CN109260462A - A kind of application of factor mutant protein and its code nucleic acid - Google Patents

Abstract

The present invention relates to the application of a kind of factor mutant protein and its code nucleic acid, it is used to prepare gene therapy medicament, including it is connect with promoter and/or termination sequence and constructs expression plasmid.Mutant protein and its code nucleic acid of the present invention have very high blood coagulation activity, can efficiently promote blood coagulation, promote body entirety coagulation function, have good gene therapy, gene editing and recombinant protein replacement therapy prospect.

Description

A kind of application of factor mutant protein and its code nucleic acid

Technical field

The invention belongs to hemorrhage therapy field, in particular to a kind of factor mutant protein and its code nucleic acid Using.

Background technique

It will lead to the generation of hemorrhagic disease when clotting factor deficiencies or other human body blood coagulation dysfunctions, wherein due to solidifying Hemorrhagic disease caused by blood factor VIII/IX (FVIII/FIX) defect is referred to as hemophilia (A type/B-mode), heavy disease The blood coagulation factor VIII of people/IX activity is often below normal 1%, and spontaneous bleeding leads to muscle hemotoncus or pass frequent occurrence Section deformity.Infusion of factors VIII/IX preparation (being at present usually blood coagulation factor VIII/IX albumen of in-vitro recombination expression) supplement is suffered from The intracorporal blood coagulation factor VIII of person/IX level is currently the only effective treatment method, but needs frequent administration.Gene is controlled Treatment is the treatment method of currently clinical test, and normal blood coagulation factor VIII/IX channel genes patient's body is expressed, from And reach raising blood coagulation factor VIII/IX level, prevent the purpose of bleeding.Although the FVIII/ in application recombination or blood plasma source FIX can effectively treat hemophilia A and B, but there are about 30% patients to generate antibody after the treatment, make failure in treatment.It is other Road blood coagulation activity drug is the optimal selection that treatment has mortifier to generate haemophiliac.But the blood coagulation clinically applied at present Factor VIIa (FVIIa), since half life is short (~2 hours), required dosage is big (90~100 μ g/kg weight), and treatment cost is high It is high, therefore how to obtain and have the characteristics that the novel bypass coagulation pathway drug of better healing effect and drug metabolism is current blood friend One urgent problem to be solved of disease treatment.

Factor is the core component of Coagulation test, when it becomes the blood coagulation with enzymatic activity by factor X activation After enzyme, catalytic pyrolysis fibrinogen is known as fibrin, makes blood clotting to achieve the purpose that hemostasis.Fibrin ferment is in blood coagulation It also plays an important role in the adjusting of reaction.Under the assistance of thrombomodulin (TM), PROTEIN C is cracked and is activated by fibrin ferment The PROTEIN C (aPC) referred to as activated, the latter is catalyzed the labile factor and VIII of inactivation activation, to inhibit the expansion of Coagulation test Increase.Therefore, under physiological environment, fibrin ferment/factor plays dual function in Coagulation test, both promotes blood clotting, Coagulation test is adjusted again, is inhibited too strong Coagulation test to occur, is led to thrombosis.Fibrin ferment is inhibited by it rapidly in the circulating cycle Object, if antithrombase (AT III) is inactivated, so that Coagulation test be made to terminate.

Prothrombin complex (including factor, plasma thromboplastin component, Stuart factor, proconvertin) is also applied In hemophilia, especially there is haemophiliachemophiliac treatment existing for mortifier, but the effect is unsatisfactory.It is applied in haemophiliac After factor, in addition to being activated and promoting Coagulation test, it can also inhibit Coagulation test with activated protein C, weaken factor Hemostatic treatment effect.Simultaneously as fibrin ferment can be inactivated by antithrombase rapidly, also limit factor in hemophilia Haemostatic effect in patient.Therefore, how factor is transformed, makes it have the selectivity for promoting solidifying function, that is to say Possess promote fibrinogen to be converted into fibrinous activity while, weaken the anticoagulant activity of activated protein C；Meanwhile The half life for extending fibrin ferment, generates resistance to the rejection ability of antithrombase, can also promote factor in hemorrhage Therapeutic effect.

