CN110240656A - A kind of fusion protein and the preparation method and application thereof - Google Patents
A kind of fusion protein and the preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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Abstract
The present invention relates to a kind of fusion proteins and the preparation method and application thereof, belong to novel drugs technical field.Fusion protein of the invention includes anchorin and PD-L1 albumen.Fusion protein of the invention can show anchorin and the function of PD-L1 simultaneously, in vitro, animal model experiment confirms that fusion protein map-PD-L1 can be combined efficiently on cell membrane (endothelial cell membrane including organs and tissues) in vivo, and the activation of T cell can be inhibited in vitro and promote t cell proliferation, and the construction of GPI-anchored mGM of isolated viscus is further realized in organ transplant model in vivo, the inflammatory reaction of graft part can be effectively improved and extend the survival of graft, can apply to the prevention and treatment of the postoperative rejection of clinical transplantation.
Description
Technical field
The present invention relates to a kind of fusion proteins and the preparation method and application thereof, and in particular to a kind of anchoring fusion protein and its
Preparation method and application.
Background technique
Transplanting is to treat the preferred approach of whole Terminal Disease patient.Acute cellular rejection is still that chronic rejection and graft lose function
Major incentive, although as the development of immunosuppressive drug, acute cellular rejection has obtained certain control, but still has nearly 20% patient
The postoperative risk for facing acute cellular rejection.Currently, clinically preventing and treating the generation of acute cellular rejection, mainly answered by early postoperation vein
With large dosage of immune induction drug, but this non-specific, systemic induction scheme excessively presses down the immune function of patient itself
System, to increase the probability of patient's complicated by postoperative severe infections.Therefore, seek that one kind can target, specific effect is in transplanting
The novel immune induction scheme of object, by the prevention and treatment to post-transplantation acute cellular rejection, the prognosis for improving transplant patient has great meaning
Justice.
Apoptosis albumen -1 (PD-1) and its ligand PD-L1 are a kind of important immunologic test point molecules, right
Immunization has negativity adjustment effect, and when tumour cell is overexpressed PD-L1, the T cell that can induce the PD-1 positive loses function, consumption
It exhausts, and then promotes the generation of immunosurveillance escape.The main effects cell of acute cellular rejection is T cell, and the T cell of activation can
Express PD-1；In addition, more the study found that B cell, macrophage and NK cell etc. have the table of PD-1 other than T cell
It reaches.The expression for kidney part PD-L1 is raised as a result, it is possible to realize that (T cell, B are thin for " cover type " induction receptor's immunocyte
Born of the same parents, macrophage and NK cell etc.) function is lost, and then peripheral immune tolerance is induced, promote the effect of transplanted kidney survived.
PD-1/PD-L1 plays important work in terms of regulating and controlling immunity of organism balance as a kind of important immunologic test point
With, and influence the key factor of tumour immunity microenvironment.The T cell of atopy how is removed, or inhibits recipient's body
The activation of interior T cell, it would be possible to as the crucial point of penetration for inhibiting post-transplantation rejection.Our in vitro study and
Having delivered report confirms, after PD-L1 is in conjunction with its receptor PD-1, can effectively activate PD-1/PD-L1 signal path, promote T cell
Apoptosis and inhibit T cell activation.And the T cell surface activated when rejection occurs can high expression PD-1.As a result, we
Guess: enhancing the expression for kidney organ PD-L1, builds the immune microenvironment for kidney part high level PD-L1, is expected to realize induction
The mistake function and apoptosis of the main effects T cell of receptor's rejection, and then mitigate rejection degree of injury, induction periphery is immune
Tolerance, promotes surviving for transplanted kidney.
Early stage is studied, and discovery can accelerate the repulsion of cardiac transplantation by blocking PD-1/PD-L1 approach, meanwhile, it utilizes
Virus transfection technology can extend a series of phenomenal researchs such as survival of cardiac transplantation, mention so that graft height expresses PD-L1
Show that the regulation of PD-1/PD-L1 approach in heart transplant tolerance, plays the part of key effect.Liu et al. is inquired into using mouse liver transplantation model
Under the premise of no immunosupress is applied, discovery comparison spleen DC cell when the mechanism of the spontaneous tolerance of allosome liver transfer operation inducing mouse,
Height expression PD-L1 molecule liver DC cell is easier to induce cd4 t cell to Treg cell transformation, and the DC of PD-L1 expression deletion
Cell is unable to cause Treg, further confirms that high expression PD-L1 molecule liver DC cell lures in liver transplantation model in vivo
Graft tolerance is led, the ability for extending survival is stronger.The organ transplants such as same heart, liver are the same, by virus transfection DC cell,
So that its height expression PD-L1 is can induce CD8+T cell tolerance, and can induce kidney transplantation tolerance.It to sum up states, PD-L1/PD-1 signal is logical
Road is inhibiting to be immunoreacted, and is of great significance in terms of inducing transplantation immune tolerance.
