CN107083366A - Express adoptive immunity cell of hirudin and its production and use - Google Patents
Express adoptive immunity cell of hirudin and its production and use Download PDFInfo
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Abstract
The invention discloses a kind of adoptive immunity cell for expressing hirudin and its production and use, adoptive immunity cell behaviour source immunocyte, the lepirudin 023 ludon of adoptive immunity cell expression includes signal peptide and hirudin.The upper people source secreting, expressing signal peptide of amino terminal connection of lepirudin 023 ludon, its encoding gene is transfected into adoptive immunity cell using technique for gene engineering, feed back after patient, continual and steady expression lepirudin 023 ludon, plays the effect in " minicell pharmaceutical factory " in patient's body.In this way, being used as carrier cell by adoptive immunity cell, lepirudin 023 ludon can be expressed steadily in the long term in vivo, hirudin is effectively solved to preparing higher purity requirement, often half-life short, the drawback such as injection patient compliance difference.
Description
Technical field
The present invention relates to biological therapy and biomedicine field, more particularly to a kind of adoptive immunity cell for expressing hirudin
And its production and use.
Background technology
Hirudin is the activity most strong natural thrombin inhibitor found so far, initially from Hementaria officianalis saliva
It is isolated in gland, it is made up of 65~66 amino acid, can be directly with fibrin ferment with l：L (mol ratio) mode combines to form non-
Covalent complex, so that fibrin ferment loses the ability of cracking fibrinogen, suppresses the formation of thrombus.Natural hirudin is present
More than ten kinds variants, mainly there is referred to as tri- kinds of homologys of HV1, HV2, HV3 very high isomers (Hirudin Variant).
At present, external existing two lepirudin 023 ludon products approval listing：1.Desirudin (trade names：Revasc, it is auspicious
Scholar Novartis products)；2.Lepirudin (trade names：Refludan, Britain Pharmion and U.S. Berlex
Laboratories products).Only there is fine difference at N- ends in both structures, Desirudin is Val1-Val2, and
Lepirudin is Leu1-Tyr2.The prevention and treatment of DVT (DVT) when Desirudin is used to perform the operation,
Lepirudin is used for the anticoagulant therapy of heparin-induced thrombocytopenic disease patient.In addition, hirudin is preventing and treating unstable
Property angina pectoris, disseminated intravascular coagulation, brain blood coagulation, thrombophlebitis and coronary artery thrombosis in terms of all have it is huge latent
In clinical value.
High blood coagulation state, such as lung cancer, breast cancer, stomach cancer, cancer of pancreas are there is in Several Kinds of Malignancy patient.Dislike
Property tumor patient blood in hypercoagulative state, can not only promote internal thrombosis, and with tumour growth, invasion and attack and shifting
It is substantially related.Coagulation function change can cause tumour cell phenotype and activity change, promote tumour cell and blood platelet, endothelium thin
Born of the same parents, fibronectin (Fibrinectin) and Von Willebrand factor (vWF) stick, so that tumour is thin
Born of the same parents even shift in local multiplication, infiltration to other positions.Thrombotic diseases are the malignant tumor patients for being only second to metastases
Second largest lethal factor.The tumor patient high-risk to thrombosis carries out Drug intervention early, for extension patient survival,
Reduce the death rate significant.Research shows that hirudin can also play a role in oncotherapy.Lepirudin 023 ludon passes through it
Anticoagulation, suppress it is fibrinous formed, tumour cell and fibrin or platelet aggregation can be prevented, make NK cells or
The activity of other cytotoxic effector cells is played.Be proved leech extract for treating can play curative effect tumour have fibrosarcoma,
Osteosarcoma, angiosarcoma, melanoma and leukaemia etc..Hirudin can also coordinate chemotherapy and radiation, by promoting the blood in tumour
Stream improves anoxia state and heightened the effect of a treatment.
Animal experiment and clinical research show that vein or hypodermic injection hirudin are without obvious toxic-side effects, semilethal agent
Amount is easily accepted by much larger than dosage needed for treatment, body, and immunity is weak, and blood platelet, fibrinogen level and blood red egg are not influenceed
Bai Hanliang, not easily passs through blood-CSF barrier.No matter acute, subacute toxicity test, hirudin is to breathing, blood pressure, heart rate
Do not influence.
Hirudin as a kind of efficient, direct, special thrombin inhibitor, with very strong anti-freezing, antithrombotic and
A variety of pharmacological activity such as anti-inflammatory.Compared with traditional anticoagulant such as heparin, its therapeutic dose is small, curative effect is high, will not cause
Quick reaction.Leech have antigenicity, but its antibody does not do harm to, and prolongs long elimination half-life on the contrary, is that a kind of rare plus effect resists
Body.Therefore the application of hirudin will be continuously available developing and promote, and lepirudin 023 ludon will turn into the anti-freezing of a new generation, anti-bolt
And anti-cancer agent.
One of defect of hirudin is that clinical practice has certain hemorrhage risk.Hirudin can cause blood coagulation correlation ginseng
Number, such as activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) are significantly raised, from
And whole body or systemic bleeding risk may be caused.Therefore, country Wu Zuze etc. passes through in the connection of the amino terminal of hirudin
The oligopeptides that can be recognized and be cracked by plasma thromboplastin antecedent a (a of F Ⅺ) and Ⅹ a (a of F Ⅹ).When internal blood coagulation system is activated generation thrombus
When, the characteristics of using the biochemical change triggered in thrombus generating process, the hirudin that amino terminal is closed is returned again
It is hirudin original shape again, local in the thrombus that may occur or occur plays specific anticoagulation, so as to reduce bleeding
Side effect, as a class is new, safe and effective anti-coagulants.
Hirudin further disadvantage is that half-life short.Hirudin is in vivo without degraded, with active component through kidney excretion, water
Leech element eliminates very fast from blood plasma.Research shows that the anticoagulation of hirudin depends entirely on its blood plasma level, because it not only exists
Blood is transported, and site of action is also only in circulatory system.Hirudin administering mode is mainly drug administration by injection, when maintaining drug effect
Between many hours of only one, it is difficult to the need for meeting chronic hypercoagulative state Disease long-term anticoagulant therapy, giving needs long-term anti-freezing
Patient bring very big painful and inconvenience, therefore the clinical non-injection administration method waited in expectation more easily and effectively.In addition, from
Hirudin is extracted in natural leech tissue or in genetic engineering fermentate, is limited by material source, purified product often band
There is the pollution of foreign protein, be unfavorable for clinic and used through vein.
