CN1940066A - Production of expression vermis kinase tr-gene plant and its products - Google Patents

Abstract

Production of expression vermis kinase transfer gene plant and its product are disclosed. It can be used to inhibit or dissolve heart, brain and limb blood vessel thrombus of human or mammal by directly eating or intestinal tract medicine feeding.

Description

Express the production method and the product of vermis kinase tr-gene plant

The present invention relates to express earthworm Lumbrukinase and derivative production method of transgenic plants thereof, thereby and and then utilize these transgenic plant or derivatives thereofs that contain the Lumbrukinase recombinant protein to cause that people or Mammals heart and brain and limb vessel thrombosis are suppressed or thromboclastic method by direct mode edible or enterally administering.

Thrombus (thrombus) be since human body or Mammals heart and brain and the intraluminal blood of limb vessel solidifies or blood in some formed elements mutually sticking gather together afterwards formed.Under normal physiological status, some thrombin in the blood constantly are activated and under the effect of zymoplasm, form the scleroproein of trace and attached on the tunica intima, but the fibrinolytic system that these micro-scleroproeins constantly have been activated again dissolves, meanwhile, those thrombin that are activated are also constantly engulfed by mononuclear phygocyte system.The blood coagulation system and the anticoagulation system (fibrinolytic system) that are in the mutual antagonism process are keeping a kind of dynamic balance, have guaranteed that promptly blood has the potential solidifiable to guarantee the fluid state of blood again all the time.Yet under the effect of some factor, above-mentioned running balance is broken sometimes, makes its side's run-off the straight to coagulation process, and blood just begins accelerated solidification and the final thrombus that forms in heart and brain and limb vessel tube chamber.For example, bed after operation, wound, the easy thrombus that forms when the terminal cancer whole body shifts.

The intravital fibrinolytic system of people (plasmin system) is a proteolysis system with multiple physiological function, and it mainly acts on is sedimentary scleroproein on the degraded vessel wall.This system has mainly comprised three integral parts, and they are respectively Profibrinolysin, plasminogen activator and fibrinolysis inhibition.Human plasminogen (plasminogen) is that a molecular weight is 92, and the strand glycoprotein of 000Da is by at arginine ₅₆₀-Xie Ansuan ₅₆₁Rupture in the place, it can be activated and become to have the plasmin of two peptide chains, energy hydrolysis of fibrin.The activator of Profibrinolysin has multiple, and most important plasminogen activator is tissue plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA), and they all can act on Profibrinolysin makes it change activated plasmin into.The fibrinolysis inhibition mainly contains α ₂Antiplasmin (α ₂-antiplasmin) and the inhibition (PAI-1) of plasminogen activator, their effect is opposite with above-mentioned albumen just, α ₂The molecular weight of antiplasmin is about 70,000Da, and it is the deactivation plasmin rapidly.PAI-1 directly reacts with t-PA or u-PA, makes their lose activation capability.

The continuous quickening with working speed of improving constantly along with people's living standard, the probability of heart and brain and limb vessel generation thrombus is also more and more higher, therefore, the material that maybe can activate human plasminogen of the albumen with plasmin activity all might become the thrombolysis preparation of a new generation and play a significant role on clinical treatment.Nineteen eighty-two, Japanese Mihara extracts one group of albumen with plasmin activity first from the powder earthworm (Lumbricus rubellus) of Lumbricidae (Lumbricidae), and with its called after Lumbrukinase (lumbrokinase) ^[1]Now know, in the positive earthworm of powder, contain six kinds of different Lumbrukinase components at least, be named as F-I-0, F-I-1, F-I-2, F-II, F-III-1 and F-III-2 respectively, their molecular weight is respectively 29662,29667,24664,24220,24196 and 23013Da, all have plasmin activity, and all be single peptide chain structure.These six kinds of enzymes are not by same coded by said gene, but from the isozyme of at least 4 genes ^[2,3]In addition, people find that also in the earthworm of some other kind, (Eisenia foetida) also exists multi-component Lumbrukinase phenomenon as Eisenia foetida ^[4,5]

Up to now, there are many Lumbrukinases that studies have shown that to have a very strong plasmin activity external.What be worth to propose especially is, different is that it can play a role by the mode of oral or enterally administering with other plasmins.1992, people such as Mihara begin the Lumbrukinase composition to the oral purification of aspiration patient, the mensuration of multinomial physiology and biochemical indicator is carried out in every day blood drawing subsequently, found that: 1) fibrin degradation product (FDP) just began to raise behind oral administration in 1-2 days, remained to active disappearance the after the 17th day; 2) content and the plasmin activity of tissue plasmin activator alive (t-PA) increase during administration always, and this is indicating that Lumbrukinase not only has the activity of plasmin, and can bring into play fibrinolytic by activating t-PA ^[6]

The Lumbrukinase preparation is now succeeded in developing by Instite of Biophysics, Chinese Academy of Sciences, now by the (trade(brand)name: of pharmaceutical factory in the river Bo Luoke) with the two imperial pharmaceutical factories in Qingdao (trade(brand)name: the multiple enteric coated capsule of general grace) produce and sell, total effective rate is 93.73%, obvious effective rate is 7.6%, and the recovery effects of the special wind-induced quadriplegia of centering, facial hemiparalysis, aphasis is more obvious ^[7]Have to be solvedly but the production of Lumbrukinase preparation still is faced with many problems at present, be subjected to the influence of earthworm growth cycle as industrial scale, leaching process is numerous and diverse from earthworm, non-single composition effect etc.Calendar year 2001, two kinds of plasmin activity composition F-III-1 that Sugimoto etc. use pichia spp (Pichia pastoris) successfully to express respectively to extract in the positive earthworm of powder first and F-III-2, its activity is the same with the natural component that separates in Lumbrukinase ^[8], this has brought new hope for the exploitation and the Lumbrukinase Its Mechanisms of the genetically engineered preparation of Lumbrukinase single component undoubtedly.