Can be realized the method that cell membrane surface expresses new albumen at present mainly includes technique for gene engineering and GPI anchoring egg
White technology, and the former mode based on gene transfer, there are still many problems not can solve: gene transfer method first needs to rely on
Carrier, such as the carriers such as virus and plasmid, and this kind of carrier does not have targeting selectivity, into internal rear host cell and tissue
Position has uncertainty, is unable to control, and thus bring adverse consequences is unable to estimate；Secondly, the complexity of internal microenvironment,
The transfection efficiency of gene is largely limited, the load capacity of carrier is increased, undoubtedly aggravates toxic side effect；Again, at soft-boiled eggs
White expression need to be time-consuming and complicated by the serial procedures such as protein modified after the transcription of nucleic acid, translation and translation；Finally,
Exogenous gene will generally be integrated into stablize in host chromosome and play a role after carrier enters host cell,
But this integration is simultaneously unstable, is easily degraded, gene transfer is caused to fail.Just because of above-mentioned problem, gene transfer method is being faced
Application on bed is still pessimistic.Meanwhile the method for existing induction PD-L1 expression up-regulation, no matter gene infection protocol or gene are compiled
Volume technology, based on principle be all viral vectors and nucleic acid editing technique, and not only transfection efficiency is low for this kind of technology, but also not
Have organ and targets selectivity, once application in vivo, there is no the ability of targeting host cell and tissue site at present, and it is this not true
It is qualitative currently effectively to solve, it is systemic using brought risk and unpredictable as a result,.
In short, having the model animal by virus transfection and genetic engineering transformation, in existing report to realize internal PD-
The high expression of L1, but there are still many problems in clinic for these method distance services, such as: primary cell is not easy to transfect, transfection
The disadvantages of low efficiency, unstable expression, potential oncogenicity；Therefore, there is no a kind of safe and effective method can internal organs or other
Graft realizes the high efficient expression of PD-L1 in ex vivo stage." targeting is for organ anchor for protection for key point of the invention and protection point
Determining map-PD-L1 improves the damage of graft caused by post-transplantation acute rejection " intellectual property, protection scope includes anchoring
Fusion protein map-PD-L1 is in organ transplantation (heart, liver, lung, kidney, skin and cornea etc.) and cell transplantation (pancreas islet, stem cell
With marrow etc.) etc. graft rejection caused by post-transplantations protective effect.
Summary of the invention
There is provided that a kind of to improve post-transplantation thin it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art
The fusion protein and the preparation method and application thereof that born of the same parents mediate graft caused by rejection to damage.
To achieve the above object, the technical scheme adopted by the invention is as follows:
In a first aspect, the fusion protein includes anchorin and PD-L1 egg the present invention provides a kind of fusion protein
It is white.
In view of virus transfection and gene editing technology in terms of clinical application existing deficiency, the present invention is based on cell surfaces
Engineering philosophy successfully constructs using recombinant protein technology while including the recombinant protein of PD-L1 and film anchorin APT542
Membrane anchored protein-PD-L1 (map-PD-L1), the albumen not only remain PD-L1 combination PD-1 activation
The ability of PD-1/PD-L1 access, while APT542 function is increased, it can be achieved that quickly, PD-L1 is efficiently anchored to cell
Film surface；Cell in vitro is horizontal, internal renal transplantation model, confirms the biological function of the controllable T cell of map-PD-L1, together
When can improve the function of transplanted kidney, mitigate postoperative acute and repel the transplanting injury of kidney mediated, reduce transplanted kidney local inflammatory cells
Infiltration and inflammatory factor expression, the final survival for extending transplanted kidney, which is expected to as post-transplantation acute cellular rejection
Prevention and treatment new approach is provided, there is important clinical meaning and social value.
As the preferred embodiment of fusion protein of the present invention, the anchorin is APT542.
Second aspect, the present invention provides the preparation methods of above-mentioned fusion protein comprising following steps:
(1) using codon optimization software to the protein amino acid sequence of big source of mouse PD-L1 and anchorin APT542 into
Row optimization synthesizes the fusion of PD-LI and APT542, and fusion is inserted into carrier for expression of eukaryon, obtains containing melting
Close the expression vector of gene；
(2) expression vector containing fusion obtained by step (1) is transferred in escherichia coli cloning bacterial strain, extracts transfection
Grade plasmid, then carries out transient expression into mammalian cell HEK293 for plasmid transfection；
(3) it extracts and purifies, obtain the fusion protein.
The preferred embodiment of preparation method as fusion protein of the present invention, the carrier for expression of eukaryon are
PcDNA3.1 (-), the escherichia coli cloning bacterial strain are DH5a clone strain.
The third aspect treats or prevents post-transplantation rejection in preparation the present invention provides above-mentioned fusion protein and causes
Graft damage drug in application.