To solve leech essence injecta The book of Changes renal excretion half-life short, to preparing, purity requirement is higher, often inject patient
The problems such as compliance is poor, the present invention is therefore.
The content of the invention
It is an object of the invention to provide a kind of adoptive immunity cell and its production and use for expressing hirudin, at least
It can solve the problem that one of above mentioned problem.
To achieve the above object, according to an aspect of the invention, there is provided a kind of adoptive immunity for expressing hirudin is thin
Born of the same parents, adoptive immunity cell behaviour source immunocyte, the lepirudin 023 ludon of adoptive immunity cell expression includes signal peptide and hirudin.
The upper signal peptide of amino terminal connection of lepirudin 023 ludon, its encoding gene is adopted using technique for gene engineering transfection
Immunocyte, is fed back after patient, and continual and steady expression lepirudin 023 ludon, is played in " minicell pharmaceutical factory " in tumor patient body
Effect.In this way, being used as carrier cell by adoptive immunity cell, lepirudin 023 ludon can be expressed steadily in the long term in vivo, effectively solution
Certainly the drawbacks of hirudin half-life short.
In some embodiments, signal peptide behaviour source secreting signal peptide,
In some embodiments, hirudin is hirudin isomers, hirudin mutant, hirudin chimera, truncation
HIRULOG, the hirudin or hirudin fusion protein of genetic modification.
In some embodiments, adoptive immunity cell be α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK,
One or more in TCR-T, above-mentioned adoptive immunity cell is immunocyte that is natural or manually cultivating.
In some embodiments, hirudin is hirudin isomers HV1, and the amino acid sequence of lepirudin 023 ludon is：SEQ
ID NO.1, gene order is：SEQ ID NO.2；
Or, hirudin is hirudin isomers HV2, and the amino acid sequence of lepirudin 023 ludon is：SEQ ID NO.3, base
Because sequence is：SEQ ID NO.4；
Or, hirudin is hirudin isomers HV3, and the amino acid sequence of lepirudin 023 ludon is：SEQ ID NO.5, base
Because sequence is：SEQ ID NO.6；
Or, hirudin is hirudin mutant, by the Ser32-Asn33- of wild type hirudin (HV-3) peptide chain
Gly34-Lys35 replaces with Arg32-Gly33-Asp34-Ser35 (RGDS), and the amino acid sequence of lepirudin 023 ludon is：SEQ
ID NO.7, gene order is：SEQ ID NO.8；
Or, hirudin is hirudin mutant, by the Ser32-Asn33- of wild type hirudin (HV-3) peptide chain
Gly34-Lys35 replaces with Arg32-Gly33-Asp34-Met35 (RGDM), and the amino acid sequence of the lepirudin 023 ludon is：
SEQ ID NO.9, gene order is：SEQ ID NO.10；
Or, hirudin is hirudin chimera, by wild type hirudin HV-1 and HV-3 chimeric expression, the restructuring water
Leech element amino acid sequence be：SEQ ID NO.11, gene order is：SEQ ID NO.12.
Or, the hirudin is hirudin fusion protein, and hirudin amino terminal, which is connected with, to be known by factor Ⅹa
Not and the oligopeptides that cracks, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.13, gene order is：SEQ ID
NO.14；
Or, the hirudin is hirudin chimera, by wild type hirudin HV-1 and HV-3 chimeric expression, is fitted together to water
Leech element amino terminal is connected with can be by the oligopeptides that factor Ⅹa is recognized and is cracked, the amino acid sequence of the lepirudin 023 ludon
For：SEQ ID NO.15, gene order is：SEQ ID NO.16；
Or, the hirudin is hirudin mutant, by the Ser32-Asn33- of wild type hirudin (HV-3) peptide chain
Gly34-Lys35 replaces with Arg32-Gly33-Asp34-Met35 (RGDM), hirudin amino terminal be connected with can by blood coagulation because
The oligopeptides that Ⅹ a of son is recognized and cracked, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.17, gene order is：
SEQ ID NO.18；
Or, the hirudin is hirudin isomers HV1, and hirudin amino terminal is connected with can be by factor Ⅹa
The oligopeptides for recognizing and cracking, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.19, gene order is：SEQ ID
NO.20。
In some embodiments, lepirudin 023 ludon constructive expression or by be coupled inducible expression carry out induction table
Reach.
In some embodiments, adoptive immunity cell expression suicide gene/prodrug system.
Understand at present, the amino terminal of hirudin molecule is its core, containing three couples of disulfide bond (Cys6-
Cys14, Cys16-Cys28, Cys32-Cys39), make amino terminal peptide chain around dense core cyclic peptide structures are changed into, with blood coagulation
The active site of enzyme is combined；And carboxyl terminal is the chain structure of a random stretching, extension, wherein Gln49~Gly54 has time
Between and hydrophobic effect, and Asp55~Pro60 is rich in acidic amino acid, and with negative electrical charge, the substrate easily with fibrin ferment alkalescence is recognized
Site is combined.HV1/HV2 Ser32-Asp33-Gly34-Glu35 or HV3 Ser32-Asn33-Gly34-Lys35 are one prominent
For the Loop structures between beta sheet, conformation is free, to activity without obvious effect.Research discovery, HV3 carboxyl terminal
(HV354-66) carboxyl terminal (HV154-65) of the binding ability than HV1 with fibrin ferment is stronger.According to more than hirudin special
Property, the Ser32-Asn33-Gly34-Lys35 of wild type hirudin HV3 peptide chains is replaced with into Arg32-Gly33-Asp34-
Ser35(RGDS).RGD sequence is made up of arginine, glycine and aspartic acid, is present in various kinds of cell epimatrix, can be with
11 kinds of integrin specific bindings.The sequence peptide being capable of various attachment proteinses and blood of the Reverse transcriptase including fibrin
The combination of platelet, can reach and suppress the purpose that blood platelet is combined with fibrin.RGD modifying hirudins (HV3-RGD) have anti-
Platelet aggregation ability.
