CN1928096A - Method of producing recombination hirudin in transgene soybean with oil body protein as carrier - Google Patents

Abstract

The present invention relates to method of producing recombinant hirudin in transgenic soybean by using oil-body protein as vector. Through the first extracorporeal splicing of artificially synthesized and plant preferred codon optimized hirudin gene, protease cleaving identification signal and oil-body protein gene, the subsequent expression of the fusion protein in soybean via transgenic way and location in the surface of soybean oil-body, and the final separation of oil-body and fusion reformed oil-body protein from soybean seed and the specific cleavage with protease, active recombinant hirudin protein may be released.

Description

Make carrier is produced lepirudin 023 ludon in genetically engineered soybean method with oil body protein

The present invention relates to make carrier is produced lepirudin 023 ludon in genetically engineered soybean method with oil body protein.With synthetic, according to plant have a preference for codon optimized after hirudin gene and proteolytic enzyme cutting recognition signal and oil body protein gene carry out external splicing, in soybean, express and be positioned at the surface of soybean oil body by the transgenosis approach in the mode of fusion rotein then, from soybean seeds, isolate oil body and merge improved oil body protein and carry out the specificity cutting and can discharge activated lepirudin 023 ludon albumen through proteolytic enzyme.

R-hirudin is the anticoagulated blood polypeptides that is contained in hirudinaria manillensis (being commonly called as leech) saliva or the health, and molecular weight ratio is less, is about 7000Da, belongs to acidic protein (iso-electric point pI=3.9) ^[1], they can form 1: 1 non-covalent bonded mixture and become the most effective known up to now zymoplasm protein inhibitor with the zymoplasm protein molecular.Eighties of last century eighties, the aminoacid sequence of three kinds of natural hirudins (HV1, HV2 and HV3) obtains report in succession ^[2,3,4], these three r-hirudins respectively contain 65-66 amino-acid residue, have the sequence homology more than 85% each other.(between the 1-47 residue) contains 6 cysteine residues in the conserved regions of these r-hirudin protein moleculars N end, the disulfide linkage that forms between these cysteine residues makes the N end become a compactness, nonpolar structural domain, and it can combine with the catalytic site of zymoplasm; The C end (between the 49-66 residue) of r-hirudin can combine with the fibrinogen recognition site of zymoplasm by a large amount of static and hydrophilic interaction, and these two structural domain actings in conjunction are all main biologic activity of Trombin inhibiting effectively ^[5]

R-hirudin has great pharmaceutical use, and this is not only because it can act on zymoplasm very specifically, and it also has numerous characteristics and makes it more more superior than many anticoagulins of present use such as heparin.Each leech head approximately contains the 20ug r-hirudin ^[6], therefore, it is very limited to extract the natural proteic amount of r-hirudin from leech, can not satisfy people's demand fully.The lepirudin 023 ludon isomer protein gene of biologically active has been cloned and intestinal bacteria ^[3,4], yeast ^[7,8], the insect cell that infects of polyhedrosis virus ^[9]And streptomycete (Streptomyces lividans) successful expression ^[10], the lepirudin 023 ludon that these expression systems are produced does not wait from several milligrams to tens milligrams, and degree of purification can reach more than 99%.

Oil body is to store triacylglycerol (triacylglycerols in some plant seeds, TAG) ubcellular organ, spherical in shape, portion storage has liquid TAG within it, and the outside is then by phosphatide unimolecular layer (PL) and embed half unit membrane that the oil body protein (oleosin) in it forms and wrapped up.In all produce oil seeds, all contain a large amount of oil bodies and oil body protein as soybean, rape and peanut ^[11]

The oil body protein molecular structure is relatively unique, and the middle part is a hydrophobic amino acid long-chain, and is all longer than known other albumen, has very strong hydrophobicity, so this part aminoacid sequence can firmly be embedded in the oil body lipid film.Hydrophobic core skeleton both sides are that hydrophilic N end and C hold, and are free in the outside of oil body lipid film usually, and these hydrophilic segments are different on aminoacid sequence and the length in different plants.

Oil body protein distinctive molecular structure and biochemical characteristic make it be suitable for being applied in the production of recombinant exogenous protein, at first, oil body protein is very high at produce oil seed intensive amount, for example in soybean, estimate that oil body protein accounts for about the 2%-10% of seed protein, the accumulating level of this content and same plant seed storage protein is suitable ^[12]Secondly, when seed grinds the back when using buffer extraction, oil body and oil body protein can float on the surface, and be easy to other albumen composition of flush away and oil body protein is concentrated to a very high stage through centrifuge washing repeatedly ^[12,13]Oil body protein is easy to separate with seed storage protein and other albumen composition in this way, if may observe same separation principle during with the N of other foreign protein and oil body protein end or C end amalgamation and expression, this just brings great convenience for the downstream of recombinant exogenous protein processes.

Although soybean is a kind of dicotyledons, but as the expert of the art is known, the genetic transformation of soybean is very difficult, have only only a few kind genetically engineered soybean achieving successes such as antiweed both at home and abroad so far, therefore, before the present invention, also nobody is by genetically modified mode, utilize the soybean oil body to produce other external source recombinant proteins, especially r-hirudin albumen, the codon of having a preference for according to soybean of also having no talent is modified and is transformed hirudin gene, so that farthest improve the expression efficiency of this foreign protein in plant.

The present invention utilizes the molecular structure characteristics of oil body protein, in intraseminal high expression level characteristic and be easy to separate and the characteristics of purifying are produced in genetically engineered soybean and had active lepirudin 023 ludon albumen.According to a preferential embodiment, the present invention relates to the codon that r-hirudin 1 protein gene (HV1) is had a preference for according to soybean is modified and transformed, so that improved gene can be expressed in soybean seeds more efficiently.

The invention provides the method for making carrier, producing lepirudin 023 ludon in the mode of fusion rotein with oil body protein, according to a preferential embodiment, the N end of hirudin gene and oil body protein gene or C end encoding sequence carry out external splicing, but this splicing can't change reading frame each other, also can not influence the location of oil body protein on the soybean oil body after the fusion.By extracting soybean oil body, be easy to lepirudin 023 ludon and other soybean seeds albumen sepn are come, can simplify purification process thus and reduce production costs greatly.

