CN109439679A - A kind of high activity recombinant human plasma thromboplastin component fusion protein and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of high activity recombinant human plasma thromboplastin component fusion proteins and preparation method thereof.Including promoter, Human factor IX protein gene, the Fc section gene and terminator of human immunoglobulin(HIg) IgG1.People's FIX-Fc antigen-4 fusion protein gene expression cassette is transferred in mammalian genome using carrier and obtains transgenic animals by the present invention, and people's FIX-Fc amalgamation and expression albumen is obtained from transgenic animals milk.Method according to the present invention, the people FIX activity expressed in animal milk can be used for the production of Human factor IX than about 12 times of natural FIX high up to 6IU/ml, average specific up to 3000IU/mg living.

Description

A kind of high activity recombinant human plasma thromboplastin component fusion protein and preparation method thereof

Technical field

The invention belongs to field of transgenic technology, and in particular to a kind of high activity recombinant human plasma thromboplastin component fusion protein and Preparation method.

Background technique

Hemophilia B is a kind of recessive hemorrhagic disease of X-linkage, and the disease incidence in male neonate is about It is 1/25000, caused by causing internal FIX albumen synthesis to lack because of patient's plasma thromboplastin component (FIX, Factor IX) gene mutation, Patient's Yi Fasheng hematostaxis, patient with severe symptoms often lead to deformity because of arthrorrhagia, cerebral hemorrhage etc., influence quality of life and longevity Life.Currently, hemophilia B patient relies primarily on venoclysis supplemented with exogenous plasma thromboplastin component to treat, this kind of product includes The plasma thromboplastin component product of blood sources and recombinant sources.

The Fc segment of human immunoglobulin(HIg) IgG can by mediated in conjunction with FcRn with the process recycling of its fusion protein, To extend the half-life period of target protein, extensive use is had been obtained in this method in the production of drug.Hundred strong companies Alprolix is the FIX-Fc fusion protein drug of first listing, clinical studies show its have compared with the FIX of wild type etc. Same activity, but half-life period dramatically increases 2-3 times, effectively shortens the medication interval (NCT01425723) of patient.

There are a series of functions acquired (gain-of-function mutation) mutation can make blood coagulation for plasma thromboplastin component Activity obtains different degrees of increase.For example, the saltant type FIX albumen found in an Italian families for 2009,384 Position is become leucine (R384L, also referred to as FIX Padua) from arginine, which causes FIX blood coagulation activity to increase 5-10 Times.The saltant type has been used for gene therapy research at present, and clinical test achieves good effect (PCT/EP2009/ 061935).Separately have in the article for being published in the Journal of Biological Chemistry for 1999 and describes Another function gain mutation (R384A) of FIX, in vitro study show that the mutation increases 2-3 times compared to wild-type activity [Jinli Chang,Jianping Jin,Pete Lollar,et.al.Changing Residue 338in Human Factor IX from Arginine to Alanine Causes an Increase in Catalytic Activity [J].The Journal of Biological Chemistry,1998,273(20):12089–12094]；Transgenic animals The inside and outside blood coagulation activity of the mutant protein of mammary carcinoma model can be improved 2.5-3 times [YAN J-B, WANG S, HUANG W-Y, et al.Transgenic Mice Can Express Mutant Human Coagulation Factor IX with Higher Level of Clotting Activity[J].Biochemical Genetics,2006,44(7-8):347- 358.].Coagulation factor can be improved in the application that Gene Therapy for Hemophilia B and recombinant protein medicine are researched and developed in function gain mutation body The activity expression of IX is horizontal, has important application value.

Currently, the usual yield of recombinant human blood coagulation factor is lower, half-life short, expensive.And it is solidifying with recombinant cell production Blood factor IX is compared, the production cost relative moderate of animal bioreactor, is a potentiality side of cheap mass production FIX To.The pig galactophore biological reactor of cow's milk gland bioreactor and ProGenes company that inventor utilizes can people FIX table It reaches, in clinical precursor and activity test in vitro confirms that generated FIX has efficient blood coagulation activity.But in published any report The precedent using galactophore biological reactor expression FIX fusion protein is not had been reported that in road also, unknown galactophore biological reactor table yet Whether still there is FIX activity up to FIX fusion protein and high expression level can be maintained.

Summary of the invention

The purpose of the present invention is to provide a kind of high activity recombinant human plasma thromboplastin component fusion proteins and preparation method thereof.

A kind of high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene, which is characterized in that including promoter, people's blood coagulation because Sub- IX protein gene, the Fc section gene and terminator of human immunoglobulin(HIg) IgG1.