In some embodiments, the Membrane surface expression of adoptive immunity cell has target molecules, and target molecules pass through cross-film
Sequence is positioned at cell membrane surface.It is convenient to detect foreign gene transfection efficiency, isolate and purify recombinant immune cell, in-vivo monitoring mistake
After immunocyte proliferative conditions and pass through corresponding target antibody targeting inactivation adoptive immunity cell when necessary.
In some embodiments, target molecules are CD19 total length or its truncated segment, or CD20 total length or its section
Short-movie section.
Correspondingly, present invention also offers the preparation method of the adoptive immunity cell of above-mentioned expression hirudin, including it is following
Step：Vitro culture of human source immunocyte, prepares the genophore of coding lepirudin 023 ludon and membrane-type molecules target, genophore
By signal peptide sequence-leech prime sequences and signal peptide sequence-target molecules sequence-Linker sequences-cross-film section combined sequence
Into；Above-mentioned coding lepirudin 023 ludon and the genophore of target molecules are transfected into adoptive immunity cell by gene.
Correspondingly, present invention also offers the purposes of the adoptive immunity cell of above-mentioned expression hirudin, expression hirudin
Adoptive immunity cell is used for anticoagulant therapy, antithrombotic treatments, antineoplaston, is protected for antilipemic healthy, anti-freezing health care, anti-bolt
It is strong, the prevention and treatment of DVT during for performing the operation, for the anti-of heparin-induced thrombocytopenic Disease
Solidifying treatment, preventing and treating unstable angina, disseminated intravascular coagulation, brain blood coagulation, thrombophlebitis and coronary artery thrombosis, prevents
Control high fat of blood, atherosclerosis.
Beneficial effects of the present invention are：
1st, using adoptive immunity cell as carrier, in vivo continuous expression lepirudin 023 ludon or by genetic modification restructuring water
Leech element, it is possible to resolve the drawbacks of hirudin half-life short, without repetitively administered in the short time, is more suitable for needing long-term anti-freezing, anti-
The patient of bolt.
2nd, using adoptive immunity cell as carrier, continuous expression lepirudin 023 ludon, can avoid repetitively administered from causing in vivo
Quick or induction of antibodies is produced.
3rd, the adoptive immunity cell of expression lepirudin 023 ludon is while film expression target molecules, can be used as target detection external source base
Because of transfection efficiency, isolate and purify recombinant immune cell, the proliferative conditions of in-vivo monitoring adoptive immunity cell and pass through when necessary
Corresponding target antibody targeting inactivation adoptive immunity cell, makes this technology Clinical practice more safely controllable.
4th, the adoptive immunity cell of expression lepirudin 023 ludon can target tumor local expression, the solidifying shape of improvement tumor patient height
State, dissolves microthrombus, prevents metastases, promotes immunocyte to enter inside tumor and plays killing ability, can be used alone or
Clinic is used with combination/radiotherapy.
Embodiment
The present invention is described in detail below.
The adoptive immunity cell of the expression hirudin of the present invention, adoptive immunity cell behaviour source immunocyte, adoptive immunity
The lepirudin 023 ludon of cell expression includes signal peptide and hirudin.Signal peptide behaviour source signal peptide.Signal peptide can be people's acid amides
Change one kind in enzyme (APM) signal peptide, human IL-2's signal peptide, the signal peptide of human IL-2 1.
The amino acid sequence of people's amidating enzyme (PAM) signal peptide is：
MAGRVPSLLVLLVFPSSCLA,
Gene order is：
ATGGCTGGCCGCGTCCCTAGCCTGCTAGTTCTCCTTGTTTTTCCAAGCAGCTGTTTGGCT。
The expression of adoptive immunity cell membrane surface has target molecules, and target molecules are positioned at cell membrane table by cross-film sequence
Face, the lepirudin 023 ludon of adoptive immunity cell expression includes signal peptide and hirudin.Hirudin is hirudin isomers, hirudin
Mutant, hirudin chimera, the HIRULOG truncated, the hirudin or hirudin fusion protein of genetic modification.
Total length or its truncated segment that target molecules are CD19, or CD20 total length or its truncated segment.Adoptive immunity is thin
Born of the same parents are the natural or immunocyte manually cultivated in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T.It is excellent
The adoptive immunity carrier cell of choosing is CIK, NK, NKT or gamma delta T cells, and the Nonspecific immunity that tumour can be played simultaneously is controlled
Treatment is acted on.
The hirudin amino terminal of the lepirudin 023 ludon of adoptive immunity cell expression is connected with can be by clotting factor identification simultaneously
The oligopeptides of cracking.Clotting factor is Ⅺ a (a of F Ⅺ) and Ⅹ a (a of F Ⅹ).
Above-mentioned can have a variety of by the oligopeptides that clotting factor is recognized and is cracked, for example, EPR (gene order GAACCTAGG),
IEGR (gene order ATCGAAGGTCGT), LGPR (gene order TTGGGTCCAAGA), LEKRLGPR (gene orders
CTCGAGAAAAGATTGGGTCCAAGA), LEKRIEGR (gene order CTCGAGAAAAGAATCGAAGGTCGT).
Lepirudin 023 ludon can also be coupled inducible expression and be carried out induced expression with constructive expression.Induced expression
System be tetracycline inducible expression, moulting hormone (ecdysone) inducible expression, tacrolimus (tacrolimus,
FK506 one kind in)/rapamycin (rapamycin) inducible system or RU486 inducible systems.
The adoptive immunity cell of expression hirudin also expresses suicide gene/prodrug system.Suicide gene/prodrug system is single
Cyclic guanosine (HSV-tk/GCV) system of the pure type thymidine kinase of herpesviral 1/penta, herpes zoster virus thymidine kinase/
Flucytosine (CD/5FC) system of arabinose methoxypurine (VZV-tk/Ara-M) system, cytosine deaminase/5, cytochromes
P450 microsomal enzyme CYP2B1/ endoxan (CYP2B1/CPA) system；Cytochrome P450 CYP4B1/ amino anthracenes (CYP4B1/
2AA) system, deoxycytidine kinase/cytarabine (dCK/Ara-C) system, crow glycosides-xanthine phosphoribosyltransferase/6-
Sulfur purine (gpt/6TX) system, nitroreductase/CB1954 (NTR/CB1954) system, purine nucleoside phosphorylase/6- first
Base purine deoxyriboside (PNP/6-MeP-dR) system, thymidine phosphorylase/5 '-deoxidation -5 FU 5 fluorouracil (TP/5 ' -
DFUR) one in system, carboxypeptidase G2/CMDA systems (CPG2/CMDA), carboxy-lesterase/CPT-11 (CE/CPT-11) system
Kind.