According to an embodiment, the present invention relates under the fusion rotein state, to discharge active lepirudin 023 ludon method, because between oil body protein and r-hirudin encoding sequence, contain proteolytic enzyme specificity cutting recognition signal, handle through protease digestion, activated recombinant exogenous protein is released.According to a preferential embodiment, this recognition signal can be discerned by erepsin or Xa factor, after from soybean seeds, isolating soybean oil body and oil body protein (fusion rotein), utilize erepsin or Xa factor to handle this fusion rotein and just lepirudin 023 ludon can be discharged, become activated protein polypeptide.

To the relevant item with other of the method used in the present invention structure of some plasmid vectors or dna fragmentation very importantly, they are at the genetically engineered soybean that obtains indication of the present invention and have in the process of active lepirudin 023 ludon and play an important role.According to a preferential embodiment, the plasmid vector or the dna fragmentation that are used for soybean transformation are made up of the dna sequence dna of coding oil body protein-proteolytic enzyme cutting recognition signal-hirudin gene and the plant promoter that this dna sequence dna of manipulation is expressed soybean.In some specific embodiments, plasmid vector comprises that also one is selected or marker gene, is mainly used in the screening of genetically engineered soybean.In some specific embodiments, plant promoter used in the present invention is the promotor of cauliflower mosaic virus 35S promoter or oil body protein gene.Plasmid vector provided by the invention or dna fragmentation can be used for the particle bombardment soybean transformation.

The present invention also provides the method that obtains the genetically engineered soybean cell, and it may further comprise the steps: 1. hirudin gene is codon optimized; 2. make up a plasmid vector or section of DNA sequence, wherein all contain the antigen-4 fusion protein gene of coding oil body protein-proteolytic enzyme cutting recognition signal-r-hirudin, and this gene is placed under the manipulation of a plant promoter; 3. with plasmid vector or above-mentioned dna fragmentation soybean transformation cell.According to a preferential embodiment, also comprise the method that bears complete genetically engineered soybean from the soya cells that transforms again.

The invention provides the various genetic transforming methods of soybean.Although only utilized specific method for transformation in the preferential embodiment of some that is described below, but it is evident that, other methods for plant transformation that the expert of the art knew already also should be included within the claim scope of the present invention, and these methods comprise that microtubule injection, PEG merge and electro fusion method.According to some preferential embodiment, use be agriculture bacillus mediated method for transformation, refer in particular to the Agrobacterium transformation system that Ti-plasmids participates in.In some other preferential embodiment, can also use particle bombardment soybean transformation cell or plant.

In order to describe some preferential embodiment of the present invention in detail, and for referencial use with corresponding drawing:

The building process of Fig. 1 bacterial plasmid vector pGEMHV1.

The clone of Fig. 2 soybean oil body protein gene and the building process of bacterial plasmid vector pGEMO.

The building process of Fig. 3 plant expression vector pBI0H.Wherein GUS represents β-Glucuronidase (glucuronidase) gene; Formed antigen-4 fusion protein gene after on behalf of 1 splicing of soybean oil body protein gene-erepsin cutting recognition signal-r-hirudin, antigen-4 fusion protein gene finish.

Contained 2 structure genes and regulating and expressing sequence thereof among the T-DNA of Fig. 4 plasmid pBIOH.Wherein Nos-Pro represents rouge alkali synthetase promoter; Npt-II represents neomycin phosphotransferase gene; Nos-Ter represents the rouge alkali synthetase terminator; 35S Pro represents the cauliflower mosaic virus 35S promoter; RB representation DNA right border sequence; LB representation DNA left margin sequence.

Fig. 5 PCR detects the integration situation of oil body protein-hirudin fusion protein gene in different genetically engineered soybean strains are.Wherein 1 is λ DNA/EcoRI+HindIII Marker, and 2 positive contrast amplifications are template with the pBIOH plasmid DNA, and 3 for being the negative control amplified production of template with the non-transgenic soy bean DNA, and 4-8 is the amplified production that different genetically engineered soybean strains are.

Fig. 6 not homophyletic is the Northern results of hybridization of oil body protein in the genetically engineered soybean blade-hirudin fusion protein gene mRNA.1 is the non-transgenic soybean, and 2-6 is the genetically engineered soybean plant.

The SDS-PAGE of the reorganization HV1 that Fig. 7 is purified into behind the extracting of soybean oil body, erepsin cutting and ammonium sulfate precipitation analyzes.Wherein 1 is standard molecular weight albumen (Amersham Biosciences); 2 is non-transgenic soybean seeds total soluble protein extract; 3 is genetically engineered soybean seed total soluble protein extract; 4 is the partially purified reorganization HV1 that obtains behind the extracting of soybean seeds oil body, erepsin cutting and ammonium sulfate precipitation.

Fig. 8 suppresses the activity that the colour developing quantitative analysis detects the soybean lepirudin 023 ludon by zymoplasm.Wherein 1 is to add the blank damping fluid (negative control) of 2ul in the working buffer liquid; 2 is to add 2ul soybean lepirudin 023 ludon in the working buffer liquid; 3 is to have added 2ul natural hirudin (positive control) in the working buffer liquid; 4 is to have added 2ul erepsin (negative control) in the working buffer liquid.

The present invention includes following part: 1. the codon of having a preference for according to plants, to the hirudin base Because modifying and transforming, improved gene will be expressed in soybean more stable and more efficiently; 2 utilize DNA restructuring skill wood makes up plasmid expression vector, it contain coding oil body protein-protease cutting identification signal-The plant promoter that the dna sequence dna of hirudin gene and this dna sequence dna of manipulation are expressed in soybean forms. 3. soybean is carried out genetic transformation, make the above-mentioned fusion of coding stable gene be integrated into soybean chromosome In and can entail the offspring; 4. from the soya cells that transforms, bear again complete transfer-gen plant, and should The fusion that oil body protein and hirudin consist of can be stablized, be produced efficiently to plant; The restructuring leech Plain separation and purifying, it is characterized in that from the seed of described genetically engineered soybean maturation, extracting oil body, Add protease and digest so that hirudin discharges from oil body, use then ammonium sulfate precipitation and cold Freeze-drying is dry, obtains at last activated lepirudin 023 ludon albumen.

1. the modification of hirudin gene and transformation

Although leech and soybean all belong to eucaryote, they still exist aspect the gene codon preference Certain difference, this species diversity can cause foreign gene not enough stable in soya cells, in addition gene Transcribe with translation efficiency and all can be subjected to certain impact.

