejection of TIGIT-ECD recombinant protein

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 120

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys

1 5 10 15

Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln

20 25 30

Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys

35 40 45

Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val

50 55 60

Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn

65 70 75 80

Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr

85 90 95

Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu

100 105 110

His Gly Ala Arg Phe Gln Ile Pro

115 120

<210> 2

<211> 360

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

atgatgacag gcacaataga aacaacgggg aacatttctg cagagaaagg tggctctatc 60

atcttacaat gtcacctctc ctccaccacg gcacaagtga cccaggtcaa ctgggagcag 120

caggaccagc ttctggccat ttgtaatgct gacttggggt ggcacatctc cccatccttc 180

aaggatcgag tggccccagg tcccggcctg ggcctcaccc tccagtcgct gaccgtgaac 240

gatacagggg agtacttctg catctatcac acctaccctg atgggacgta cactgggaga 300

atcttcctgg aggtcctaga aagctcagtg gctgagcacg gtgccaggtt ccagattcca 360

Claims (10)

1. a kind of recombinant protein TIGIT-ECD, which is characterized in that the amino acid sequence such as SEQ of recombinant protein TIGIT-ECD Shown in ID NO:1.

2. encoding the nucleotide of recombinant protein TIGIT-ECD described in claim 1, which is characterized in that nucleotide sequence is such as Shown in SEQ.ID.NO:2.

3. application of the recombinant protein TIGIT-ECD described in claim 1 as graft-rejection inhibitor.

4. application of the recombinant protein TIGIT-ECD described in claim 1 in preparation prevention and treatment graft-rejection drug.

5. application as claimed in claim 4, which is characterized in that the drug is by simulating, activating TIGIT and CD155 The intermolecular signal path regulation antigen presenting cells state of activation, thus the drug for inducing allograft to be resistant to.

6. application as claimed in claim 4, which is characterized in that the drug is up-regulation macrophage IL-10 and Arg1 table The drug reached.

7. application as claimed in claim 4, which is characterized in that the drug is to lower iNOS, IL-6 and IL-1 β factor table The drug reached.

8. application as claimed in claim 4, which is characterized in that the drug is to promote macrophage ERK phosphorylation intracellular, Increase STAT3 nuclear translocation, promotes the drug of IL10 transcription.

9. application as claimed in claim 4, which is characterized in that the drug is to promote Treg differentiation, depression effect T cell The drug of proliferation.

10. application as claimed in claim 9, which is characterized in that the drug is that can be improved Treg cell proportion, is reduced M1 type macrophage ratio, and increase the drug of M2 type macrophage ratio.