Summary of the invention

Technical problem to be solved by the invention is to provide answering for a kind of factor mutant protein and its code nucleic acid With the mutant blood coagulation activity is high, has good gene therapy and recombinant protein replacement therapy prospect.

The present invention provides the applications of a kind of factor mutant protein and its code nucleic acid, are mutated using factor The nucleotides sequence of body, which is listed in, prepares gene therapy medicament, including it is connect with promoter and/or termination sequence and constructs expression Plasmid.

The present invention also provides the applications of a kind of factor mutant protein and its code nucleic acid, using mutant protein (factor R541W albumen) and its code nucleic acid are in the virus of gene editing, non-virus carrier or template.

The present invention also provides the applications of a kind of factor mutant protein and its code nucleic acid, using mutant protein (factor R541W albumen) is in the recombinant protein therapeutic agent of preparation hemophilia or other hemorrhagic diseases.

The present invention also provides the applications of a kind of factor mutant protein and its code nucleic acid, using mutant protein (factor R541W albumen) in preparing factor R541W mutant fusion protein, and be applied to hemophilia or other The recombinant protein therapeutic agent of hemorrhagic disease.

The fusion protein is human albumin, 1 antitrypsin of immunoglobulin Fc, transferrins or alpha.

The nucleotide sequence of factor mutant as shown in SEQ ID NO:1, positioned at 1621 nucleotide be T rather than C；For the amino acid sequence of factor mutant protein as shown in SEQ ID NO:2, the amino acid positioned at 541 is inhuman for Trp The Arg of wild type factor hFII；Or other any amino acid are in the change in the site.

The present invention also provides a kind of nucleic acid of factor mutant, or it is identical as the code nucleic acid length and with institute State the nucleic acid of code nucleic acid complete complementary.

The present invention also provides a kind of preparation methods of factor mutant protein, include the following steps:

(1) the human thrombin protogene that people's wild type or factor R541W are mutated is connected into carrier, obtains recombination and carries Body；

(2) above-mentioned recombinant vector is converted into host cell, obtains expressing recombination prothrombin activated R541W mutated cell clones；

(3) the above-mentioned recombinant cell clone of continuously perfused culture in serum free medium, induces recombination prothrombin activated R541W Mutant protein expression；

(4) it isolates and purifies, filter, last filling, freeze-drying obtains expressed factor R541W mutant protein.

Serum free medium in the step (3) is " SAFC Biosciences EX-CELL^TM302 " (commercialization Reagent).

Purifying in the step (4) includes just pure and mild consummate.

The present invention also provides a kind of plasmid vectors for expressing mutain (factor R541W) to carry out gene transfer, It is prepared and inspection includes the following steps:

(1) cDNA for encoding factor R541W is connected into the pcDNA3.1 plasmid containing CMV promoter.

(2) the 150 μ g of plasmid vector of the expression factor R541W of purifying is dissolved in 2mL physiological saline through caudal vein High-pressure injection enters in 4-8 weeks haemophilia A Mice Body.Injection same volume PBS is negative control.

(3) after injecting 48 hours, eye socket blood sampling detects solidifying prothrombin activity, ELISA method detection blood coagulation using freezing method Proenzyme antigen.

(4) 72 hours after injecting, dock at diameter 2mm, be placed in warm saline and observe 10 minutes internal haemorrhage situations.It is logical Cross detection hemoglobin content estimation amount of bleeding, the blood with buffer injection group amount of bleeding for 100%, after more different plasmid transductions Amount of bleeding after friendly disease mouse docking.

(5) heart extracting blood carries out thrombelastogram detection.

The pharmaceutical composition or gene therapy vector of nucleic acid or amino acid sequence of the invention, for diagnosing, prevention and/or Disease is treated, wherein the disease mainly includes bleeding caused by hemorrhagic disease or a variety of causes；Wherein, most probable bleeding Property disease be hemophilia A and B, i.e., hemorrhagic disease caused by being lacked due to hereditary coagulation factors VIII or IX, and including it In there is inhibiting antibody to generate hemophilia A and the acquired blood coagulation factor VIII caused by being generated because of mortifier B or the day after tomorrow Or IX lacks；And other hemorrhagic diseases using bypass preparation, such as neonatal coagulation disorder；Serious liver diseases；It is high Risk operations；Traumatic blood loss；Bone-marrow transplantation；Thrombopenia and dysfunction of platelet；Take orally anticoagulant urgent reverse； The defect of congenital factor V, VII, X and XI；It is obtained caused by von Willebrand disease and vWF ELISA mortifier Property von Willebrand disease, and largely damage related blood loss, cerebral hemorrhage, dysfunction of platelet.