Complete hirudin gene is manually synthesized, and in the process of synthetic gene, it is constant but codon changes the codon that soybean is had a preference for into to keep original amino acid sequence^[14] Improved hirudin gene is in plant To express more stable and more efficiently;

2. transformation of soybean method

There are many kinds of methods foreign gene can be imported in the soybean, wherein the foreign gene stable integration entered The main method of soybean chromogene group DNA comprises: 1) agrobacterium-mediated transformation; 2) DNA directly takes in method, Comprise Protoplast fusion method, electrization, the microtubule injection of PEG mediation and use particle gun that DNA is straight Pick into plant cell and tissue etc.

Agrobacterium-mediated transformation is to utilize plasmid vector that specific dna fragmentation is integrated into the plant chromosome genome DNA. The most frequently used method is Ye Panfa, and it is applicable to any plant explants that is easy to the regeneration whole plant. Conversion method for agrobacterium is specially adapted to the conversion that dicotyledon comprises soybean.

Take in method for transformation as the top various dna directs of having mentioned, electrization is protoplastis to be placed to be placed on a highfield earlier, under galvanic action foreign DNA is sent in the protoplastis.The microtubule injection is with microtubule dna direct to be injected in the cell.Particle bombardment is that DNA is adsorbed on earlier on sal epsom or the tungsten particle, and these particles are accelerated to be injected in vegetable cell or the tissue.

According to a preferential embodiment of the present invention, Agrobacterium Ti-plasmids system is selected.Contain one section in the Ti-plasmids of Agrobacterium and be called transfer DNA (T-DNA), it can enter in the soya cells and be integrated at last in the karyomit(e) of soybean.The structure of transfer vector was divided into for two steps, at first be to make up the plasmid that can in intestinal bacteria, duplicate, this plasmid contains the dna sequence dna of coding oil body protein-proteolytic enzyme cutting recognition signal-hirudin gene and handle the plant promoter composition that this dna sequence dna is expressed in soybean, the T-DNA border sequence is contained at the two ends of this dna sequence dna, and it is responsible for the insertion site of goal gene in the soybean gene group.Usually another one selected marker gene (as anti-kalamycin resistance gene) also is comprised in the left and right sides border sequence of T-DNA, can provide a selective marker to confirm whether soybean or soya cells contain the T-DNA sequence of integration after this gene is expressed in soya cells.Second step of vector construction is that plasmid is transferred to the Agrobacterium from intestinal bacteria.This can finish by the mode or the dna direct lead-in mode of triparental mating.For the transfer of T-DNA, also to contain a cover induced gene in the used agrobacterium strains, they are transferred in the soya cells at T-DNA and play an important role.

The people who is familiar with the soybean heredity conversion will be appreciated that the agrobacterium strains that transforms usefulness can have multiple choices, and plasmid construction also has multiple mode, and its purpose all is in order more to help the genetic transformation of soybean.What the conversion of soybean was the most frequently used is the c4 plant leaf discs conversion method, in some cases, also needs to add some helpers.Other method for transformation comprises protoplast transformation, goes out soya cells and whole plant by protoplast regeneration then.

The present invention is not limited only to use Agrobacterium Ti-plasmids conversion system, and foreign gene is directly imported the method for soya cells or protoplastis to the means that also should comprise other physical property and other imports foreign gene the method for soya cells.

3. genetic expression promotor

After method for transformation is determined, a composing type, grow the selected so that foreign gene of expression promotor adjustment type or the organizing specific type and can in soybean, express.

All are known can handle the promotor that foreign gene transcribes and can use in the present invention in vegetable cell.These promotors can obtain from plant or virus, as cauliflower mosaic virus 35S promoter (comprise through splicing, double and the improved 35S promoter that suddenlys change); The oil body protein gene promotor; Photoinduction gene promoter such as ribose bisphosphate carboxylase small subunit (rbcS) or adversity inducible gene such as ethanol dehydrogenase (Adhl) etc., but be not limited only to this.

The plasmid that is used for the soybean conversion contains a selected marker gene usually.Many have the gene of this function to be developed.

Be example with agriculture bacillus mediated soybean heredity method for transformation below, describe a preferential embodiment, but application of the present invention be not limited only to this.

Embodiment 1

1. the synthetic of hirudin gene (HV1)

Up to now, still nobody formally reports the nucleotide sequence that clones coding r-hirudin 1 (HV1) gene in leech, and for this reason, we can only be according to the aminoacid sequence of the HV1 that had announced already ^[15], under the situation that does not change this Argine Monohydrochloride sequence, the codon of having a preference for according to soybean ^[14], manually design and synthesized the complete genome sequence (seeing sequence table 1 and 2) of r-hirudin 1.For the ease of structure, the splicing of antigen-4 fusion protein gene and the release of expression and lepirudin 023 ludon of plasmid vector from now on, in synthetic r-hirudin 1 gene coded sequence, also synthetic simultaneously and increased erepsin cutting recognition site (seeing sequence table 3 and 4) and restriction enzyme site KpnI at 5 ' end of this gene, after ending codon, 3 ' end of HV1 gene increased restriction enzyme site SacI (said gene splicing and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing).

2.HV1 the clone of gene

The gene fragment of above-mentioned synthetic is inserted directly in the T site in the pGEM-T plasmid (Promega company product), the method that provides according to the said firm, by the white screening system of indigo plant, obtain containing the bacterial clone pGEMHV1 (see figure 1) of this gene, then, by nucleotide sequence analysis, determine that HV1 gene and erepsin cutting recognition signal is correct and complete (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

3. the clone of soybean oil body protein gene

(see GenBanK, U09118), synthesized a pair of specific primer, wherein upstream primer (5 ' end primer) sequence is: 5 '-AA according to the soybean oil body protein gene order of having announced TCTAGATCAACCATGACCACACAAGTAC-3 ', the XbaI cleavage site is contained in its inside, and downstream primer (3 ' end primer) is: 5 '-TGC GGT ACCGGTTGTTGCTGTCACTGTTGT-3 ', the KpnI cleavage site is contained in its inside.According to Wang Guanlin etc. ^[16]People's method, (kind is Century from soybean seeds, provide by the Qiu Lijuan researcher of Institute of Crop Science, Chinese Academy of Agricultural Science) extract its genomic dna, with this DNA is template, and [soybean gene group DNA1ul (containing 1ug approximately) is as masterplate, 5 ' end primer and 3 ' each 1ul of end primer for the method by PCR, 10XPCR damping fluid 3ul, dNTP (each 2.5mM) 3ul, the about 1.5U of Taq enzyme 0.5ul adds water to cumulative volume 30ul.94 ℃ 30 seconds, 50 ℃ 45 seconds, 72 ℃ 1.5 minutes, carry out 35 circulations altogether].Amplification obtains the amplified production of the about 700bp of a segment length.This product is inserted directly in the T site in the pGEM-T plasmid (Promega company product), the method that provides according to the said firm, by the white screening system of indigo plant, obtain containing the bacterial clone pGEMO (see figure 2) of this gene, then, by nucleotide sequence analysis, definite soybean oil body protein gene is correct and complete (seeing sequence table 5 and 6) (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