The preparation method of the adoptive immunity cell of above-mentioned expression hirudin, comprises the following steps：Prepare coding restructuring water
The genophore of leech element and membrane-type molecules target, genophore is by signal peptide sequence-leech prime sequences and signal peptide sequence-target
Molecular sequences-Linker sequences-cross-film section combined sequence is formed；Above-mentioned coding lepirudin 023 ludon and the gene of target molecules are carried
Body transfects immunocyte by technique for gene engineering.
Technique for gene engineering is known technology, and gene transfection system used includes slow-virus transfection system, retrovirus
Transfection system, Adenovirus Transfection system, adeno-associated virus transfection system, sleeping beauty transposon stand transfection system, plasmid electrotransfection system
System.It is preferred that, using sleeping beauty transposon stand transfection system or electrotransfection system.With transient expression vector, by lepirudin 023 ludon base
Because importing adoptive immunity cell, its exogenous gene expression amount can be made controllable, it is to avoid the risk of overexpression.
The structure of genophore of coding lepirudin 023 ludon and membrane-type molecules target is：Signal peptide-lepirudin 023 ludon-
IRES- signal peptides-target molecules-Linker- cross-films section, or, signal peptide-target molecules-Linker- cross-films section-P2A- letters
Number peptide-lepirudin 023 ludon.
The adoptive immunity cell of above-mentioned expression hirudin is used for anticoagulant therapy, antithrombotic treatments, antineoplaston, for dropping
Blood fat health care, anti-freezing health care, anti-bolt health care, the prevention and treatment of DVT during for performing the operation, for heparin-induced
The anticoagulant therapy of thrombocytopenic Disease, preventing and treating unstable angina, disseminated intravascular coagulation, brain blood coagulation, blood
Bolt phlebitis and coronary artery thrombosis.
With reference to specific embodiment, the present invention is further detailed explanation.
Embodiment 1
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface, the lepirudin 023 ludon of the adoptive immunity cell expression includes signal peptide and leech
Element.Hirudin is hirudin isomers HV1.The structure of lepirudin 023 ludon of adoptive immunity cell expression is：Signal peptide-HV1, ammonia
Base acid sequence is：SEQ ID NO.1, gene order is：SEQ ID NO.2.
Embodiment 2
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface, the lepirudin 023 ludon of the adoptive immunity cell expression includes signal peptide and leech
Element.Hirudin is hirudin isomers HV2, and the structure of the lepirudin 023 ludon of adoptive immunity cell expression is：Signal peptide-HV2, ammonia
Base acid sequence is：SEQ ID NO.3, gene order is：SEQ ID NO.4
Embodiment 3
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface, the lepirudin 023 ludon of the adoptive immunity cell expression includes signal peptide and leech
Element.Hirudin is hirudin isomers HV3, and the structure of the lepirudin 023 ludon of adoptive immunity cell expression is：Signal peptide-HV3, ammonia
Base acid sequence is：SEQ ID NO.5, gene order is：SEQ ID NO.6.
Embodiment 4
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface, the lepirudin 023 ludon of the adoptive immunity cell expression includes signal peptide and leech
Element.Hirudin is hirudin mutant, and the Ser32-Asn33-Gly34-Lys35 of wild type hirudin HV3 peptide chains is replaced with
Arg32-Gly33-Asp34-Ser35 (RGDS), the structure of lepirudin 023 ludon of adoptive immunity cell expression is：Signal peptide-
HV3-RGDS, amino acid sequence is：SEQ ID NO.7, gene order is：SEQ ID NO.8.
The lepirudin 023 ludon has anti-platelet aggregation ability simultaneously.
Embodiment 5
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface, the lepirudin 023 ludon of the adoptive immunity cell expression includes signal peptide and leech
Element.Hirudin is hirudin mutant, and the Ser32-Asn33-Gly34-Lys35 of wild type hirudin HV3 peptide chains is replaced with
Arg32-Gly33-Asp34-Met35 (RGDM), the structure of lepirudin 023 ludon of adoptive immunity cell expression is：Signal peptide-
HV3-RGDM, amino acid sequence is：SEQ ID NO.9, gene order is：SEQ ID NO.10
The lepirudin 023 ludon has anti-platelet aggregation ability simultaneously.
Embodiment 6
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface, the lepirudin 023 ludon of the adoptive immunity cell expression includes signal peptide and leech
Element.Hirudin is hirudin chimera, and by wild type hirudin HV1 and HV3 chimeric expression, the structure of the lepirudin 023 ludon is
People's amidating enzyme (PAM) signal peptide-hirudin HV1-HV3 chimeras, its amino acid sequence is：SEQ ID NO.11, gene sequence
It is classified as：SEQ ID NO.12.
Embodiment 7
The hirudin of the present embodiment is hirudin fusion protein, and hirudin amino terminal is connected with can be by factor Ⅹa
The oligopeptides for recognizing and cracking, structure behaviour amidating enzyme (PAM) signal peptide-EPR- hirudin HV1 of the lepirudin 023 ludon, ammonia
Base acid sequence is：SEQ ID NO.13, gene order is：SEQ ID NO.14.
Oligopeptides EPR can also be replaced by the short peptide sequence of equivalent efficacy.
The lepirudin 023 ludon of the present embodiment, which has, so both to be closed by the oligopeptides EPR that factor Ⅹa is recognized and is cracked
The activity of hirudin in the normal tissue, reduces the risk of systemic bleeding, lepirudin 023 ludon can be made again in thrombosis portion
Hirudin original shape of the position release with bioactivity, reaches the economic benefits and social benefits purpose of anti-freezing and anti-bolt.
Embodiment 8
The hirudin of the present embodiment is hirudin chimera, by wild type hirudin HV1 and HV3 chimeric expression, is fitted together to water
Leech element amino terminal is connected with can be by the oligopeptides that factor Ⅹa is recognized and is cracked, the structure behaviour acyl of the lepirudin 023 ludon
Aminase (PAM) signal peptide-EPR- hirudin HV1-HV3 chimeras, amino acid sequence is：SEQ ID NO.15, gene order
For：SEQ ID NO.16
Oligopeptides EPR can also be replaced by the short peptide sequence of equivalent efficacy.