Fourth aspect, the present invention provides above-mentioned fusion proteins in preparation prevention and treatment post-transplantation rejection or to improve transplanting
Application in the drug of postoperative graft function.
5th aspect, the present invention provides above-mentioned fusion proteins to prepare the medicine for extending post-transplantation graft survival time
Application in object.
As the preferred embodiment of application of the present invention, the transplantation is organs and tissues transplanting or cell transplantation.
As the preferred embodiment of application of the present invention, organs and tissues transplanting be the heart, liver, lung, kidney, skin or
Corneal transplantation etc.；The cell transplantation is islet cells or bone marrow cell transplantation etc..
6th aspect, the present invention provides above-mentioned fusion proteins to prepare the activation and/or promotion for inhibiting T cell in vitro
Application in the drug of t cell proliferation.
7th aspect, the present invention provides a kind of pharmaceutical compositions containing above-mentioned fusion protein.
Compared with prior art, the invention has the benefit that currently, donor organ from get remigrate by
In person's body, during which due to assessment, transhipment, for factors such as organ trimmings, generally there is the ex vivo stage that a few hours are not equal.For organ
In vitro feature, seek to raise the opportunity for the expression of organ PD-L1 for us and provide good point of penetration: external up-regulation supplies
The expression of organ part PD-L1 effectively reduces its whole body application bring side effect.In addition, after reopening blood supply for organ,
It is the object that donee's cells contact earliest for organ vascular endothelial cell.Endothelial cell is very quick to the reactivity of cell factor
Sense, and the area that interacts is wide, and the expression of cell membrane surface itself be combined with each other a variety of of effect with various lymphocyte ligands
Molecule, to be earliest for organ endothelial cell, and react most rapidly, acutely when a moment reopened for kidney blood supply
Position.Studies have shown that resisting the first line of defence of receptor's immunocyte as graft, receptor's lymphocyte and donor endothelium are thin
Interaction is the key factor of immune-mediated rejection of the starting including acute and chronic rejection between born of the same parents.
The present invention is based on the new albumen map-PD-L1 that cellular degeneration necrosis principle and recombinant protein technology synthesize, and are applied to
When internal, the transcription and translation of nucleic acid level are needed not move through, so that it may realize the quick expression of cell membrane, and external real
It tests, the ability for yet sufficiently confirming map-PD-L1 anchoring combination cell film is not very big by low temperature effect quickly and efficiently, and
After being anchored to aim cell film, the ability that PD-L1 ligand identifies and combines its receptor PD-1 molecule is still remained, and mix in vitro
It closes in lymphocyte culture experiment, its activation of induction of T cell apoptosis and inhibition.Therefore, the research and development of this albumen, to target for kidney
Expression provides a new effective ways.
The present invention relies on for organ ex vivo stage and transfects and realize quickly, efficiently by PD-L1 albumen independent of gene
It is expressed in the new technology of cell membrane surface, the map-PD-L1 being re-combined into is anchored to for organ endothelial cell surface, so that for
Wide expression PD-L1 in the organ endothelial cell short time reinforces graft resists host immune cell attack the to realize
The purpose of one of protecting wall induces the mistake function of host's effect immunocyte, for prevention and treatment transplanting (including the heart, liver, lung, kidney, skin
The transplanting of the organs and tissues such as skin, cornea also includes the cell transplantations such as pancreas islet, marrow) postoperative AR has great application prospect.
Detailed description of the invention
Fig. 1 is the qualification result figure of transfection grade plasmid and fusion protein map-PD-L1 described in the embodiment of the present invention 1；Wherein,
A indicates that agarose gel analysis map-PD-L1 transfects grade plasmid, and B indicates that SDS-PAGE analyzes map-PD-L1 protein purification, C table
Show the specificity using His label protein antibody verifying fusion anchorin, D indicates to melt by the verifying of PD-L1 specific antibody
Close the specificity of anchorin map-PD-L1.