Embodiment 2

1. the structure of oil body protein-hirudin fusion protein plant expression vector pBIOH

In order to make up the high-efficiency plant expression vector, when synthetic HV1 gene, added restriction enzyme site KpnI and erepsin cutting recognition signal at its 5 ' end, added restriction enzyme site SacI at its 3 ' end.When clone soybean oil body protein gene, added restriction enzyme site XbaI at its 5 ' end, added restriction enzyme site KpnI at its 3 ' end.Therefore after containing the plasmid vector pGEMHV1 of above-mentioned two genes and pGEMO and using KpnI+SacI and XbaI+KpnI double digestion respectively, through agarose gel electrophoresis, isolate the DNA of two small segments, thereafter these two gene fragments are inserted into by direct orientations in the former gus gene site of plasmid pBI121 (through XbaI and SacI double digestion), just have been built into double base plant expression vector pBIOH.

Plasmid pBI121 is available from U.S. Clonetech company, and its XbaI restriction enzyme site is between cauliflower mosaic virus 35S promoter and gus gene initiation codon, and SacI is then between gus gene terminator sequence and NOS terminator.Select for use plasmid pBI121 to be because with gus gene can being deleted behind XbaI and the SacI double digestion, and can insert the new foreign protein genes of another one subsequently, newly gene can efficiently express in vegetable cell.Plasmid pBI121 also contains neomycin phosphotransferase II gene (nptII), and the enzyme of its coding can be vegetable cell kalamycin resistance is provided, thereby can come containing the cell of T-DNA and tissue and unconverted vegetable cell or tissue division.The nptII gene has promotor and the poly gland acid signal sequence of oneself, and they all are derived from nopaline (Nopaline) synthetic enzyme (NOS) gene.

Plasmid pBIOH contains: 1) Xin Meisu synthase gene II (nptII), and it offers the vegetable cell kalamycin resistance; 2) soybean oil body protein gene-erepsin cutting recognition signal-r-hirudin 1 gene (antigen-4 fusion protein gene) and handle the cauliflower mosaic virus 35S promoter of this gene; 3) T-DNA left and right sides border sequence, it can be transferred to nptII gene and above-mentioned antigen-4 fusion protein gene in the plant materials and be incorporated in the karyomit(e) of soybean.The structure of pBIOH as shown in Figure 3 and Figure 4.

2. expression vector pBIOH is imported Agrobacterium (A.tumefaciens)

According to the method that Clonetech company is provided, the plasmid pBIOH that contains antigen-4 fusion protein gene is transferred in the Agrobacterium LBA4404 bacterial strain by the mode of triparental mating, and this bacterial strain is available from U.S. Clonetech company.Bacterial strain LBA4404 now is widely used, and it is improved agrobacterium strains, contain complete Vir gene, but T-DNA is deleted.The Vir gene can trans adjusting T-DNA from the transfer of plasmid pBIOH in vegetable cell.

Agrobacterium contain the 50ml YEP of 25mg/L Streptomycin sulphate (peptone 10 gram, yeast extract 10 grams, NaCl 5 grams, adding distil water to 1 liter, pH7.0) shaking culture in the substratum is at OD _600nmWhen value reached 0.4-0.7, the centrifugal collection bacterial cell of 4000g was suspended in 1ml again with agrobatcerium cell and does not contain in any antibiotic YEP substratum stand-by.The HB101 (available from U.S. Clonetech company) that contains the DH5 α (available from U.S. GIBCO company) of expression vector pBIOH and contain helper plasmid pRK2013 (available from U.S. Clonetech company) is seeded in respectively and contains shaking culture in the 50mg/L kantlex 50ml LB substratum, at OD _600nmWhen value reached 0.4-0.7, the centrifugal collection bacterial cell of 4000g was suspended in cell respectively 1ml then and does not contain in any antibiotic LB substratum stand-by.Get above-mentioned three kinds of each 200ul of cell suspending liquid, place same 1.5ml centrifuge tube, 28 ℃ leave standstill overnight incubation, get this culture 5-10ul, evenly spread upon on the YEP solid medium flat board, contain 25mg/L Streptomycin sulphate and 50mg/L kantlex in this substratum simultaneously, cultivate after 2 days for 28 ℃, the Agrobacterium bacterium colony that contains the pBIOH plasmid begins to produce.

From Agrobacterium, extract plasmid DNA with alkaline lysis, and can detect the pBIOH plasmid DNA with digestion with restriction enzyme and whether exist.

Embodiment 3

1. the genetic transformation of soybean

No. 28, beans are available from Institute of Crop Science, Chinese Academy of Agricultural Science in soybean (the Glycine max L.) Cultivar.Method according to people such as Horsch ^[17], utilize leaf dish method soybean transformation.The agrobacterium strains LBA4404 that contains expression vector pBIOH is seeded among the 50ml YEP, cultivates 2 days for 28 ℃, and centrifugal 5 minutes of 4000g collects thalline, is suspended in again then in the MS substratum of liquid to OD _600nm=0.4, with knife blade the aseptic cotyledon of soybean after germination 5-6 days is cut from connecting place, immersed immediately in the agrobacterium liquid after the dilution 30 minutes, take out then, to blot with filter paper, the blade face places on the MS solid medium up, and 28 ℃ of lucifuges were cultivated 4 days.[add plant hormone (Sigma company) 6-BA2mg/L and IBA0.2mg/L in the MS basic ingredient on then that the blade dislocation is the fresh selection substratum, and contain 50ug/ml kantlex and 500ug/ml carboxylic Bian penicillin] cultivate, give 25 ℃ and illumination in 16 hours.Grow callus and differentiate seedling up to the petiole wound every changing a selective medium 2 week later on.When seedling occurs and long to enough greatly the time, seedling is scaled off (cultivating 4-8 week of back altogether usually), move on to (MS contains 0.2mg/mlIBA and 50ug/ml kantlex) on the root media, after root grew, seedling was moved in the aseptic nutrition soil growth and is aided with suitable temperature and illumination condition.