The promoter is this field routine, as long as can start in mammalian cells the expression of downstream gene i.e. It can, it is preferred that start the high promoter of expression efficiency in mammalian milk glandular tissue, it is preferred that beta-casein gene opens Mover, more preferably goat B-casein gene promoter.

The Human factor IX is high blood coagulation activity mutant, and nucleotide sequence is as shown in SEQ ID NO:1, position 1960-1961 nucleotide is GC rather than CG.

The plasma thromboplastin component is high activity mutant, and amino acid sequence is as shown in SEQ ID NO:2, position 384 Amino acid be A rather than R.

The Fc section nucleic acid sequence of the human immunoglobulin(HIg) IgG1 is as shown in SEQ ID NO:3, amino acid sequence such as SEQ Shown in ID NO:4.

It include joint sequence between the Fc of the human coagulation factor IX gene and human immunoglobulin(HIg) IgG1 section gene.

The connector is the peptide indicated by amino acid sequence (GGGGS) n, and wherein n is the integer of 1-20, and the connector is also Peptide comprising one section of flexible polypeptide EFAGAAAV.

It is a kind of to produce high activity recombinant human plasma thromboplastin component fusion protein using mammary gland bioreactor of transgenic animals Method, by containing the high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene carrier through pronuclear microinjection be integrated by Smart ovum render transgenic mammal galactophore expresses and secretes the external source recombinant blood coagulation factor IX fusion protein；The lactation is dynamic Object is mouse, ox or sheep.

A kind of preparation method of the mammary gland-specific expression vector of the mutain of high activity plasma thromboplastin component, including walk as follows It is rapid:

(1) the Fc segment cDNA of the human coagulation factor IX gene of plasma thromboplastin component (R384A) mutation and human IgG1 is passed through Joint sequence connection is built into carrier, obtains recombinant plasmid；

(2) above-mentioned recombinant vector microinjection is imported into mouse fertilized egg, prepares hFIX-Fc transgenic mice；

(3) PCR method identifies positive mice, takes positive mice mating breeding, obtains positive female rat milk；

(4) the hFIX-Fc expressing fusion protein situation in mouse milk is detected.

The recombinant vector is eukaryotic expression vector pcDNA3.1 (-).

Beneficial effects of the present invention: the present invention realizes FIX fusion protein in galactophore biological reactor expression system for the first time High efficient expression, it was demonstrated that galactophore biological reactor can correctly express FIX-Fc fusion protein, and compared to non pregnant women, FIX- Fc fusion protein has higher specific activity.

Detailed description of the invention

Fig. 1 is that fusion DNA vaccine constructs Human factor IX-Fc amalgamation and expression gene schematic diagram.

Fig. 2 is vector construction schematic diagram of the present invention.

Fig. 3 is the corresponding plasmid hFIX-Fc fusion protein ELISA testing result of HepG2 cell transient transfection.

Fig. 4 is hFIX-Fc fusion protein ELISA testing result in transgenic mouse milk.

Fig. 5 is hFIX-Fc fusion protein activity expression result in transgenic mouse milk.

Fig. 6 is hFIX-Fc fusion protein specific activity result in transgenic mouse milk.

Specific embodiment

The present invention is specifically illustrated below with reference to example, please be understand before this, it is unless otherwise defined, used herein all Technical and scientific term has and the normally understood identical meaning of one skilled in the art institute of the present invention.Although with this The similar or equivalent method and material of those of text description can be used for implementing or test the present invention, but suitable side is described below Method and material.When conflict, it is subject to including present specification defined herein.In addition, it should also be understood that, material, method and example are only Illustrative, those skilled in the art can make various modifications or changes to the present invention, and such equivalent forms are equally fallen within The scope of the appended claims of the present application.

Embodiment 1

One, segment synthesizes

1) amplification of human IgG1 Fc cDNA segment

With upstream primer Fc-F:5 '-GAGCCCAAATCTTGTGACAAAACT-3 '；Downstream primer Fc-R:5 '- TTTACCCGGAGACAGGGAGAGGCT-3 ' carries out PCR amplification using human peripheral blood cell cDNA as template.Reaction system is such as Under:

4 reaction tubes are done in total, and reaction condition is 94 DEG C of 5min；(94℃45sec,56℃45sec,72℃3min)×30 A circulation；72℃10min.The hIgG1Fc cDNA segment for expanding about 0.7kb after mixing reaction solution, takes 2 μ L to send to Shanghai beauty Lucky biotech company is sequenced, and remaining product product cuts glue and with kit recycling 0.7kb's through agarose electrophoresis HIgG1Fc CDNA segment.Then sequencing gained sequence is compared with the sequence AF150959.1 in Genebank database It is right, discovery 1 base of difference is compared, being investigated the site is SNP site, does not influence the function of Fc segment.