The lepirudin 023 ludon of the present embodiment, which has, so both to be closed by the oligopeptides EPR that factor Ⅹa is recognized and is cracked
The activity of hirudin in the normal tissue, reduces the risk of systemic bleeding, lepirudin 023 ludon can be made again in thrombosis portion
Hirudin original shape of the position release with bioactivity, reaches the economic benefits and social benefits purpose of anti-freezing and anti-bolt.
Embodiment 9
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface.Hirudin is hirudin mutant, by the Ser32- of wild type hirudin HV3 peptide chains
Asn33-Gly34-Lys35 replaces with Arg32-Gly33-Asp34-Met35 (RGDM), the restructuring water of adoptive immunity cell expression
Leech element structure be：Signal peptide-oligopeptides EPR-HV-3-RGDS, amino acid sequence is：SEQ ID NO.17, gene order is：
SEQ ID NO.18。
Oligopeptides EPR can also be replaced by the short peptide sequence of equivalent efficacy.RGDS can be replaced by the short peptide sequence of equivalent efficacy.
The lepirudin 023 ludon of the present embodiment, which has, so both to be closed by the oligopeptides EPR that factor Ⅹa is recognized and is cracked
The activity of hirudin in the normal tissue, reduces the risk of systemic bleeding, lepirudin 023 ludon can be made again in thrombosis portion
Hirudin original shape of the position release with bioactivity, reaches the economic benefits and social benefits purpose of anti-freezing and anti-bolt.
Embodiment 10
The adoptive immunity cell of the expression hirudin of the present embodiment, its Membrane surface expression has target molecules, and target molecules are led to
Cross cross-film sequence and be positioned at cell membrane surface hirudins for hirudin isomers HV1, the restructuring leech of adoptive immunity cell expression
Element structure be：Signal peptide-oligopeptides EPR-HV1-RGDS, amino acid sequence is：SEQ ID NO.19, gene order is：SEQ
ID NO.20。
Oligopeptides EPR can also be replaced by the short peptide sequence of equivalent efficacy.Hirudin isomers HV1 can by HV2, HV3 etc. its
Its hirudin isomers is replaced.
The lepirudin 023 ludon of the present embodiment, which has, so both to be closed by the oligopeptides EPR that factor Ⅹa is recognized and is cracked
The activity of hirudin in the normal tissue, reduces the risk of systemic bleeding, again EPR-HV1 can be made to be released at thrombosis position
The hirudin original shape with bioactivity is put, the economic benefits and social benefits purpose of anti-freezing and anti-bolt is reached.
Embodiment 11
The either table of embodiment 1~10 is prepared up to the method for the adoptive immunity cell of hirudin, is comprised the following steps：External training
People source immunocyte is supported, the genophore of coding lepirudin 023 ludon and membrane-type molecules target is prepared, genophore is by signal peptide sequence
Row-leech prime sequences and signal peptide sequence-target molecules sequence-Linker sequences-cross-film section combined sequence are formed；By above-mentioned volume
Code lepirudin 023 ludon and the genophore of target molecules transfect immunocyte by gene.
Gene transfection system uses sleeping beauty transposon stand transfection system and electrotransfection system.With transient expression vector, by weight
Group hirudin gene imports adoptive immunity cell, its exogenous gene expression amount can be made controllable, it is to avoid the risk of overexpression.
The structure of genophore of coding lepirudin 023 ludon and membrane-type molecules target is：Signal peptide-hirudin-IRES- letters
Number peptide-target molecules-Linker- cross-films section, or, signal peptide-target molecules-Linker- cross-films section-P2A- signal peptides-water
Leech element.Specifically, membrane-type molecules target construction：GM-CSFR signal peptides-Δ CD20-Linker-CD28 cross-film section, Δ CD20 is
CD20 truncated segment, Linker length is 2~15 amino acid.The amino acid sequence of membrane-type molecules target is：SEQ ID
NO.21, gene order is：SEQ ID NO.22.
When the gene order of lepirudin 023 ludon is expressed in adoptive immunity cell, two amino acid sequences are generated：Signal peptide-
Hirudin is one, signal peptide-target molecules-Linker- cross-films Duan Weiyi.
The adoptive immunity cell of the present embodiment uses CIK cell.Specifically, the adopting for hirudin of expression of the present embodiment, exempts from
Epidemic disease cell preparation method includes step S1~S3, specific as follows.
S1, vitro culture of human source CIK cell, including step A PMNC (PBMC) are gathered with step B's
CIK cell culture.
A, PBMC collection have steps of：
A1, the extraction peripheral blood 50-100ml from patient's body；
PBMC is further purified in A2, lymphocyte separation medium density-gradient centrifugation method；
A3, serum-free medium are washed 2 times, obtain PBMC of the purity more than 90%.
B, CIK cell culture：
B1, by PBMC press 1~2 × 106/ ml concentration is suspended in serum-free medium, adds 1,000U/ml restructuring
People's IFN-γ, 37 DEG C, 5%CO2Cultivated in incubator；
50ng/ml CD3 monoclonal antibodies and 300U/ml recombinant human il-2 are added after B2,24h, CIK cell is stimulated
Growth and propagation；
Note：100U/ml recombined human IL-1 α now can be also added simultaneously.
B3, every 3 days half amounts change liquid or expand bottle once, and add recombinant human il-2 300U/ml；
B4, the 14d in culture, harvest CIK cell.
Wherein, 5%~20% autoserum can be also added in cell culture medium.
S2, Prepare restructuring genophore.
C, this implementation row use lepirudin 023 ludon isomers HV1, and lepirudin 023 ludon HV1 amino acid sequence is：SEQ ID
NO.1, gene order is：SEQ ID NO.2.Chemical synthesis genetic fragment, the N-terminal addition restriction enzyme sites of Nhe I, C-terminal adds
Plus the restriction enzyme sites of EcoR I.
D, this implementation row use target molecules structure for：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 cross-films section,
Its amino acid sequence is：SEQ ID NO.17, gene order is SEQ ID NO.18.Chemical synthesis genetic fragment, N-terminal addition
The restriction enzyme sites of BamH I, the C-terminal addition restriction enzyme sites of Not I.