2.PCR detect the integration situation of antigen-4 fusion protein gene in soybean

The extracting method of soybean gene group DNA is substantially according to the method for Chee ^[18]Get the fresh blade of 0.5g soybean transgene plant, put into the mortar liquid nitrogen grinding, in the powder dislocation 20ml centrifuge tube after grinding, (100mM Tris-HCl, pH7.5 contain 5M NaCl, 10mM EDTA to add 9ml CTAB extracting solution then, the 10ml/L beta-mercaptoethanol, 10g/L CTAB), thermal agitation 1 minute makes it abundant mixing.Temperature was bathed 90 minutes in 60 ℃ of water-baths, during vibration in per 30 minutes once.From water-bath, take out centrifuge tube, placed 4-5 minute under the room temperature, make it cooling, add the 4.5ml chloroform then, shake gently, make it abundant mixing.5000g is centrifugal 10 minutes under the room temperature.Collect supernatant liquor in another 20ml centrifuge tube, add the 4.5ml chloroform, repeat above-mentioned steps.Collect supernatant liquor, add the ice-cold Virahol of 10ml, jog 10-15 minute, centrifugal 5 minutes of 5000g, collecting precipitation contains 0.2M NaOAc and 2ml 76% ethanol contains 10mM NH with 3ml 76% ethanol ₄OAc respectively washes precipitation once, adds TE (pH8.0) damping fluid dissolving DNA at last, and DNA concentration is transferred to 1ug/ul.

Get above-mentioned soybean gene group DNA1ul (containing 1ug approximately) as masterplate, add soybean oil body protein gene 5 ' end primer and hirudin gene 3 ' each 1ul of end primer (seeing embodiment 1), 10XPCR damping fluid 3ul, dNTP (each 2.5mM) 3ul, the about 1.5U of Taq enzyme 0.5ul adds water to cumulative volume 30ul.94 ℃ 30 seconds, 50 ℃ 45 seconds, 72 ℃ 1.5 minutes, carry out 35 circulations altogether.After reaction finishes, get 5ul amplification sample and carry out the detection of 1% agarose gel electrophoresis.As shown in Figure 5, (swimming lane 3) do not find specific band in the negative control plant, in the genetically engineered soybean plant, then can amplify the specific band (swimming lane 4-8) of 1 about 900bp, consistent with expected result, this result shows that soybean oil body protein-erepsin cutting recognition signal-r-hirudin 1 antigen-4 fusion protein gene has been integrated among the soybean gene group DNA.

3. the RNA of genetically engineered soybean analyzes

Utilize the hirudin gene dna fragmentation of digoxigenin labeled to make probe (marking method is seen Roche company test kit), the part kalamycin resistance soybean plant strain RNA after the pBIOH plant expression vector is transformed has carried out hybridization analysis.

The total RNA of the blade of genetically engineered soybean plant is extracted, the method document that sees reference ^[19]Approximately the 1.0g blade is clayed into power with mortar in liquid nitrogen, adds 10ml RNA and extracts damping fluid (0.2M Tris-HCl, pH6.8; 0.2M NaCl; 20mM EDTA; 2%SDS), add 10ml saturated phenol damping fluid (10mM Tris-HCl, pH8.0 immediately; 1mM EDTA is saturated) carry out extracting, use the chloroform extracting of 10ml then.3000g is centrifugal, collect the upper strata water, the dehydrated alcohol that adds 0.3M Potassium ethanoate (pH5.2) damping fluid and 2.5 times of volumes, the 10000g centrifugal collecting precipitation, after air-dry, precipitation is suspended in the water of 2ml again, adds ammonium acetate buffer and the 10ml dehydrated alcohol of 2ml 6M subsequently, the 10000g centrifugal collecting precipitation.After being deposited in drying, be suspended in again in the 0.4ml water, RAN concentration can estimate that the RNA light absorption value of supposing 1mg/ml is 25 units by the light absorption value of measuring 260nm.

Get 10ug RNA sample, about 5ul, after 2 times of volume RNA sample buffers (10ml deionized formamide, 3.5ml 37% formaldehyde, 2ml 5X MOPS) mixing, 65 ℃ were heated 5 minutes, after the cooling, add 3ulRNA sample loading buffer (50% glycerine, 1mM EDTA, 0.4% tetrabromophenol sulfonphthalein), then through 1% RNA agarose gel electrophoresis.Nucleic acid is transferred on the nylon membrane (Hybond N+) by the hair pipette method, and used damping fluid is 20XSSC, pH7.2 (3M sodium-chlor, 0.3M Trisodium Citrate), and be 16 hours transfer time.Nucleic acid by uviolizing after by commissure on nylon membrane, film is placed prehybridization solution [5XSSC, 0.1% (w/v) N-Lauroylsarcosine, 0.02%SDS, 100ug/ml salmon sperm dna], 68 ℃ of prehybridizations 3 hours, hybridization solution [the 5XSSC that more renews then, 0.1% (w/v) N-Lauroylsarcosine, 0.02%SDS, 1% encapsulant (providing in the BM test kit)], the dna probe 60ng that adds the digoxigenin labeled of sex change in the 5ml hybridization solution is hybridized, the long 225bp of probe, it is the double digestion small segment from the KpnI of plasmid pGEMHV1 and SacI, has comprised whole encoding sequences of hirudin gene.68 ℃ of hybridization were taken out Hybond membrane after 24 hours in hybridization solution, in 200ml 2XSSC, washed film under the room temperature 2 times in the 0.1%SDS damping fluid, then in 100ml0.1XSSC, washed film 2 times for 68 ℃ in the 0.1%SDS damping fluid.Color reaction is substantially according to the method that provides in the test kit (Roche), and nylon membrane is prior to elution buffer [0.1M Maleic acid, pH7.5; 0.15M NaCl; 3%Tween 20 (w/v)] in balance 5 minutes, with 50ml 1X sealing damping fluid sealing 30 minutes, DigiTAb to the concentration that adds alkali phosphatase enzyme mark was 75mU/ml then, reaction is 30 minutes under the room temperature, with elution buffer 2 times, each 15 minutes.With film dislocation 20ml colour developing damping fluid (0.1M Tris-HCl, pH9.5; 0.1M NaCl; 50mM MgCl ₂) in balance 5 minutes, the substrate buffer solution that more renews (200ul NBT/BCIP substrate adds 10ml colour developing damping fluid), colour developing is 16 hours under the shading condition, treat that desired band occurs after, the water termination reaction.