Two, vector construction

1) building of pcDNA3.1 (-)-F9Fc plasmid

Building process is as shown in Figure 1.Restriction enzyme used in molecular cloning and ligase are purchased from NEB public affairs Department.

FIXFc is expanded by the method for fusion DNA vaccine with corresponding primer first and merges segment, Ppum is introduced in upstream primer I restriction enzyme site introduces BamH I restriction enzyme site in downstream primer, introduces joint sequence between FIX and Fc gene, we construct 3 FIXFc merge segments, according to being referred to as F9Fc1, F9Fc2, F9Fc3 after the difference of joint sequence.

Primer information is as follows:

Reaction system is as follows:

(specifying information can refer to document YAN J-B, WANG S, HUANG W-Y, et to pcDNA3.1 (-)-FIX carrier al.Transgenic Mice Can Express Mutant Human Coagulation Factor IX with Higher Level of Clotting Activity [J] .Biochemical Genetics, 2006,44 (7-8): 347-358.) conduct Skeleton carrier is separately connected above-mentioned segment with Ppum I enzyme and BamH I linearization for enzyme restriction, obtains corresponding pcDNA3.1 (-)- CMV-hFIXFc vector plasmid, hereinafter referred to as CMV-F9Fc1, CMV-F9Fc2, CMV-F9Fc3.

Digestion system are as follows: 10 μ g, Ppum I (10U/ μ L) of plasmid, 3 μ L, 10 × Cutsmart buffer10 μ L mends H₂O is extremely 100 μ L, 37 DEG C of water-bath digestions are stayed overnight.Digestion products recycle the DNA fragmentation of 7.5Kb with kit, in TE through agarose electrophoresis Dissolution.

Coupled reaction system is the 0.7kb IgG1-Fc cDNA segment of 200ng, the carrier segments of 100ng 7.5kb, 5 μ L 2 × ligation buffer, adds H₂O supplies volume to 10 μ L.16 DEG C of connections overnight, are transformed into E.coli DH 5alpha sense By state cell, Amp resistant panel is applied, chooses spot, amplification cultivation, alkaline lysis extracts plasmid, digestion identification, sequencing confirmation.

Vector construction process of the present invention is as shown in Figure 2.

Three, cell transfecting

(1) cell strain: HepG2 cell strain is purchased from Chinese Academy of Sciences Shanghai OEG cell institute；

(2) cell culture medium: DMEM in high glucose, calf serum are purchased from Gibco, Min Hai company respectively.Lipofectamine 3000 are purchased from Invitrogen company；

(3) cell culture processes: taking HepG2 cell cryopreservation tube in liquid nitrogen, quickly sets freeze thawing in 37 DEG C of water.Frozen-thawed cell 1000rpm 5min, incline supernatant, it is seen that cell precipitation, then plus about 5mL full nutrient solution, cell is gently resuspended.Culture bottle is added In, visible finely dispersed cell under mirror.37 DEG C are placed, 5%CO₂And it is cultivated under the conditions of saturated humidity.HepG2 cell is with containing The DMEM culture medium culture of 10% calf serum.Two days later with 0.25% pancreatin/0.01%EDTA by cell dissociation in culture bottle Under, it is transferred to and continues to cultivate in 6 orifice plates, culture medium 1.5mL is added in every hole, and next day is for transfecting.

(4) gene transfects

1) preparation of transfection liquid:

Sterilizing EP pipe is used to prepare transfection liquid A liquid and B liquid.

A liquid: the corresponding plasmid of 2.5 μ g (is EGFP control plasmid, CMV-FIX control plasmid, CMV-F9Fc1, CMV- respectively F9Fc2, CMV-F9Fc3) and 5 μ L P3000 with DMEM in high glucose culture solution, constant volume is diluted to 100 μ L respectively, jog mixes.

B liquid: 3.75 μ L Lipofectamine, 3000 plasmalogen transfection reagent (Invitrogen company) is cultivated with DMEM Liquid constant volume is diluted to 100 μ L, and jog mixes.

A liquid and B liquid are mixed, gently shakes up, is stored at room temperature 5min to get transfection liquid.