Fig. 2 is fusion protein in the embodiment of the present invention 2 and control group in conjunction with the kidney endothelial cell of in vitro culture
Verification result figure；
Fig. 3 is the kidney of the fusion protein and control group and in vitro culture in the embodiment of the present invention 3 after fluorescein label
The verification result figure that endothelial cell combines；
Fig. 4 is flow cytometer showed result of the fusion protein map-PD-L1 in conjunction with kidney endothelial cell in the embodiment of the present invention 4
Figure；Wherein, various concentration gradient under the conditions of A indicates 37 degree, various concentration gradient under the conditions of B indicates 4 degree, C indicate 37 degree of conditions
Lower difference incubation time, D different incubation times under the conditions of indicating 4；
Fig. 5 is the result figure that streaming technology detects t cell proliferation and Activation in the embodiment of the present invention 5；Wherein, A table
Show t cell proliferation situation, B indicates T cell activation situation；
Fig. 6 is the result for detecting endothelial cell apoptosis situation in the embodiment of the present invention 6 using TUNEL apoptosis detection kit
Figure；
Fig. 7 is that fusion anchorin map-PD-L1 influences the function of transplanted kidney and time-to-live in the embodiment of the present invention 7
Result figure；
Fig. 8 is that pathology HE and PAS are dyed and observed using TUNEL apoptosis detection kit each in the embodiment of the present invention 8
The result figure of apoptosis situation in group transplanting kidney specimen；
Fig. 9 is to detect inflammatory cell cell and its hypotype in transplanted kidney using immunohistochemistry technique in the embodiment of the present invention 9
With the result figure of the Infiltrating of macrophage；
Figure 10 is to detect the expression of inflammatory factor in transplanted kidney using RT-PCR technology in the embodiment of the present invention 10
Result figure；
Figure 11 be the embodiment of the present invention 11 investigate fusion anchorin map-PD-L1 anchoring for after kidney in the intracorporal table of receptor
Up to the result figure of level variation；
Figure 12 is the group of other internal organs (heart, liver, lung and spleen) in 12HE of embodiment of the present invention dyeing observation rat model
Knit morphological result figure；
Figure 13 is that map-PD-L1 of the present invention is anchored kidney endothelial cell, realizes " implanted " high expression PD-L1, mitigation office
Portion's inflammatory reaction improves the ideograph of transplanted kidney prognosis.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with attached drawing and specific embodiment pair
The present invention is described further.
In following embodiments, map-PD-L1 is that the present invention can express APT542 anchoring structure again while express PD-L1's
Fusion protein PD-L1-APT542.
Map-PD-L1 of the present invention is anchored kidney endothelial cell, realizes " implanted " high expression PD-L1, mitigates local inflammation
Reaction, the mode for improving transplanted kidney prognosis are as shown in figure 13.
Synthesis, purifying and the identification of 1 fusion protein map-PD-L1 of embodiment
A kind of embodiment of fusion protein of the present invention, the fusion protein of the present embodiment include APT542 anchorin and
PD-L1 albumen.
The map-PD-L1's the preparation method comprises the following steps: using codon optimization software of fusion protein described in the present embodiment
Argine Monohydrochloride of the MaxCodonTM Optimization Program (V13) to big source of mouse PD-L1 and anchorin APT542
Sequence optimizes, and is synthesized using full genome and passes through double digestion method PD-LI+APT542 fusion is inserted into pcDNA3.1
In (-) expression vector, followed by the accuracy of enzyme cutting method and sequencing technologies confirmation expression vector, DH5a clone bacterium is moved be finally made to
In strain, transfection grade plasmid is extracted by the big pumping kit of plasmid, it is thin that plasmid is transfected into mammal by transfection reagent later
Transient expression is carried out in born of the same parents HEK293, then passes through affinitive layer purification map-PD-L1 albumen.
The qualification result of transfection grade plasmid and fusion protein map-PD-L1 described in the present embodiment are as shown in Figure 1.In Fig. 1, A
Indicate that agarose gel analysis map-PD-L1 transfects grade plasmid: Lane, M:DNA marker；Lane 1: transfection grade plasmid；B table
Show that SDS-PAGE analyzes map-PD-L1 protein purification: Lane M:SDS-PAGE Protein marker, Lane 1: after centrifugation
Supernatant, Lane 2: efflux after supernatant is incubated for Ni-IDA, Lane 3-4:50mM Imidazole elution fraction, Lane 5-
8:100mM Imidazole elution fraction, Lane 9-12:500mM Imidazole elution fraction；C indicates to utilize His label
The specificity of protein antibodies verifying fusion anchorin: Lane 1:BSA (1.0ug), Lane 2:map-PD-L1protein
(0.4ug)(Reduced),Lane 3:map-PD-L1protein(0.4ug)(Non-Reduced),M1:SDS-PAGE
Marker,M2:Western Blot Marker；D is indicated through PD-L1 specific antibody verifying fusion anchorin map-PD-
The specificity of L1.
Embodiment 2
The present embodiment demonstrates fusion protein described in embodiment 1 by experiment in vitro can be thin with the kidney endothelial of in vitro culture
The combination of born of the same parents.Specific verification step is as follows:
Using the big source of mouse kidney endothelial cell (rgEC) of in vitro culture, experimental group is anchored using the fusion of 5ug/mL concentration
Albumen map-PD-L1 is incubated for 1 hour in 37 degree of cultures, and control group uses isometric medium treatment, incubation conditions and time
It is identical；It takes pictures through secondary antibody fluorescence developing after being incubated for the anti-PD-L1 of primary antibody followed by cellular immunofluorescence technology, takes pictures result such as
Shown in Fig. 2.Data analysis, display endothelial cell can show corresponding fluorescence color (red) after map-PD-L1 anchoring is incubated for,
And control group occurs without obvious fluorescence color (red), illustrates that map-PD-L1 can be anchored with the kidney endothelial cell of in vitro culture
In conjunction with.