The RNA results of hybridization that transforms the transfer-gen plant of back acquisition and negative control plant through plasmid pBIOH as shown 6.The RNA hybridization signal of different transfer-gen plants is variant, and this may be because the insertion site of gene and different the causing of copy number of gene.Compare with the standard rna molecular weight, the mRNA that transcribes has 900bp long approximately, with pre-estimate consistent.Do not observe hybridization signal in the RNA swimming lane of negative control plant.Stable, the great expression of mRNA also can show that the stability of mRNA can not become the problem that r-hirudin 1 gene is expressed in soybean with the result of coding r-hirudin 1 proteic DNA nucleic acid probe specific hybrid.

Embodiment 4

1. the separation and Extraction of soybean oil body

Get genetically engineered soybean seed 1g, fully grind in mortar, the fine powder after grinding all is transferred in the 50ml centrifuge tube, adding 0.5M sucrose solution (contains 10mMKCl with 0.1M Tris-HCl, 1mM MgCl in the centrifuge tube then ₂, the pH7.5 preparation) and 30ml, after at room temperature fully vibrating, centrifugal 15 minutes of 5000rpm.Be divided into three parts after homogenate is centrifugal, the liquid level upper strata mainly is oil body and the albumen that links with it, and liquid portion contains the seed albumen of solubility, and precipitation is insoluble albumen and some particulate materials.Collect the part of top eleoplast, in order to remove the loose bonded soluble proteins of oil body remained on surface, oil body is suspended in the more low-density sucrose extracting solution again (contains 0.5M NaCl) in the 0.3M sucrose, after centrifugal, oil body after the washing swims in the surface of extract again, this process can be carried out 2-3 time repeatedly, is fusion rotein fully up to the contained albumen of oil body part, and oil body is suspended in 5ml 50mM Tris-HCl (pH7.5) at last and contains 2.5mM CaCl ₂Damping fluid in.

2. the release of lepirudin 023 ludon

Add 20ng erepsin (available from New England Biolabs company) in the above-mentioned damping fluid and carry out the digestion of fusion rotein.Digestion is at room temperature spent the night, the centrifugal albumen of removing oil body and linking with it, collection contains the liquid portion of lepirudin 023 ludon, it is about 80% to saturation ratio to add the 3g solid ammonium sulfate, stirs 1 hour centrifugal 15 minutes of 10000rpm under the room temperature, collecting precipitation, add 1ml 10mM Tris-HCl (pH7.5) damping fluid and suspend, 4 ℃ of following dialysed overnight, dialysis buffer liquid is 2 liters of 10mMTris-HCl (pH7.5) in the dialysis tubing of packing into then.The dialysis finish after from dialysis tubing the sucking-off dialysate, after lyophilize, be dissolved in again in 1ml10mM Tris-HCl (pH7.5) damping fluid, protein content is about about 300ug/ml.Therefrom take out 10ul then and carry out the SDS-PAGE electrophoresis, the protein extract behind the coomassie brilliant blue staining behind detection seed protein extract and the purifying is by finding that relatively the foreign protein composition more than 95% is removed (see figure 7).

3. the active mensuration of lepirudin 023 ludon

The active mensuration of lepirudin 023 ludon adopts zymoplasm to suppress colour developing quantitative analysis method [20], test used zymoplasm (Thrombin) available from Sigma company, it is dissolved in (50mM Tris-HCl, pH8.0 contain 100mM/L NaCl and 5mM/L CaCl in the working buffer liquid ₂) be that 0.12-0.14u/ml is stand-by to final concentration.[the employed natural hirudin of positive control is available from Sigma company for lepirudin 023 ludon (concentration is 1ug/ml) in the above-mentioned working buffer liquid of every 200ul behind the above-mentioned purifying of adding 2ul or contrast damping fluid, article No. H7380, being dissolved in 10mM Tris-HCl damping fluid (pH7.5) to working concentration is 1ug/ml], hatched under 25 ℃ 10 minutes, add the 1ul substrate buffer solution then and [produce look substrate p-tosyl-gly-pro-arg-notroanilide available from Sigma company, it is dissolved in the substrate buffer solution that is mixed with 50pM/L in the distilled water], detect the variation of 405nm absorbance value within 2 minutes.Found that the activity that the lepirudin 023 ludon that is derived from soybean can Trombin inhibiting, its active reduction is reached more than 94% at least, its specific activity is more taller than positive control.Negative control (the 10mM Tris-HCl damping fluid that adds the blank damping fluid of 2ul, pH7.5) do not show any inhibition activity in, in addition, do not show any active (see figure 8) in the negative control of adding 2ul erepsin [be dissolved in 10mMTris-HCl damping fluid (pH7.5), working concentration is 1ug/ml] yet.

Reference

1.Stone?S?R?and?J?Hofsteenage，Biochemistry，1986，25：4622-4628.

2.Harvey?R?P，et?al.，Proc.Natl.Acad.Sci.USA，1986，83：1084-1088.

3.Rydel?T?J，et?al.，Science，1990，249：277-280.

4.Markwardt?F，Thromb.Res.，1994，74：1-23.

5.Markwardt?F，et?al.，Haemostasis，1991，(Suppl?1)，21：133-136.

6.Riechl-Bellon?N，et?al.，Biochemistry，1989，28：2941-2949.

7.Benatti?L，et?al.，Gene，1991，101：255-260.

8.Bender?E，et?al.，Appl.Microbiol.，Biotech.，1990，34：203-207.

9.Chang?J，et?al.，FEBS?Letters，1983，164：307-313.

10.Klocking?H?P，et?al.，Folia?Haematol.，1988，115：75-82.

11.Jacks?T?J，et?al.，JACOCS，1990，67：353-361.

12.Qu?R?D?and?Huang?A?H?C，J.Biol.Chem.，1990，265：2238-2243.

13.Parmenter?D?L，et?al.，In：Transgenic?plants：a?production?system?for?industrialand?pharmaceutical?protein，Owen?M?R?L?and?Pen?Jen(eds)，1996，JohnWiley&Sons?Ltd，Chichester?England，pp.261-280.

14.Stone?S?R?and?J?M?Maraganore，Meth.Enzymol.，1993，223：312-336.

15.Chang?J?Y，FEBS?Letters，1983，164：307-313.

16. the king General Guan Yu's Tomb and the Fang Hong skin of bamboo, plant genetic engineering and principle, Beijing Science Press, 1998,598-599.

17.Horsch?R?B，et?al.，Science，1985，227：1229-1231.

18.Chee?P?P，Drong，R.F.and?Slightom，J.L.，Plant?Molecular?Biology?Manual，1991，C3：1-28.

19.Mason?H，et?al.，Plant?Molecular?Biology，1988，11：845-856.