2) it transfects:

HepG2 cell changes 1mL fresh culture, and 200 μ L transfection liquids are added in every hole, rock back and forth several times.Keep culture solution complete All standing cell.37 DEG C, 5%CO₂Lower culture 48h.

3) culture solution is collected, with the content of ELISA measurement Human factor IX.

Four, Human factor IX-Fc expressing fusion protein measures in HepG2 cell

Select the expression of FIX-Fc fusion protein in ELISA method detection cell liquid supernatant.

ELISA detection kit chooses the Factor IX Human from Abcam company, the U.S. (Cambridge, MA) ELISA Kit (ab108831), detection method is referring to product description.Testing result is as shown in Fig. 3, plasmid CMV-F9Fc2 Compared to wild type expression quantity highest, chooses the plasmid and carry out subsequent research.

Five, the preparation of hFIX-Fc amalgamation and expression transgenic mice and the acquisition of milk

According on HepG2 cell as a result, we select the subsequent transgenic mice of CMV-F9Fc2 plasmid construction.It will CMV-F9Fc2 plasmid is linearized with Sal I, then obtains transgenic mice with microinjection mode.By to rat-tail DNA's PCR detection, confirms positive mice.The specific method is as follows:

One) fertilized eggs donor mouse

Using intraperitoneal injection method, to inject 10U respectively to female mice pregnant on 1st for female mice (7 week old) during selection emotionally Horse serum promoting sexual gland hormone (PMS), in third day, the female mice intraperitoneal injection 10U people to oneself injection PMS promotees chorion sex hormone (HCG), it is then mated with male mouse with 1:1.Check female mice yin bolt in the morning on the 4th, has negative bolt to illustrate that oneself, through mating, will there is negative bolt Female mice as fertilized eggs donor mouse.

Two) pseudopregnant recipients mouse

On the donor female rat HCG injection same day, select the male mouse of estrous female mice and vasectomy with 1:1 or 2:1 ratio Example mates.Second day checks negative bolt with donor mouse together, that is, regard the female mice for having negative bolt as pseudopregnant recipients mouse.

Three) acquisition and microinjection of fertilized eggs

1) acquisition of fertilized eggs

Before experiment, article high pressure sterilization used, experimental room disinfection.

Culture solution prepares:

In vitro culture: under super-clean bench environment, KSOM culture solution (being free of hyaluronic acid) is drawn in disposable modeling with liquid-transfering gun Expect to drip several drops (culture dish back side corresponding position number) of about 40 μ L on different location in culture dish, add embryo Grade paraffin oil all covers KSOM drop, culture dish is then placed in 37 DEG C, 5%CO₂Incubator balance its humidity and pH Value, so that the fertilized eggs placement after washing is carried out in vitro culture in this culture dish.

In vitro operation: M2 culture solution, the drop of M2 containing hyaluronidase are added dropwise in culture dish, the fallopian tubal of clip is put into Wherein.

Cervical dislocation puts to death fertilized eggs donor mice, and back side is placed in a culture dish upward and covers, and 75% ethanol disinfection waits for The fur of incision is longitudinally cut off in the nearly middle part vertebra side of mouse back side, is separated skin with stomach wall with eye scissors, then by abdomen Wall equally cuts off an osculum, is proposed ovary, fallopian tubal and uterus with ophthalmic tweezers, then completely cut fallopian tubal with eye scissors Come, is placed in the drop for filling hyaluronidase culture solution, equally takes the fallopian tubal of opposite side and other donor female mice.

Ampulla of uterine tube is torn with ophthalmic tweezers under stereoscope, it successively will fertilization with ovum shifting tube (Transfer pipette) Ovum collecting is transferred in M2 culture solution.Fertilized eggs are picked up one by one in M2 drop, are moved into next drop, it so will be each Fertilized eggs clean for several times, will also remove hyaluronic acid as far as possible while selecting suitable egg cell in this way and (note: on objective table There is thermal light source or place constant temperature micro-hotplate, be in cell in suitable temperature environment).

From CO₂The culture dish containing culture drop and paraffin oil is taken out in incubator, with ovum shifting tube by washed fertilization Ovum is transferred in KSOM drop, if fertilization female pronucleus development is still not up to perfect condition, can be put into CO₂One timing of incubator culture Between.

This every oviductus lateralis of process donor mouse can obtain 20,80 pieces of super row's fertilized eggs and differ.Ideal fertilized egg cell's shape State is full normal, and oolemma is complete, and male pronucleus and female pronucleus are clear.