Beneficial effect

Blood coagulation selectivity highly-active thrombin original mutant of the invention has selective procoagulant activity, claims being converted After fibrin ferment, still there is very strong blood coagulation activity, but the effect of anticoagulation system is obviously reduced, the inactivation of antithrombase is produced It is raw to resist, to generate higher facilitation to Coagulation test, promote body entirety coagulation function.The factor mutant It can be used for including the hemorrhagic diseases such as hemophilia (plasma thromboplastin component/Factor VIII deficiency) (presence or absence of mortifier) The treatment of disease has good gene therapy and recombinant protein replacement therapy prospect.

Detailed description of the invention

Fig. 1 is the schematic diagram of carrier structure of the present invention；

Fig. 2 a and b are that recombination prothrombin activated R541W promotees solidifying and activated protein C schematic diagram；

Fig. 3 a and b correct blood coagulation factor VIII/IX defect Thrombin plasma generation for recombination prothrombin activated R541W in vitro and show It is intended to；

Fig. 4 is survival rate schematic diagram after the docking of hemophilia A mouse after recombination prothrombin activated R541W processing.

Specific embodiment

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Range.

Embodiment 1

Present embodiments provide a kind of amino acid sequence of blood coagulation selection type highly-active thrombin original mutant protein such as Shown in SEQ ID NO:2.

Preparation method includes the following steps:

(1) the human thrombin original encoding gene of people's wild type or factor mutant R541W are connected into carrier, are obtained Recombinant vector；(see Fig. 1)

(2) above-mentioned recombinant vector is converted into host cell, obtains recombinant expression cell clone；

(3) above-mentioned cell clone is cultivated in serum free medium, expresses the mutant protein of high activity plasma thromboplastin antecedent Expression；

Serum free medium is " SAFC Biosciences EX-CELL^TM302 " (reagents of commercialization).To guarantee to produce Product safety, prevent blood sources preparation propagate infectious diseases, therefore apply serum free medium for mammaliancellculture, Protein expression, cell maintain cell density in target interval after logarithmic phase growth reaches stable state, maintain plasma thromboplastin antecedent Height expression.

(4) it isolates and purifies and is lyophilized, to obtain expressed factor mutain and related fusion.

After culture medium collection, isolate and purify through depth filter clarification filtration and further.Purification step is divided into just pure and mild It is consummate two stages, just pure: will to filter after clear culture solution is concentrated by ultrafiltration at 10 times through organic solvent/Detergent method inactivation Lipid-coated virus, that is, HIV1/2, HCV and HBV etc.；It is consummate: with chromatographies such as ion exchange (anion and cation) and molecular sieves Chromatography method further removes the impurity based on other albumen for remaining in product and secreting with host cell.Purifying protein is by super Liquid is changed in filter, adjusting carries out i.e. 20nm nanometer film again and removes virus filtration and be lyophilized after being formulated.Freeze-drying process be it is quick-frozen, quenching, freezing, Vacuum, trunk be dry, rear drying.Freeze-drying formula is with the inertia such as glycine, mannitol, sodium chloride, calcium chloride carbohydrate and inorganic salts Based on (group becomes glycine, mannitol, sodium chloride, calcium chloride etc.；Freeze-drying time is 30 hours).

(5) factor mutant promotees solidifying, anticoagulating active and antigen detection method.The specific clotting activity of factor It is calculated compared with antigen by blood coagulation activity and is obtained, protein activation measures factor R541W as shown in Figure 4 by Chromogenic assay and coagulates Blood selectivity highly-active thrombin original mutant.