20.Parmenter?D?L，et?al.，Plant?Molecular?Biology，1995，29：1167-1180.

Just some the special preferential embodiments that further describe of the present invention are according to the patent application requirement and in order to explain and illustrate the content of this patent.Obviously, do not deviating within the spirit and scope of the present invention, can do further improvement and variation on this basis.

Sequence table

＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences

＜120〉make carrier is produced lepirudin 023 ludon in genetically engineered soybean method with oil body protein

<160>6

<210>1

<211>225

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(226)

<400>1

ggt?acc?gat?gat?gat?gat?aag?gtc?gtc?tac?aca?gat?tgc?aca?gag?tcc?ggg?cag?aac?57

Gly?Thr?Asp?Asp?Asp?Asp?Lys?Val?Val?Tyr?Thr?Asp?Cys?Thr?Glu?Ser?Gly?Gln?Asn

1 5 10 15

ctc?tgc?ctc?tgc?gaa?ggg?tcc?aac?gtc?tgc?ggg?cag?ggg?aac?aag?tgc?atc?ctc?ggg?114

Leu?Cys?Leu?Cys?Glu?Gly?Ser?Asn?Val?Cys?Gly?Gln?Gly?Asn?Lys?Cys?Ile?Leu?Gly

20 25 30 35

tcc?gat?ggg?gag?aag?aac?cag?tgc?gtc?aca?ggg?gag?ggg?aca?cca?aag?cca?cag?tcc?171

Ser?Asp?Gly?Glu?Lys?Asn?Gln?Cys?Val?Thr?Gly?Glu?Gly?Thr?Pro?Lys?Pro?Gln?Ser

40 45 50 55

cac?aac?gat?ggg?gat?ttc?gag?gag?atc?cca?gag?gag?tac?ctc?cag?tag?gag?ctc 225

His?Asn?Asp?Gly?Asp?Phe?Glu?Glu?Ile?Pro?Glu?Glu?Tyr?Leu?Gln?***

60 65 70

<210>2

<211>72

<212>PRT

＜213〉artificial sequence

<400>2

Gly?Thr?Asp?Asp?Asp?Asp?Lys?Val?Val?Tyr?Thr?Asp?Cys?Thr?Glu?Ser

1 5 10 15

Gly?Gln?Asn?Leu?Cys?Leu?Cys?Glu?Gly?Ser?Asn?Val?Cys?Gly?Gln?Gly

20 25 30

Asn?Lys?Cys?Ile?Leu?Gly?Ser?Asp?Gly?Glu?Lys?Asn?Gln?Cys?Val?Thr

35 40 45

Gly?Glu?Gly?Thr?Pro?Lys?Pro?Gln?Ser?His?Asn?Asp?Gly?Asp?Phe?Glu

50 55 60

Glu?Ile?Pro?Glu?Glu?Tyr?Leu?Gln

65 70

<210>3

<211>15

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(15)

<400>3

gat?gat?gat?gat?aag

Asp?Asp?Asp?Asp?Lys

<210>4

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉erepsin cutting recognition sequence

<400>4

Asp?Asp?Asp?Asp?Lys

<210>5

<211>681

<212>DNA

＜213〉soybean (Glycine max L.) oil body protein gene

<220>

<221>CDS

<222>(1)...(678)