Embodiment 3
The present embodiment confirms that the fusion anchorin FITC-map-PD-L1 after fluorescein marks is same by experiment in vitro
Sample retains the ability of anchoring kidney endothelial cell.Specific verification step is as follows:
Using the big source of mouse kidney endothelial cell (rgEC) of in vitro culture, experimental group is marked using the FITC of 5ug/mL concentration
Fusion anchorin FITC-map-PD-L1 is incubated for 1 hour in 37 degree of cultures, control group using isometric medium treatment,
Incubation conditions and time are identical；It then observes and takes pictures using DAPI dye core, result of taking pictures is as shown in Figure 3.Data analysis, display
Endothelial cell can be shown corresponding fluorescence color (green) after FITC-map-PD-L1 anchoring is incubated for, and control group is without obvious glimmering
Light color (green) occurs, and illustrates that the fusion anchorin FITC-map-PD-L1 after fluorescein marks equally retains anchoring kidney
The ability of dirty endothelial cell.
Embodiment 4
The present embodiment has investigated concentration, temperature and time to 1 fusion protein map-PD-L1 of embodiment by experiment in vitro
Influence in conjunction with kidney endothelial cell.The step of the present embodiment is investigated are as follows: thin using the big source of mouse kidney endothelial of in vitro culture
Born of the same parents (rgEC), when to proper density, digestion centrifugation prepares cell suspension, then according to various concentration, temperature, time point
Group:
A group: under the conditions of 37 degree, the binding ability of the FITC-map-PD-L1 and rgEC of various concentration gradient, through streaming point
The map-PD-L1 concentration for analysing combination and the incubation of map-PD-L1 and endothelial cell as the result is shown is positively correlated；1ng/mL,10ng/
FITC-map-PD-L1 and rgEC under mL, 100ng/mL, 1ug/mL, 10ug/mL, 100ug/mL concentration conditions are incubated for 1 hour
Percentage bound be respectively as follows: 0.07%, 0.08%, 0.09%, 4.22%, 22.1% and 98.9%.
B group: under the conditions of 4 degree, the binding ability of the FITC-map-PD-L1 and rgEC of various concentration gradient, through flow cytometer showed
The map-PD-L1 concentration of combination and the incubation of map-PD-L1 and endothelial cell is positively correlated as the result is shown；1ng/mL,10ng/
FITC-map-PD-L1 and rgEC under mL, 100ng/mL, 1ug/mL, 10ug/mL, 100ug/mL concentration conditions are incubated for 1 hour
Percentage bound be respectively as follows: 0.05%, 0.16%, 0.14%, 4.53%, 19.9% and 95.7%.
C group: under the conditions of 37 degree, using the FITC-map-PD-L1 of 50ug/mL concentration, through flow cytometer showed map- as the result is shown
The time of combination and the incubation of PD-L1 and endothelial cell is positively correlated；It is incubated for 10min, 30min, 60min, 90min, 120min
Under the conditions of FITC-map-PD-L1 and rgEC hatching combination rate be respectively as follows: 87.3%, 94.6%, 98.9%, 99.4% and
99.9%, illustrate that the combination of the albumen and endothelial cell is quickly and stable.
D group: under the conditions of 4 degree, using the FITC-map-PD-L1 of 50ug/mL concentration, through flow cytometer showed map- as the result is shown
The time of combination and the incubation of PD-L1 and endothelial cell is positively correlated；It is incubated for 10min, 30min, 60min, 90min, 120min
Under the conditions of FITC-map-PD-L1 and rgEC hatching combination rate be respectively as follows: 19.0%, 78.2%, 79.0%, 89.9% and
90.6%, illustrate that the combination of the albumen and endothelial cell is same quickly and stable under low temperature environment.
The above results are as shown in figure 4, result confirms: fusion protein map-PD-L1 it is rapid with kidney endothelial cell in conjunction with and
Joint efficiency and map-PD-L1 concentration and incubation time are proportional, and cryogenic conditions influence its joint efficiency little.
Embodiment 5
The present embodiment is tested by external heart xenotransplantaion confirms that map-PD-L1 can promote t cell proliferation and suppression
Make its activation.Specific experiment step are as follows: utilize 48 orifice plates, the big source of mouse kidney endothelial cell (rgEC) of in vitro culture, to suitable
When density, experimental group uses 50ug/mL map-PD-L1 albumen, and control group uses isometric medium treatment, and 37 degree incubate
It educates 1 hour, then abandons supernatant, the spleen lymphocyte of 1-2millionSD rat is added in every hole；Apoptosis experiment, co-cultures 3
It, is through apoptosis detection kit after, two groups of machine testing of apoptosis situation in streaming, as a result as shown in A in Fig. 5；T cell activation
Experiment is stimulated using anti-CD3+anti-CD28, is co-cultured 3 days, detects CD3/CD25 index observing followed by streaming technology
T cell activation situation, as a result as shown in B in Fig. 5.