2) microinjection of fertilized eggs

Embryo's fixing pipe (holding pipette) of the fixation fertilized eggs first prepared is placed in microinjection instrument Left side.DNA solution is added from the prefabricated injection needle tail portion (Microinjection needle), is checked under stereoscope There is bubble-free to generate (Plasmid DNA is diluted to 4ng/ μ L, is centrifuged 1-2min after thawing before injecting) in DNA solution.It then will injection Needle is placed in the right side of microinjection instrument, and makes in injection needle in barotropic state.

20-30 μ L M2 is dripped in shrinkage pool glass slide center, paraffin oil is covered, fertilized eggs to be injected is moved into ovum shifting tube.It will Glass slide moves on the objective table of micromanipulation instrument.

It is focused under low power lens, fertilized eggs is placed in the appropriate location in the visual field with ovum holding tube, then by ovum holding tube and note It penetrates needle and is adjusted to suitable position (with slide bottom plane in 10 ° of angles), make ovum holding tube, injection needle and fertilization by finely tuning left and right arms Ovum is in straight line and high-visible.

The tip of injection needle is gently collided on ovum holding tube, to touch a little tip of disconnected injection needle, to allow DNA solution to overflow Out.

Under high power lens, with ovum holding tube, pressure-vaccum fertilized eggs are adjusted to suitable position repeatedly, are intended to injection by negative pressure Fertilized eggs are on the top of left side ovum holding tube.The optimum position of fertilized eggs is that polar body is directly on top, and the position of male pronucleus is corresponding Center and nearly right side in ovum holding tube.

Injection needle is quickly pierced into male pronucleus, does not touch kernel, it is appropriate to pressurize, DNA is injected, is expanded to core for original body Injection needle is extracted rapidly after long-pending one times, fertilized eggs are shifted.Repeatedly, other fertilized eggs are injected one by one, to the greatest extent It may complete within a short period of time.

The fertilized eggs that injection finishes can be placed in 37 DEG C, 5%CO₂In incubator cultivate a period of time, pick out form compared with Good fertilized eggs are for transplanting.

3) transplanting of fertilized eggs

Intraperitoneal injection method, it is by the dosage of 1mg/10g weight that false pregnancy female rat is numb with Nembutal sodium solution (10mg/mL) It is liquor-saturated.Oneself back of mice through anaesthetizing is placed in culture dish upwards to cover, in last root rib cage by recess unhairing, 75% ethyl alcohol Disinfection.Routinely surgical operation row at mouse disinfection with the longitudinal incision parallel with vertebra, surgical forceps protrudes into abdominal cavity, by fat and Ovary, fallopian tubal and the uterus being wrapped in membrane vesicle propose together, and are fixed with fatty tweezer.

Fertilized eggs suck in same ovum shifting tube after injecting, and bubble is sucked in pipe as mark.

The cyst membrane between ovary and fallopian tubal is gently torn with ophthalmic tweezers at the sparse place of blood vessel, tweezers are protruded into cyst membrane, Lightly ovary and fallopian tubal are separated, fimbriae tubae portion is clamped, mentions at breach, find free-end.It will be filled with fertilized eggs Ovum shifting tube is gently inserted into pars infundibularis from fimbriae tubae portion, fertilized eggs is blown into fallopian tubal at leisure with mouth, it is seen that fallopian tubal is bright Expand aobviously and ovum shifting tube in bubble move in umbrella opening.

Ovum shifting tube is gently extracted from fallopian tubal, ovary and fallopian tubal are put back into abdominal cavity, puts back to cleaning after sewing up a wound In mouse cage, pay attention to keeping warm, routinely be raised after its revival.19,20 days after implantation, fertilized eggs can develop into mouse birth.

Four) identification of transgenic mice

1) rat-tail tissue DNA extracts

Three weeks clip l cm tail tissues are placed in 1.5mL EP pipe after mouse birth.

In EP pipe plus 400 μ L digestive juice buffer, 10 μ L (20mg/mL) Proteinase Ks, 56 DEG C of digestion are stayed overnight.

Add isometric phenol, vibrate, 13000rpm is centrifuged 12min after mixing.

Supernatant is taken to add isometric phenol: chloroform: 13000rpm centrifugation 6min after isoamyl alcohol (25:24:1) oscillation mixes.It takes Supernatant adds isometric chloroform: 13000rpm centrifugation 4min after isoamyl alcohol (24:1) oscillation mixes.

After taking supernatant that 1/10 volume 3mol/L NaAC and 2 times of volume dehydrated alcohol mixings is added to be placed at room temperature for 10min, 13000rpm is centrifuged 10min.