E, the genophore using technique for gene engineering preparation coding lepirudin 023 ludon and membrane-type molecules target, by signal peptide
Sequence-lepirudin 023 ludon sequence and signal peptide sequence-target molecules sequence-Linker sequences-cross-film section sequence two sections of genes point
Two cloning sites of pIRES plasmids are not cloned into.Technique for gene engineering used is known technology, is summarized as follows：
The digestion with restriction enzyme system of standard is set up according to existing method, is then handled using Nhe I and EcoR I
PIRES plasmids, reclaim large fragment；Handled using identical restriction enzyme Nhe I and EcoR I and to reclaim lepirudin 023 ludon different
Structure body HV1 genetic fragments, then the carrier pIRES large fragment good with above-mentioned digestion be connected, construct recombinant hirudin expression matter
Grain pIRES-HV1；Convert after bacillus coli DH 5 alpha, filter out positive colony and expand.
PIRES-HV1 plasmids are purified, then using BamH I and Not I processing pIRES-HV1 plasmids, large fragment are reclaimed；Profit
Handled with identical restriction enzyme BamH I and Not I and reclaim leukine R signal peptide-Δ CD20-Linker2-
CD28 cross-films section genetic fragment, then the pIRES-HV1 carrier large fragment good with above-mentioned digestion be connected, construct while expression is weighed
The double expression plasmid pIRES-HV1/CD20 of group hirudin and membrane-type molecules target CD20.Convert after bacillus coli DH 5 alpha, screening
Go out positive colony and expand, purify pIRES-HV1/CD20 plasmids.
The pIRES-HV1/CD20 recombinant plasmids that S3, purifying are obtained through step S2, with the recombinant vector with plasmid electrotransfection
The CIK cell that method transfection procedure S1 is obtained, carries out more than cell culture 36h afterwards, and the CIK for obtaining expressing lepirudin 023 ludon is thin
Born of the same parents, transfection efficiency is obtained through flow cytomery, is needed positive cell quantity needed for calculating according to clinic, is fed back tumor patient.
The CIK cell of the expression lepirudin 023 ludon, for anticoagulant therapy, antithrombotic treatments, antineoplaston, for reducing blood lipid
Health care, anti-freezing health care, anti-bolt health care, the prevention and treatment of DVT during for performing the operation are small for heparin-induced blood
The anticoagulant therapy of plate reduction property Disease, preventing and treating unstable angina, disseminated intravascular coagulation, brain blood coagulation, thrombus are quiet
Arteries and veins inflammation and coronary artery thrombosis, preventing and treating high fat of blood, atherosclerosis.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not
On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection model of the present invention
Enclose.
Sequence table
<110>He Xiangfeng
<120>Express adoptive immunity cell of hirudin and its production and use
<160>22
<210>1
<211>84
<212>PRT
<213>Artificial sequence
<400>1
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys
5 10 15
Gly Val Gln Cys Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln
20 25 30
Asn Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly Asn
35 40 45
Lys Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn Gln Cys Val Thr
50 55 60
Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe
65 70 75
Glu Glu Ile Pro Glu Glu Tyr Leu Gln
80
<210>2
<211>252
<212>DNA
<213>Artificial sequence
<400>2
atggagtttt ggctgagctg ggttttcctt gttgctattt taaaaggtgt ccagtgtgtt 60
gtttacacgg attgtacaga atcgggtcaa aatttgtgcc tctgcgagga tagcaatgtt 120
tgcggtcaag gcaataagtg catattgggt tctaatggag agaaaaacca atgtgtcact 180
ggcgaaggta caccgaagcc tcaaagccat aatgacggcg atttcgaaga aattccagaa 240
gaatatttac aa 252
<210>3
<211>84
<212>PRT
<213>Artificial sequence
<400>3
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys
5 10 15
Gly Val Gln Cys Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln
20 25 30
Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn
35 40 45
Lys Cys Ile Leu Gly Ser Asn Gly Glu Glu Asn Gln Cys Val Thr
50 55 60
Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asn Gly Asp Phe
65 70 75
Glu Glu Ile Pro Glu Glu Tyr Leu Gln
80
<210>4
<211>255
<212>DNA
<213>Artificial sequence
<400>4
atggagtttt ggctgagctg ggttttcctt gttgctattt taaaaggtgt ccagtgtatt 60
acttacactg attgtacaga atcgggtcaa aatttgtgcc tctgcgaggg aagcaatgtt 120
tgcggtaaag gcaataagtg catattgggt tctaatggag aggaaaacca atgtgtcact 180
ggcgaaggta caccgaagcc tcaaagccat aataacggcg atttcgaaga aattccagaa 240
gaatatttac aatga 255
<210>5
<211>86
<212> PRT
<213>Artificial sequence
<400>5
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys
5 10 15
Gly Val Gln Cys Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly Gln
20 25 30
Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn
35 40 45
Lys Cys Ile Leu Gly Ser Asn Gly Lys Asp Asn Gln Cys Val Thr
50 55 60
Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe
65 70 75
Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys
80 85
<210>6
<211>261
<212> DNA
<213>Artificial sequence
<400>6
atggagtttt ggctgagctg ggttttcctt gttgctattt taaaaggtgt ccagtgtatt 60
acttacactg attgtataga atcgggtcaa aatttgtgcc tctgcgaggg aagcaatgtt 120
tgtggtaaag gcaataagtg catattgggt tctaatggaa aggacaacca atgtgtcact 180
ggcgaaggta caccgaagcc tcaaagccat aatcaaggcg atttcgaacc aattccagaa 240
gacgcttatg atgaaaaatg a 261
<210>7
<211>87
<212> PRT
<213>Artificial sequence
<400>7
Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys Ile
5 10 15
Cys Met Ser Gln Ala Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly
20 25 30
Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly
35 40 45
Asn Lys Cys Ile Leu Gly Arg Gly Asp Ser Asp Asn Gln Cys Val
50 55 60
Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp
65 70 75
Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys
80 85
<210>8
<211>264
<212> DNA
<213>Artificial sequence
<400>8
atgttctctc tgaagctgtt cgttgtcttc ttggcggttt gcatctgcat gtctcaagca 60
attacttaca ctgattgtat agaatcgggt caaaatttgt gcctctgcga gggaagcaat 120
gtttgtggta aaggcaataa gtgcatattg ggtcgcggag attctgacaa ccaatgtgtc 180