By A in Fig. 5 as it can be seen that endothelial cell of the fluidic cell apoptosis experiment detection discovery after map-PD-L1 is protein modified
Experimental group t cell proliferation rate is 12.6%, and control group is 5.98%, it was demonstrated that the endothelium after map-PD-L1 is protein modified is thin
Born of the same parents can promote t cell proliferation；By B in Fig. 5 as it can be seen that Flow cytometry experiments detection T cell activation index discovery is through map-PD-L1 egg
The bis- positive activated rates of endothelial cell experimental group T cell activation CD3/CD25 after white modification are 10.1%, and control group is
5.05%, it was demonstrated that the endothelial cell after map-PD-L1 is protein modified can inhibit T cell activation.The above results confirm: fusion anchor
It can induce t cell proliferation after determining albumen map-PD-L1 in conjunction with kidney endothelial cell, and T cell activation can be inhibited.
Embodiment 6
The present embodiment confirms that map-PD-L1 can realize the guarantor of Human Umbilical Vein Endothelial Cells by the experiment of external heart xenotransplantaion
Shield.Specific experiment step are as follows: mix with lymphocyte co-cultivation treated endothelial cell for above-mentioned, utilize the inspection of TUNEL apoptosis
The apoptosis situation of endothelial cell in test agent box test experience group and control group.As a result as shown in Figure 6.In Fig. 6, external mixing leaching
Bar cell culture experiments, protective effect of the observation map-PD-L1 to the effector cell killing endothelial cell after activation, as a result
It was found that the experimental group endothelial cell apoptosis situation after the map-PD-L1 construction of GPI-anchored mGM of 50ug/mL concentration is (referring to green fluorescence
Intensity) obviously mitigated than control group, it was demonstrated that map-PD-L1 can realize the protection of Human Umbilical Vein Endothelial Cells.
Embodiment 7
The present embodiment, which merges anchorin map-PD-L1 by rat kidney transplantation model validation, can improve the function of transplanted kidney
The time-to-live of energy and energy lengthening model.
The present embodiment successfully constructs each group model according to experimental group, and before renal transplantation 0d and post-transplantation 1d,
3d, 5d leave and take the blood specimen of each group model, and serum creatinine is total in the model being anchored through detection discovery experimental group through map-PD-L1
Body level is declined compared with control group and blank group, and on day 3 with map-PD-L1 anchoring group in 5 days blood specimens compared with
APT542 anchoring group and blank group are and poor between APT542 anchoring group and the group of blank group there are obvious statistical difference (p < 0.05)
Different little (p > 0.05)；Urea nitrogen entire change level is also experimental group lower than control group and blank group, and the 5th day after surgery obtains
In the blood specimen taken map-PD-L1 anchoring group compared with APT542 anchoring group and blank group there are obvious statistical difference (p < 0.05),
And the group difference of APT542 anchoring group and blank group is less (p > 0.05)；It analyzes, passes through from the survival outcome of transplantation model
The experiment group model of map-PD-L1 anchoring, the time-to-live is considerably longer than control group and blank group (p < 0.05), and APT542 is anchored
Group and blank group group difference are unobvious (p > 0.05).
It is as shown in Figure 7 to the function of transplanted kidney and the influence of time-to-live to merge anchorin map-PD-L1.Wherein, A table
Show the level of serum creatinine and blood BUN in blood specimen between group；Between B expression group transplantation model survivorship curve figure (* indicate p <
0.05).As seen from Figure 7, fusion anchorin map-PD-L1 can improve the function of transplanted kidney and extend the survival of transplantation model.
Embodiment 8
The present embodiment by rat kidney transplantation model validation merges anchorin map-PD-L1, and can to improve transplanted kidney acute
The pathology damage of repulsion.The specific method is as follows: according to experimental group, successfully constructing each group model, every group of 5 models, observation group
Between pathologic condition, and in Post kidney transplantation acquisition in the 5th day transplant kidney specimen.Pathology HE and PAS then are carried out to the sample of acquisition
Dyeing, and utilize apoptosis situation in TUNEL apoptosis detection kit observation each group transplanting kidney specimen.It is found through data analysis: warp
Capillaritis and renal damage degree caused by the transplanting kidney specimen acute cellular rejection of the experimental group of map-PD-L1 anchoring compared with
Have clear improvement in APT542 anchoring group and blank group, and by Banff standards of grading calculate scoring, statistical analysis discovery disease
Degree of impairment is managed, there are statistical difference (p < 0.05) compared with APT542 anchoring group and blank group for map-PD-L1 anchoring group, and
The group difference of APT542 anchoring group and blank group is not significant (p > 0.05).TUNEL apoptosis detection kit detects each group model
Paraffin specimen equally prompt through map-PD-L1 be anchored experimental group transplanting kidney specimen in Apoptosis degree compared to
APT542 anchoring group and blank group have clear improvement, through sxemiquantitative calculate score, statistical analysis discovery map-PD-L1 anchoring group compared with
There are statistical difference (p < 0.05) for APT542 anchoring group and blank group, and the group difference of APT542 anchoring group and blank group is not
Significantly (p > 0.05).