Prothrombin activity and antigen detection method:

1. freezing method detects prothrombin activity:

Normal pooled plasma carries out 1:10,1:20,1:40,1:80,1:160,1:320 dilution, blood with OV Buffer respectively It starches sample to be tested and carries out 1:10 and 1:20 dilution, cell supernatant is not handled.Take 50 microlitres of diluted normal pooled plasmas, to be measured Plasma sample or the cell supernatant for transfecting factor expression vector, are added 50 microlitres of factor matrix plasmas, and PT is added Reagent records setting time on the semi-automatic coagulo meter of ST4 (Stago company, France).It is diluted solidifying with normal pooled plasma 1:10 Blood zymogen activity is 100%, establishes mark with the log value of the corresponding setting time of different dilutions and the corresponding active log value that obtains Directrix curve brings the value of sample to be tested into calculating if coefficient R 2 is greater than 0.95, and the factor for obtaining sample to be tested is living Property.

2. the antigen of double-antibody method detection factor:

With coating buffer (1.59g/L sodium carbonate and 2.94g/L sodium bicarbonate, pH 9.6) by coated antibody (F9ELISA reagent Box, Affinity Biologicals, EIA9-0035R1) 1:100 dilution, the dilution hole antibody 100ul/, incubation at room temperature 2 is added Hour.Repeated washing 3 times.By normal pooled plasma sample diluting liquid (23.8g HEPES (free acid)/L, 5.84g/L NaCl, 3.72g/L Na2EDTA, 10g/L BSA, 0.1% Tween-20, Ph 7.2) 1:100 two-fold dilution is distinguished to 1:3200. For test plasma sample with 1:200,1:400 and 1:800 dilution, cell supernatant is respectively stoste, 1:10 and 1:100 dilution.Often The just mixed-blood slurry or sample to be tested that 100ul has diluted is added in hole, is placed at room temperature for 90min.Repeated washing 3 times.It will test antibody use Sample diluting liquid 1:100 dilution, every hole are added the detection antibody of 100ul diluted, are placed at room temperature for 90min.Repeated washing 3 It is secondary.The OPD substrate of 100ul is added in every hole, and after stable yellow to appear (about 5-10min), the termination of 100ul is added in every hole Liquid.Absorbance is read under the wavelength of 450nm with microplate reader.Standard curve is established, and calculates the antigen value of sample to be tested.

Factor and thrombin activity testing result are as shown in Figure 2 a, the wild type and R541W mutant of recombinant expression The tired antigen levels of factor are seemingly 60% or so, and two kinds of albumen are living to the cracking of small molecule substrates (chromophoric substrate) Property is also close, is 65~66%, but blood coagulation activity R541W mutant is decreased slightly as that low (wild type 65.1%, R541W are 38.6%).

(6) catalyzed by thrombin cracks activated protein C

Factor is climbed very much snake venom activation and is known as the fibrin ferment with enzymatic activity, by the albumen of fibrin ferment and various concentration Reaction product and chromophoric substrate S2765 are incubated for, after 37C incubation by detecting its cracking energy to chromophoric substrate by C jointly Power estimates PROTEIN C by the ability of activated by thrombin.As a result as shown in Figure 2 b, wild type fibrin ferment can efficient catalytic protein C It is changed into the PROTEIN C (aPC) of activation, but the activity of factor mutant R541W crack protein C is substantially less than wild blood coagulation Enzyme, Km is higher than wild fibrin ferment, and Kcat is lower than wild fibrin ferment.

(7) factor mutant corrects hemophilia A patients' Thrombin plasma and generates defect

Fibrin ferment generates test (thrombin generation test, TGT): being raw for monitoring fibrin ferment in blood plasma At the comprehensive experiment of ability.Activator (containing tissue factor and phosphatide) is added in blood plasma and starts Coagulation test, is added solidifying The catalyzed by thrombin substrate of the fluorogenic substrate of hemase specificity, generation releases fluorophor, is read using FLUOROSKAN fluorescence The fluorescence signal that number instrument dynamic monitoring generates generates experiment software using matched fibrin ferment and converts the signal into a fibrin ferment Formation curve.The main several parameter evaluation fibrin ferment generative capacities for passing through curve: (1) delay time (lag time), i.e., from The time required to reaction starts to fibrin ferment to start to generate；(2) peak value (peak), that is, the fibrin ferment maximum generated；(3) when reaching peak Between (time to peak, ttpeak), i.e., to the time required to fibrin ferment reach to peak value since reaction；(4) fibrin ferment generates potentiality (endogenous thrombin potential, ETP), i.e. area under fibrin ferment formation curve, reaction fibrin ferment generate total Amount.