actggcgaag gtacaccgaa gcctcaaagc cataatcaag gcgatttcga accaattcca 240
gaagacgctt atgatgaaaa atga 264
<210>9
<211>87
<212> PRT
<213>Artificial sequence
<400>9
Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys Ile
5 10 15
Cys Met Ser Gln Ala Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly
20 25 30
Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly
35 40 45
Asn Lys Cys Ile Leu Gly Arg Gly Asp Met Asp Asn Gln Cys Val
50 55 60
Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp
65 70 75
Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys
80 85
<210>10
<211>264
<212> DNA
<213>Artificial sequence
<400>10
atgttctctc tgaagctgtt cgttgtcttc ttggcggttt gcatctgcat gtctcaagca 60
attacttaca ctgattgtat agaatcgggt caaaatttgt gcctctgcga gggaagcaat 120
gtttgtggta aaggcaataa gtgcatattg ggtcgcggag atatggacaa ccaatgtgtc 180
actggcgaag gtacaccgaa gcctcaaagc cataatcaag gcgatttcga accaattcca 240
gaagacgctt atgatgaaaa atga 264
<210>11
<211>87
<212> PRT
<213>Artificial sequence
<400>11
Met Ala Gly Arg Val Pro Ser Leu Leu Val Leu Leu Val Phe Pro
5 10 15
Ser Ser Cys Leu Ala Val Val Tyr Thr Asp Cys Thr Glu Ser Gly
20 25 30
Gln Asn Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly Gln Gly
35 40 45
Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn Gln Cys Val
50 55 60
Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp
65 70 75
Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys
80 85
<210>12
<211>264
<212> DNA
<213>Artificial sequence
<400>12
atggctggcc gcgtccctag cctgctagtt ctccttgttt ttccaagcag ctgtttggct 60
gttgtttaca cggattgtac agaatcgggt caaaatttgt gcctctgcga ggatagcaat 120
gtttgcggtc aaggcaataa gtgcatattg ggttctaatg gagagaaaaa ccaatgtgtc 180
actggcgaag gtacaccgaa gcctcaaagc cataatcaag gcgatttcga accaattcca 240
gaagacgctt atgatgaaaa atga264
<210>13
<211>88
<212> PRT
<213>Artificial sequence
<400>13
Met Ala Gly Arg Val Pro Ser Leu Leu Val Leu Leu Val Phe Pro
5 10 15
Ser Ser Cys Leu Ala Glu Pro Arg Val Val Tyr Thr Asp Cys Thr
20 25 30
Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Asp Ser Asn Val Cys
35 40 45
Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn
50 55 60
Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn
65 70 75
Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln
80 85
<210>14
<211>267
<212> DNA
<213>Artificial sequence
<400>14
atggctggcc gcgtccctag cctgctagtt ctccttgttt ttccaagcag ctgtttggct60
gaacctaggg ttgtttacac ggattgtaca gaatcgggtc aaaatttgtg cctctgcgag 120
gatagcaatg tttgcggtca aggcaataag tgcatattgg gttctaatgg agagaaaaac180
caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatgacgg cgatttcgaa240
gaaattccag aagaatattt acaatag267
<210>15
<211>90
<212> PRT
<213>Artificial sequence
<400>15
Met Ala Gly Arg Val Pro Ser Leu Leu Val Leu Leu Val Phe Pro
5 10 15
Ser Ser Cys Leu Ala Glu Pro Arg Val Val Tyr Thr Asp Cys Thr
20 25 30
Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Asp Ser Asn Val Cys
35 40 45
Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn
50 55 60
Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn
65 70 75
Gln Gly Asp Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys
80 85
<210>16
<211>273
<212> DNA
<213>Artificial sequence
<400>16
atggctggcc gcgtccctag cctgctagtt ctccttgttt ttccaagcag ctgtttggct 60
gaacctaggg ttgtttacac ggattgtaca gaatcgggtc aaaatttgtg cctctgcgag 120
gatagcaatg tttgcggtca aggcaataag tgcatattgg gttctaatgg agagaaaaac 180
caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatcaagg cgatttcgaa 240
ccaattccag aagacgctta tgatgaaaaa tga 273
<210>17
<211>90
<212> PRT
<213>Artificial sequence
<400>17
Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys Ile
5 10 15
Cys Met Ser Gln Ala Glu Pro Arg Ile Thr Tyr Thr Asp Cys Ile
20 25 30
Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys
35 40 45
Gly Lys Gly Asn Lys Cys Ile Leu Gly Arg Gly Asp Ser Asp Asn
50 55 60
Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn
65 70 75
Gln Gly Asp Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys
80 85 90
<210>18
<211>273
<212> DNA
<213>Artificial sequence
<400>18
atgttctctc tgaagctgtt cgttgtcttc ttggcggttt gcatctgcat gtctcaagca 60
gaacctagga ttacttacac tgattgtata gaatcgggtc aaaatttgtg cctctgcgag 120
ggaagcaatg tttgtggtaa aggcaataag tgcatattgg gtcgcggaga ttctgacaac 180
caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatcaagg cgatttcgaa 240
ccaattccag aagacgctta tgatgaaaaa tga273
<210>19
<211>87
<212> PRT
<213>Artificial sequence
<400>19
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys
5 10 15
Gly Val Gln Cys Glu Pro Arg Val Val Tyr Thr Asp Cys Thr Glu
20 25 30
Ser Gly Gln Asn Leu Cys Leu Cys Glu Asp Ser Asn Val Cys Gly
35 40 45
Gln Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu Lys Asn Gln
50 55 60
Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp
65 70 75
Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln
80 85
<210>20
<211>264
<212> DNA
<213>Artificial sequence
<400>20
atggagtttt ggctgagctg ggttttcctt gttgctattt taaaaggtgt ccagtgtgaa 60
cctagggttg tttacacgga ttgtacagaa tcgggtcaaa atttgtgcct ctgcgaggat 120
agcaatgttt gcggtcaagg caataagtgc atattgggtt ctaatggaga gaaaaaccaa 180
tgtgtcactg gcgaaggtac accgaagcct caaagccata atgacggcga tttcgaagaa 240
attccagaag aatatttaca atag 264
<210>21
<211>83
<212> PRT
<213>Artificial sequence
<400>21
Met Val Leu Ala Gln Gly Leu Leu Ser Met Ala Leu Leu Ala Leu
5 10 15
Cys Trp Glu Arg Ser Leu Ala Asn Ile Tyr Asn Cys Glu Pro Ala
20 25 30
Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gln Tyr Cys Tyr Ser
35 40 45
Ile Gln Ser Gly Gly Ser Gly Gly Pro Phe Trp Val Leu Val Val
50 55 60
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
65 70 75
Phe Ile Ile Phe Trp Val Arg Ser
80
<210>22
<211>252
<212> DNA
<213>Artificial sequence
<400>22
atggtgctgg cccaggggct gctctccatg gccctgctgg ccctgtgctg ggagcgcagc 60
ctggcaaaca tatacaactg tgaaccagct aatccctctg agaaaaactc cccatctacc 120
caatactgtt acagcataca atctggcgga agcggaggcc ccttttgggt gctggtggtg 180
gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 240
gtgaggagtt aa 252
Claims (11)
1. express the adoptive immunity cell of hirudin, it is characterised in that adoptive immunity cell behaviour source immunocyte, it is described
The lepirudin 023 ludon of adoptive immunity cell expression includes signal peptide and hirudin.