The present embodiment is dyed using pathology HE and PAS and observes each group transplanted kidney using TUNEL apoptosis detection kit
The result of apoptosis situation is as shown in Figure 8 in sample.In Fig. 8, A. from top to bottom shows HE, PAS and TUNEL experiment knot between group respectively
Fruit；Figure B, figure C and figure D respectively show the sxemiquantitative knot of pipe week Capillaritis, renal damage and transplanted kidney Apoptosis
Fruit (* indicates p < 0.05).As seen from Figure 8, fusion anchorin map-PD-L1 can improve shifting caused by the acute cellular rejection of transplanted kidney
It plants injury of kidney pathology and the whole apoptosis situation of cell in transplanted kidney can be mitigated.
Embodiment 9
The present embodiment confirms that fusion anchorin map-PD-L1 can reduce infiltration and the inhibition office of inflammatory cell in transplanted kidney
Portion's inflammatory reaction.The specific method is as follows: the transplanted kidney of each group model is obtained within the 5th day in Post kidney transplantation, every group 5, and using exempting from
Epidemic disease group technology detects the Infiltrating of inflammatory cell cell and its hypotype and macrophage in transplanted kidney, and the marker of detection divides
Son is respectively as follows: CD3, CD4, CD8, CD68.Data analysis is found: in the transplanting kidney specimen of the experimental group through map-PD-L1 anchoring
CD3+T cell, the Infiltrating also including CD4+ and CD8+T cell and macrophage have bright compared to control group and blank group
It is aobvious to improve, statistical analysis discovery, experimental group than between control group and blank group group there are significant difference (p < 0.05), and control group and
Blank group group difference is unobvious (p > 0.05).The above results confirm: fusion anchorin mp-PD-L1 can improve transplanted kidney art
The inflammatory cell infiltration of kidney part afterwards inhibits inflammatory reaction.
The present embodiment utilizes inflammatory cell cell and its hypotype and macrophage in immunohistochemistry technique detection transplanted kidney
The result of Infiltrating is as shown in Figure 9.In Fig. 9, A. by showing blank group, control group and experimental group transplanted kidney group respectively from left to right
The Infiltrating of middle CD3+ cell is knitted, and quantitative divided data analyzes result；B. by showing blank group, control group respectively from left to right
Result is analyzed with the Infiltrating of CD4+ cell in experimental group transplanting nephridial tissue, and quantitative divided data；C. by distinguishing from left to right
Show that blank group, control group and experimental group transplant the Infiltrating of CD8+ cell in nephridial tissue, and quantitative divided data analysis knot
Fruit；D. by show respectively from left to right blank group, control group and experimental group transplanting nephridial tissue in CD3+ cell Infiltrating, and
Quantitative divided data analysis result (* indicates p < 0.05)；As seen from Figure 9, fusion anchorin map-PD-L1 can reduce in transplanted kidney
The infiltration and improvement local inflammation reaction of inflammatory cell.
Embodiment 10
The present embodiment confirms that fusion anchorin map-PD-L1 can lower the expression of inflammatory factor in transplanted kidney.Specific side
Method are as follows: obtain the transplanted kidney of each group model on the 5th day in Post kidney transplantation, every group 5, after RNA is extracted in homogenate, utilize RT-PCR skill
Art detects the expression of inflammatory factor in transplanted kidney, and the molecule of detection is respectively as follows: IFN-γ, TNF-α, IL-2, IL-4, IL-6
With IL-17 result figure, as shown in Figure 10.Data analysis is found: in the transplanting nephridial tissue of the experimental group through map-PD-L1 anchoring
The expression of IFN-γ, TNF-α, IL-2, IL-4, IL-6 and IL-17 is in a degree of downward compared with control group and blank group, system
Meter analysis finds, IFN-γ in experimental group, TNF-α, IL-2, IL-4, IL-6 expression than control group and blank group table
Up to level, there are group difference (p < 0.05).The above results confirm: fusion anchorin mp-PD-L1 can improve post-operative transplantation of kidney
The inflammatory factor expression of kidney part is horizontal, inhibits inflammatory reaction.
As seen from Figure 10, fusion anchorin map-PD-L1 can lower the expression of inflammatory factor in transplanted kidney.Through RT-PCR
Technology detection, respectively shows transplanted kidney group between inflammatory factor IFN-γ, TNF-α, the group of IL-2, IL-4, IL-6 and IL-17A
Expression in knitting (* indicates p < 0.05).