Factor mutant is added in (weary blood coagulation factor VIII) in the blood plasma of hemophilia A patients, and (concentration is normal Physiological concentration 1.5 or 3 times of 150ug~300/mL), fibrin ferment, which is carried out, in the case where TM exists and PROTEIN C is activated generates inspection It surveys, sees Fig. 3.After 5pM tissue factor starting Coagulation test is added, the starting of external source coagulation pathway promotes fibrin ferment to generate (F8- def)；But when promoting to coagulate anticoagulant comprehensive effect in addition thrombomodulin (TM) analogue body, the blood coagulation of exogenous route activation Enzyme generation is suppressed significantly (F8-def with TM)；It is subject to 1.5 times (Fig. 3 a) and 3 times in the blood plasma of weary blood coagulation factor VIII (Fig. 3 b) is after the wild type factor or R541W mutant of physiological activity, even if fibrin ferment generation still has with the presence of TM It improves, but the generation of the fibrin ferment of R541W is more significant compared with wild type proenzyme.

Embodiment 2

Factor mutant R541W treats hemophilia A mouse

(1) the wild type factor of purifying or mutant R541W are injected into A type with 1.5 times of physiological amounts (150ug/mL) Hemophilia mouse.Select 4-8 weeks hemophilia mouse, by tail vein by albumen be dissolved in 2mL physiological saline inject respectively it is 3 small Mouse.It is negative control that PBS, which injects 3 mouse,.

(2) by after mouse anesthesia, it is to dock at 2mm in diameter, is placed in cage and observes its survival rate.As shown in Figure 4, it coagulates Blood active selectable factor mutant R541W can correct clotting defect caused by Factor VIII deficiency in vivo, Bleeding is reduced, survival rate is improved.

SEQUENCE LISTING

<110>the unrestrained Ding Qiulan of the military text of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine Wang Xue cutting edge of a knife or a sword