<400>5

atg?acc?aca?caa?gta?cca?cca?cac?agt?gtc?caa?gtc?cac?aca?aca?aca?aca?cac?cgc 57

Met?Thr?Thr?Gln?Val?Pro?Pro?His?Ser?Val?Gln?Val?His?Thr?Thr?Thr?Thr?His?Arg

1 5 10 15

tac?gaa?gct?ggc?gtc?gtc?ccc?ccc?gga?gcc?cgt?ttc?gaa?acg?agt?tat?gaa?gca?ggc 114

Tyr?Glu?Ala?Gly?Val?Val?Pro?Pro?Gly?Ala?Arg?Phe?Glu?Thr?Ser?Tyr?Glu?Ala?Gly

20 25 30 35

gtc?aag?gcg?gcc?tcc?att?tac?cat?tcc?gag?aga?ggt?cca?acg?act?tcc?cag?gtc?ctc 171

Val?Lys?Ala?Ala?Ser?Ile?Tyr?His?Ser?Glu?Arg?Gly?Pro?Thr?Thr?Ser?Gln?Val?Leu

40 45 50 55

gcc?gtc?ctc?gcc?ggc?ctc?ccg?gtc?ggt?ggc?atc?ctg?ctc?ctc?ctg?gca?gga?ttg?acc 228

Ala?Val?Leu?Ala?Gly?Leu?Pro?Val?Gly?Gly?Ile?Leu?Leu?Leu?Leu?Ala?Gly?Leu?Thr

60 65 70 75

ctg?gca?gga?acc?ctc?acc?gga?ctg?gca?gtt?gcc?acg?ccg?ctc?ttc?gtc?ctc?ttc?agc 285

Leu?Ala?Gly?Thr?Leu?Thr?Gly?Leu?Ala?Val?Ala?Thr?Pro?Leu?Phe?Val?Leu?Phe?Ser

80 85 90 95

ccg?gtg?ctg?gtt?cca?gcc?acg?gtc?gcc?atc?ggc?ctg?gcc?gtg?gcc?ggg?ttc?ctg?acg 342

Pro?Val?Leu?Val?Pro?Ala?Thr?Val?Ala?Ile?Gly?Leu?Ala?Val?Ala?Gly?Phe?Leu?Thr

100 105 110

tcg?ggg?gcc?ttc?ggg?ctc?acg?gcg?ctg?tct?tcc?ttc?tcc?tgg?atc?ctg?aac?tac?atc 399

Ser?Gly?Ala?Phe?Gly?Leu?Thr?Ala?Leu?Ser?Ser?Phe?Ser?Trp?Ile?Leu?Asn?Tyr?Ile

115 120 125 130

cgg?gag?acc?cag?cca?gcc?tcg?gag?aac?ctc?gcc?gcc?gcc?gcg?aag?cat?cac?ctc?gcc 456

Arg?Glu?Thr?Gln?Pro?Ala?Ser?Glu?Asn?Leu?Ala?Ala?Ala?Ala?Lys?His?His?Leu?Ala

135 140 145 150

gag?gcg?gcg?gag?tac?gtg?ggg?cag?aag?acg?aag?gaa?gtg?ggg?cag?aag?acc?aag?gag 513

Glu?Ala?Ala?Glu?Tyr?Val?Gly?Gln?Lys?Thr?Lys?Glu?Val?Gly?Gln?Lys?Thr?Lys?Glu

155 160 165 170

gtt?ggg?cag?gat?att?cag?agc?aag?gca?cag?gat?aca?agg?gag?gca?gct?gca?agg?gat 570

Val?Gly?Gln?Asp?Ile?Gln?Ser?Lys?Ala?Gln?Asp?Thr?Arg?Glu?Ala?Ala?Ala?Arg?Asp

175 180 185 190

gca?agg?gag?gca?gct?gca?agg?gat?gca?agg?gaa?gct?gct?gca?agg?gat?gca?aag?gtg 625

Ala?Arg?Glu?Ala?Ala?Ala?Arg?Asp?Ala?Arg?Glu?Ala?Ala?Ala?Arg?Asp?Ala?Lys?Val

195 200 205

gag?gca?agg?gat?gta?aag?aga?aca?aca?gtg?aca?gca?aca?acc?gca?acc?gca?tga 681

Glu?Ala?Arg?Asp?Val?Lys?Arg?Thr?Thr?Val?Thr?Ala?Thr?Thr?Ala?Thr?Ala *

210 215 220 225

<210>6

<211>226

<212>PRT

＜213〉soybean (Glycine max L.) oil body protein

<400>6

Met?Thr?Thr?Gln?Val?Pro?Pro?His?Ser?Val?Gln?Val?His?Thr?Thr?Thr

1 5 10 15

Thr?His?Arg?Tyr?Glu?Ala?Gly?Val?Val?Pro?Pro?Gly?Ala?Arg?Phe?Glu

20 25 30

Thr?Ser?Tyr?Glu?Ala?Gly?Val?Lys?Ala?Ala?Ser?Ile?Tyr?His?Ser?Glu

35 40 45

Arg?Gly?Pro?Thr?Thr?Ser?Gln?Val?Leu?Ala?Val?Leu?Ala?Gly?Leu?Pro

50 55 60

Val?Gly?Gly?Ile?Leu?Leu?Leu?Leu?Ala?Gly?Leu?Thr?Leu?Ala?Gly?Thr

65 70 75 80

Leu?Thr?Gly?Leu?Ala?Val?Ala?Thr?Pro?Leu?Phe?Val?Leu?Phe?Ser?Pro

85 90 95

Val?Leu?Val?Pro?Ala?Thr?Val?Ala?Ile?Gly?Leu?Ala?Val?Ala?Gly?Phe

100 105 110

Leu?Thr?Ser?Gly?Ala?Phe?Gly?Leu?Thr?Ala?Leu?Ser?Ser?Phe?Ser?Trp

115 120 125

Ile?Leu?Asn?Tyr?Ile?Arg?Glu?Thr?Gln?Pro?Ala?Ser?Glu?Asn?Leu?Ala

130 135 140

Ala?Ala?Ala?Lys?His?His?Leu?Ala?Glu?Ala?Ala?Glu?Tyr?Val?Gly?Gln

145 150 155 160

Lys?Thr?Lys?Glu?Val?Gly?Gln?Lys?Thr?Lys?Glu?Val?Gly?Gln?Asp?Ile

165 170 175

Gln?Ser?Lys?Ala?Gln?Asp?Thr?Arg?Glu?Ala?Ala?Ala?Arg?Asp?Ala?Arg

180 185 190

Glu?Ala?Ala?Ala?Arg?Asp?Ala?Arg?Glu?Ala?Ala?Ala?Arg?Asp?Ala?Lys

195 200 205

Val?Glu?Ala?Arg?Asp?Val?Lys?Arg?Thr?Thr?Val?Thr?Ala?Thr?Thr?Ala

210 215 220

Thr?Ala

225

Claims (11)

1. one kind is utilized oil body protein to make carrier is produced lepirudin 023 ludon in genetically engineered soybean method.

2. as the method in the claim 1, wherein said oil body protein can't influence the location of this oil body protein on the soybean oil body after forming fusion rotein with r-hirudin.

3. as the method in the claim 1, contain the recognition site that can be cut by other proteolytic enzyme between wherein said oil body protein and the r-hirudin, as can be by the recognition site of erepsin or Xa factor cutting.

4. a plasmid vector that is used for soybean transformation comprises:

One segment structure is the dna sequence dna of coding r-hirudin-proteolytic enzyme cutting recognition site-oil body protein for the dna sequence dna or the structure of coding oil body protein-proteolytic enzyme cutting recognition site-r-hirudin.

A plant promoter that is connected with said dna sequence dna, for example 35S promoter or soybean oil body protein gene promoter, this promotor is handled said dna sequence dna and can be expressed in soybean.

5. as the plasmid vector in the claim 4, it contains a selection or marker gene.

6. one section dna sequence dna that is used for the particle bombardment soybean transformation comprises:

One segment structure is the dna sequence dna of coding r-hirudin-proteolytic enzyme cutting recognition site-oil body protein for the dna sequence dna or the structure of coding oil body protein-proteolytic enzyme cutting recognition site-r-hirudin.

A plant promoter that is connected with said dna sequence dna, for example 35S promoter or soybean oil body protein gene promoter, this promotor is handled said dna sequence dna and can be expressed in soybean.

7. as the dna sequence dna in the claim 6, it contains a selection or marker gene.

8. a method that obtains the genetically engineered soybean cell comprises:

Make up a plasmid vector or section of DNA sequence, wherein all containing a segment structure is the dna sequence dna of coding r-hirudin-proteolytic enzyme cutting recognition site-oil body protein for dna sequence dna or the structure that coding oil body protein-proteolytic enzyme cuts recognition site-r-hirudin.

A plant promoter that is connected with said dna sequence dna, for example 35S promoter or soybean oil body protein gene promoter, this promotor is handled said dna sequence dna and can be expressed in soybean.

With this plasmid vector or this dna sequence dna soybean transformation cell.

9. as the method in the claim 8, it comprises bears complete genetically engineered soybean plant again from the genetically engineered soybean cell.

10. as the method in the claim 8, wherein said soya cells transforms by the Agrobacterium system, and contains the binary vector system of Ti-plasmids in this Agrobacterium, and wherein said plasmid vector is a conformability carrier.

11. the separation of lepirudin 023 ludon and purifying, it is characterized in that from the sophisticated seed of described genetically engineered soybean, extracting oil body, add proteolytic enzyme and digest so that r-hirudin discharges from oil body, use ammonium sulfate precipitation and lyophilize then, obtain activated lepirudin 023 ludon albumen at last.

Images