Embodiment 11
The present embodiment has investigated fusion anchorin map-PD-L1 anchoring for becoming after kidney in the intracorporal expression of receptor
Change, as a result as shown in figure 11.Method particularly includes: PD-L1 in nephridial tissue was transplanted using the 3rd day after immunohistochemistry technique detection technique
Expression finds that the expression of the experimental group PD-L1 of map-PD-L1 anchoring is higher than APT542 anchoring group and blank through analysis
Group, and there are group difference (p < 0.05), and the PD-L1 of anchoring expression is prompted to may persist in transplanted kidney the 3rd day；Further inspection
Survey the expression of PD-L1 in postoperative 5th day transplanting nephridial tissue, the expression of the PD-L1 of discovery experimental group, control group and blank group
Horizontal indifference (p > 0.05).
Embodiment 12
The present embodiment is the pathological observation of the other internal organs HE dyeing of rat model.Method particularly includes: in Post kidney transplantation
The heart, liver, lung and the spleen for obtaining each group model on the 5th day dye the morphological change of observation internal organs using HE, as a result such as Figure 12 institute
Show.Analysis is found between contrast groups: the heart, liver, lung and spleen tectology had no between experimental group, control group and blank group it is bright
Significant difference is different, prompts fusion anchorin mp-PD-L1 less to systemic influence, has certain safety.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of fusion protein, which is characterized in that the fusion protein includes anchorin and PD-L1 albumen.
2. fusion protein as described in claim 1, which is characterized in that the anchorin is APT542.
3. the preparation method of fusion protein as claimed in claim 1 or 2, which comprises the following steps:
(1) it is carried out using protein amino acid sequence of the codon optimization software to big source of mouse PD-L1 and anchorin APT542 excellent
Change, synthesizes the fusion of PD-LI and APT542, and fusion is inserted into carrier for expression of eukaryon, obtain containing fusion base
The expression vector of cause；
(2) expression vector containing fusion obtained by step (1) is transferred in escherichia coli cloning bacterial strain, extracts transfection grade matter
Grain, then carries out transient expression into mammalian cell HEK293 for plasmid transfection；
(3) it extracts and purifies, obtain the fusion protein.
Graft caused by 4. fusion protein as claimed in claim 1 or 2 treats or prevents post-transplantation rejection in preparation
Application in the drug of damage.
5. fusion protein as claimed in claim 1 or 2 is in preparation prevention and treatment post-transplantation rejection or improves post-transplantation transplanting
Application in the drug of object function.
6. fusion protein as claimed in claim 1 or 2 answering in the drug that preparation extends post-transplantation graft survival time
With.
7. such as the described in any item applications of claim 4~6, which is characterized in that the transplantation is organs and tissues transplanting or thin
Born of the same parents' transplanting.
8. the use as claimed in claim 7, which is characterized in that the organs and tissues transplanting is the heart, liver, lung, kidney, skin or angle
Film transplanting；The cell transplantation is islet cells or bone marrow cell transplantation.
9. fusion protein as claimed in claim 1 or 2 inhibits the activation of T cell in preparation in vitro and/or promotes t cell proliferation
Drug in application.
10. a kind of pharmaceutical composition containing fusion protein as claimed in claim 1 or 2.
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| CN (1) | CN110240656A (en) |
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| WO2017079080A1 (en) * | 2015-11-02 | 2017-05-11 | The Johns Hopkins University | Method of preventing organ transplant rejections using agonists to the pd-1 checkpoint pathway |
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2019
- 2019-04-24 CN CN201910333580.9A patent/CN110240656A/en active Pending
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| US6713606B1 (en) * | 1996-07-15 | 2004-03-30 | Adprotech Limited | Conjugates of soluble peptidic compounds with membrane-binding agents |
| US20040106544A1 (en) * | 2000-11-03 | 2004-06-03 | Cooper Matthew Allister | Antibacterial agents comprising conjugates of glycopeptides and peptidic membrane associating elements |
| WO2017079080A1 (en) * | 2015-11-02 | 2017-05-11 | The Johns Hopkins University | Method of preventing organ transplant rejections using agonists to the pd-1 checkpoint pathway |
| CN107365361A (en) * | 2017-08-21 | 2017-11-21 | 中国药科大学 | Duplicate domain anchorin combined with PD L1 and application thereof |
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| C.E BOWLES等: "Membrane Reinsertion of a Myristoyl-Peptidyl Anchored Extracellular Domain Growth Hormone Receptor", 《ENDOCRINOLOGY》 * |
| 梁明等: "PD-L1-GPI分子膜表面修饰胰岛细胞抑制反应性T细胞的攻击", 《中国病理生理杂志》 * |
| 王雅文: "肾移植受者PD-1/PD-L1信号通路在基础和临床研究", 《中国博士学位论文全文数据库》 * |
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