<120>application of a kind of factor mutant protein and its code nucleic acid

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1869

<212> DNA

<213>artificial sequence

<400> 1

atggcgcacg tccgaggctt gcagctgcct ggctgcctgg ccctggctgc cctgtgtagc 60

cttgtgcaca gccagcatgt gttcctggct cctcagcaag cacggtcgct gctccagcgg 120

gtccggcgag ccaacacctt cttggaggag gtgcgcaagg gcaacctgga gcgagagtgc 180

gtggaggaga cgtgcagcta cgaggaggcc ttcgaggctc tggagtcctc cacggctacg 240

gatgtgttct gggccaagta cacagcttgt gagacagcga ggacgcctcg agataagctt 300

gctgcatgtc tggaaggtaa ctgtgctgag ggtctgggta cgaactaccg agggcatgtg 360

aacatcaccc ggtcaggcat tgagtgccag ctatggagga gtcgctaccc acataagcct 420

gaaatcaact ccactaccca tcctggggcc gacctacagg agaatttctg ccgcaacccc 480

gacagcagca ccacgggacc ctggtgctac actacagacc ccaccgtgag gaggcaggaa 540

tgcagcatcc ctgtctgtgg ccaggatcaa gtcactgtag cgatgactcc acgctccgaa 600

ggctccagtg tgaatctgtc acctccattg gagcagtgtg tccctgatcg ggggcagcag 660

taccaggggc gcctggcggt gaccacacat gggctcccct gcctggcctg ggccagcgca 720

caggccaagg ccctgagcaa gcaccaggac ttcaactcag ctgtgcagct ggtggagaac 780

ttctgccgca acccagacgg ggatgaggag ggcgtgtggt gctatgtggc cgggaagcct 840

ggcgactttg ggtactgcga cctcaactat tgtgaggagg ccgtggagga ggagacagga 900

gatgggctgg atgaggactc agacagggcc atcgaagggc gtaccgccac cagtgagtac 960

cagactttct tcaatccgag gacctttggc tcgggagagg cagactgtgg gctgcgacct 1020

ctgttcgaga agaagtcgct ggaggacaaa accgaaagag agctcctgga atcctacatc 1080

gacgggcgca ttgtggaggg ctcggatgca gagatcggca tgtcaccttg gcaggtgatg 1140

cttttccgga agagtcccca ggagctgctg tgtggggcca gcctcatcag tgaccgctgg 1200

gtcctcaccg ccgcccactg cctcctgtac ccgccctggg acaagaactt caccgagaat 1260

gaccttctgg tgcgcattgg caagcactcc cgcaccaggt acgagcgaaa cattgaaaag 1320

atatccatgt tggaaaagat ctacatccac cccaggtaca actggcggga gaacctggac 1380

cgggacattg ccctgatgaa gctgaagaag cctgttgcct tcagtgacta cattcaccct 1440

gtgtgtctgc ccgacaggga gacggcagcc agcttgctcc aggctggata caaggggcgg 1500

gtgacaggct ggggcaacct gaaggagacg tggacagcca acgttggtaa ggggcagccc 1560

agtgtcctgc aggtggtgaa cctgcccatt gtggagcggc cggtctgcaa ggactccacc 1620

tggatccgca tcactgacaa catgttctgt gctggttaca agcctgatga agggaaacga 1680

ggggatgcct gtgaaggtga cagtggggga ccctttgtca tgaagagccc ctttaacaac 1740

cgctggtatc aaatgggcat cgtctcatgg ggtgaaggct gtgaccggga tgggaaatat 1800

ggcttctaca cacatgtgtt ccgcctgaag aagtggatac agaaggtcat tgatcagttt 1860

ggagagtag 1869

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<212> PRT

<213>artificial sequence

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Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu

35 40 45

Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr

50 55 60

Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala Thr

65 70 75 80

Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro

85 90 95

Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu

100 105 110

Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile Glu

115 120 125

Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn Ser

130 135 140

Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro

145 150 155 160

Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val

165 170 175

Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr

180 185 190

Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro

195 200 205

Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg

210 215 220

Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala

225 230 235 240

Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln

245 250 255

Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val

260 265 270

Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu

275 280 285

Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp

290 295 300

Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr

305 310 315 320

Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys

325 330 335

Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu

340 345 350

Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser

355 360 365

Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys

370 375 380

Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp

385 390 395 400

Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn

405 410 415

Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr

420 425 430

Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr

435 440 445

Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala

450 455 460

Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp Tyr Ile His Pro

465 470 475 480

Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gln Ala Gly

485 490 495

Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr

500 505 510

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

515 520 525

Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr Trp Ile Arg Ile

530 535 540

Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg

545 550 555 560

Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser

565 570 575

Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu

580 585 590

Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg

595 600 605

Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe Gly Glu

610 615 620

Claims (6)

1. the application of a kind of factor mutant protein and its code nucleic acid, it is characterised in that: apply factor mutant Nucleotides sequence be listed in and prepare gene therapy medicament, including it is connect with promoter and/or termination sequence and constructs expression matter Grain.

2. the application of a kind of factor mutant protein and its code nucleic acid, it is characterised in that: using mutant protein and its Code nucleic acid is in the virus of gene editing, non-virus carrier or template.

3. the application of a kind of factor mutant protein and its code nucleic acid, it is characterised in that: using mutant protein in system The recombinant protein therapeutic agent of standby hemophilia or other hemorrhagic diseases.

4. the application of a kind of factor mutant protein and its code nucleic acid, it is characterised in that: using mutant protein in system Standby factor R541W mutant fusion protein, and the recombinant protein for being applied to hemophilia or other hemorrhagic diseases is controlled Treat drug.

5. the application of a kind of factor mutant protein according to claim 4 and its code nucleic acid, it is characterised in that: The fusion protein is human albumin, 1 antitrypsin of immunoglobulin Fc, transferrins or alpha.

6. the application of a kind of factor mutant protein according to any one of claims 1-4 and its code nucleic acid, Be characterized in that: the nucleotide sequence of factor mutant as shown in SEQ ID NO:1, positioned at 1621 nucleotide be T and Non- C；The amino acid sequence of factor mutant protein as shown in SEQ ID NO:2, positioned at 541 amino acid be Trp rather than The Arg of people's wild type factor hFII；Or other any amino acid are in the change in the site.