80% ethyl alcohol is washed 2 times, and vacuum is dissolved in 200 μ L pH 8.0TE after draining.

1) PCR method identifies hFIX-Fc positive mice, and PCR system see the table below.

Detection primer information:

PCR system:

Six, the detection that hFIX is expressed in transgenic mouse milk

Transgenosis female rat is taken to breed, after giving a birth, using intraperitoneal injection method, with Nembutal sodium solution (10mg/mL) Nursing period (8-10 days or so) female rat is anaesthetized by the dosage of 1mg/10g weight.Mammary gland injection oxytocins (0.03IU) after anesthesia, After waiting 5min, mammary gland is squeezed with thumb index finger, overflows milk from nipple, collected lotion with liquid-transfering gun and managed in 0.5ml EP It is interior.

Select the Factor IX Human ELISA Kit for being purchased from U.S. Abcam company (Cambridge, MA) (ab108831) kit and Instrumentation Laboratory Company company APTT activity detection kit (HeemosILSynthAsil, 0020006800, USA)；Prothrombin assay instrument is Instrumentation Laboratory Company, Products (ACL TOP720)；Detect the Human factor IX protein expression in mouse milk Kit specification is shown in situation, concrete operations, measures result referring to attached drawing 4-6.

By attached drawing 4-6 as it can be seen that positive female rat 20 is obtained in experiment, hFIX activity reaches as high as 6.2IU/ in mouse milk Ml, much higher than the 1IU/ml of human plasma.Specifically, the specific activity of hFIX is significantly increased, the ratio compared to wild type hFIX Active 200-250IU/mg, specific activity of the invention is up to 2300-3500IU/mg, mean value 3000IU/mg.

Claims (10)

1. a kind of high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene, which is characterized in that including promoter, human blood coagulation IX protein gene, the Fc section gene and terminator of human immunoglobulin(HIg) IgG1.

2. high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene according to claim 1, which is characterized in that the starting Son is goatβcasein gene promoter.

3. high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene according to claim 1, which is characterized in that the people is solidifying Blood factor IX is high blood coagulation activity mutant, and nucleotide sequence is as shown in SEQ ID NO:1, position 1960-1961 core Thuja acid is GC rather than CG.

4. high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene according to claim 1, which is characterized in that described is solidifying Blood factor IX is high activity mutant, and for amino acid sequence as shown in SEQ ID NO:2, the amino acid of position 384 is A rather than R.

5. high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene according to claim 1, which is characterized in that the people exempts from The Fc section nucleic acid sequence of epidemic disease globulin IgG1 is as shown in SEQ ID NO:3, and amino acid sequence is as shown in SEQ ID NO:4.

6. high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene according to claim 1, which is characterized in that the people is solidifying It include joint sequence between the Fc of blood factor IX gene and human immunoglobulin(HIg) IgG1 section gene.

7. high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene according to claim 6, which is characterized in that the connector It is the peptide indicated by amino acid sequence (GGGGS) n, wherein n is the integer of 1-20, and the connector also includes one section of flexible polypeptide The peptide of EFAGAAAV.

8. a kind of side using mammary gland bioreactor of transgenic animals production high activity recombinant human plasma thromboplastin component fusion protein Method, which is characterized in that by the carrier containing high activity recombinant human plasma thromboplastin component antigen-4 fusion protein gene described in claim 1 through original Core microinjection is integrated into fertilized eggs render transgenic mammal galactophore and expresses and secrete the external source recombinant blood coagulation factor IX Fusion protein；The mammal is mouse, ox or sheep.

9. a kind of preparation method of the mammary gland-specific expression vector of the mutain of high activity plasma thromboplastin component, which is characterized in that Include the following steps:

(1) the Fc segment cDNA of the human coagulation factor IX gene of plasma thromboplastin component (R384A) mutation and human IgG1 is passed through into connector Sequence connection is built into carrier, obtains recombinant plasmid；

(2) above-mentioned recombinant vector microinjection is imported into mouse fertilized egg, prepares hFIX-Fc transgenic mice；

(3) PCR method identifies positive mice, takes positive mice mating breeding, obtains positive female rat milk；

(4) the hFIX-Fc expressing fusion protein situation in mouse milk is detected.

10. the preparation side of the mammary gland-specific expression vector of the mutain of high activity plasma thromboplastin component according to claim 9 Method, which is characterized in that the recombinant vector is eukaryotic expression vector pcDNA3.1 (-).