2. the adoptive immunity cell of expression hirudin according to claim 1, it is characterised in that the signal peptide behaviour source
Secreting signal peptide.
3. the adoptive immunity cell of expression hirudin according to claim 1, it is characterised in that the hirudin is leech
Plain isomers, hirudin mutant, hirudin chimera, the HIRULOG truncated, the hirudin of genetic modification or hirudin fusion
Albumen.
4. the adoptive immunity cell of expression hirudin according to claim 1, it is characterised in that the adoptive immunity cell
For the one or more in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T, the adoptive immunity cell is
Immunocyte that is natural or manually cultivating.
5. the adoptive immunity cell of expression hirudin according to claim 3, it is characterised in that the hirudin is leech
Plain isomers HV1, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.1, gene order is：SEQ ID NO.2；
Or, the hirudin is hirudin isomers HV2, and the amino acid sequence of the lepirudin 023 ludon is：SEQ ID
NO.3, gene order is：SEQ ID NO.4；
Or, the hirudin is hirudin isomers HV3, and the amino acid sequence of the lepirudin 023 ludon is：SEQ ID
NO.5, gene order is：SEQ ID NO.6；
Or, the hirudin is hirudin mutant, by the Ser32-Asn33-Gly34- of wild type hirudin HV3 peptide chains
Lys35 replaces with Arg32-Gly33-Asp34-Ser35, and the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.7,
Gene order is：SEQ ID NO.8；
Or, the hirudin is hirudin mutant, by the Ser32-Asn33-Gly34- of wild type hirudin HV3 peptide chains
Lys35 replaces with Arg32-Gly33-Asp34-Met35, and the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.9,
Gene order is：SEQ ID NO.10；
Or, the hirudin is hirudin chimera, by wild type hirudin HV1 and HV3 chimeric expression, the restructuring leech
Element amino acid sequence be：SEQ ID NO.11, gene order is：SEQ ID NO.12；
Or, the hirudin is hirudin fusion protein, and hirudin amino terminal is connected with can be by factor Ⅹa identification simultaneously
The oligopeptides of cracking, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.13, gene order is：SEQ ID NO.14；
Or, the hirudin is hirudin chimera, by wild type hirudin HV1 and HV3 chimeric expression, is fitted together to hirudin ammonia
Base end, which is connected with, to be by the oligopeptides that factor Ⅹa is recognized and is cracked, the amino acid sequence of the lepirudin 023 ludon：SEQ
ID NO.15, gene order is：SEQ ID NO.16；
Or, the hirudin is hirudin mutant, by the Ser32-Asn33-Gly34- of wild type hirudin HV3 peptide chains
Lys35 replaces with Arg32-Gly33-Asp34-Met35 (RGDM), and hirudin amino terminal is connected with can be by factor Ⅹa
The oligopeptides for recognizing and cracking, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.17, gene order is：SEQ ID
NO.18；
Or, the hirudin is hirudin isomers HV1, and hirudin amino terminal, which is connected with, to be recognized by factor Ⅹa
And the oligopeptides cracked, the amino acid sequence of the lepirudin 023 ludon is：SEQ ID NO.19, gene order is：SEQ ID
NO.20。
6. the adoptive immunity cell of expression hirudin according to claim 1, it is characterised in that the lepirudin 023 ludon group
Become second nature and express or carry out induced expression by being coupled inducible expression.
7. the adoptive immunity cell of expression hirudin according to claim 1, it is characterised in that the adoptive immunity cell
Express suicide gene/prodrug system.
8. the adoptive immunity cell of the expression hirudin according to any one of claim 1~7, it is characterised in that the mistake
Membrane surface expression after immunocyte has target molecules, and target molecules are positioned at cell membrane surface by cross-film sequence.
9. the adoptive immunity cell of expression hirudin according to claim 8, it is characterised in that the target molecules are
CD19 total length or its truncated segment, or CD20 total length or its truncated segment.
10. the preparation method of the adoptive immunity cell of hirudin is expressed described in claim 9, it is characterised in that including following step
Suddenly：Vitro culture of human source immunocyte；The genetic fragment of difference chemical synthesis coding lepirudin 023 ludon and membrane-type molecules target, and
Restriction enzyme site is added at the two ends of restructuring encoding gene；The encoding gene of lepirudin 023 ludon and membrane-type molecules target is inserted
Enter to be prepared into recombination carrier in carrier, genophore contains signal peptide sequence-leech prime sequences and signal peptide sequence-target
Molecular sequences-Linker sequences-two sections of restructuring foreign genes of cross-film section sequence；Said gene carrier is passed through into technique for gene engineering
Adoptive immunity cell is transfected, the adoptive immunity cell of expression lepirudin 023 ludon and membrane-type molecules target is obtained.
11. the application of the adoptive immunity cell of hirudin is expressed described in claim 8, it is characterised in that the expression hirudin
Adoptive immunity cell be used for anticoagulant therapy, antithrombotic treatments, antineoplaston, for antilipemic healthy, anti-freezing health care, anti-bolt protect
It is strong, the prevention and treatment of DVT during for performing the operation, for the anti-of heparin-induced thrombocytopenic disease patient
Solidifying treatment, preventing and treating unstable angina, disseminated intravascular coagulation, brain blood coagulation, thrombophlebitis and coronary artery thrombosis, prevents
Control high fat of blood, atherosclerosis.
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