The latest developments of genetically engineered research field make the expression of plants foreign gene become possibility.Contain that the vegetable cell of foreign gene is renewable to go out whole plant, and foreign gene is stably entailed the offspring.Some unifacial leaf and dicotyledons have all carried out successful conversion so far.For example tobacco, potato, tomato, soybean, clover, wheat and corn etc.

The dicotyledons of Agrobacterium (Agrobacterium tumefaciens) mediation transforms existing numerous reports.Agriculture bacillus mediated leaf disc transformation method can carry out the importing of foreign gene, the screening of genetic transformation cell and the regeneration of transfer-gen plant effectively.

Monocotyledons is relatively more difficult through agrobacterium mediation converted, but also can utilize other method for transformation such as PEG and electro fusion method.Someone successfully imports foreign gene in the protoplastis of corn, paddy rice and wheat.Go out complete plant from the protoplast regeneration that transforms then.Method for transformation that some are new such as microtubule injection and particle bombardment may be more effective in monocotyledons transforms and regenerates.

Plant is a kind of variation, low cost and reproducible material resources, developing rapidly of biotechnology then makes us further widen the use range of plant, in plant, adopt the method for this " molecule agricultural " abroad, successfully produced increasing useful matter, comprising some high value pharmaceutical protein polypeptide, as people's cell growth factor, erythropoietin, Interferon, rabbit, tethelin, monoclonal antibody with can be used as antigen protein that vaccine uses etc. ^[9-28], some research institutions and company have begun to obtain huge economic benefit from the production of these pharmaceutical proteins.

Before the present invention, also nobody utilizes transgenic plant to express the Lumbrukinase composition of any earthworm, and this proteinoid might the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed by the mode of edible or enterally administering or thrombus is dissolved.

The present invention proposes to contain the edible method that partly is processed into capsule, electuary or other preparation of transgenic plant of earthworm Lumbrukinase recombinant protein first, owing to can carry out the quantitative of strictness to wherein contained Lumbrukinase composition, thereby people or mammiferous edible interval and amount there have been clear and definite regulation, have overcome the shortcoming that directly edible transgenic plant may exist thus.

The invention provides the construction process of expressing the earthworm vermis kinase tr-gene plant and how to use the method that contains these recombinant protein transgenic plant.This invention is specially adapted to China or other developing country.

The present invention utilizes edible transgenic plant to produce the earthworm Lumbrukinase of one-component, and this recombinant protein is similar with native protein aspect the solution fibrin activity.According to a preferential embodiment, the invention provides exactly and express the earthworm kinase single component production method of transgenic plants and how specifically to use this method that contains Lumbrukinase recombinant protein transgenic plant.

The present invention utilizes transgenic plant to produce the earthworm Lumbrukinase of one-component, if be processed into oral capsule, electuary or other preparation, then has cheapness, characteristics safe and easy to use, and more human or animal is protected.According to a preferential embodiment, people or Mammals only need eat the other parts of fruit, juice, root, stem, leaf or the plant of these transgenic plant, or utilize capsule, electuary or other preparation of these transgenic plant or transgenic plant crude extract (can quantitatively) preparation just can obtain resistivity to people and Mammals heart and brain and limb blood tubing thrombus disease in the mode of enterally administering.The plant here is meant that people are daily and eats, owing to avoided loaded down with trivial details purification process, reduced some other unfavorable factor of production cost and existence simultaneously.

According to an embodiment, the present invention relates to utilize transgenic plant to produce the method that contains one-component earthworm lumbrukinase capsule, electuary or other preparation.The transgenic plant material of producing this capsule, electuary or other preparation can have multiple mode, it can be complete plant, or the part of plant such as fruit, leaf, stem and stem tuber, or a part of crude extract of plant or through purifying and partial purification are derived from the Lumbrukinase albumen of the one-component of transgenic plant fully.In a word, adopt which kind of mode to depend primarily on the plasmin activity of this albumen itself, produce the dosage of the required this recombinant protein that is derived from transgenic plant of thrombolytic effect and in this composition what of contained interference thrombolytic effect material, as carbohydrate, pyrogen and toxin etc.

The Lumbrukinase recombinant protein of indication of the present invention is meant to be produced in transgenic plant, the gene that at first is the earthworm Lumbrukinase of coding one-component is separated, be inserted into then on the plasmid that contains selectable marker gene, promotor is positioned at the upstream of Lumbrukinase gene, and it handles the expression of this gene in plant.After foreign gene is expressed in each organ of plant or edible part, this part plant tissue of human or animal's edible.

The invention provides the method for in vegetable cell, producing the earthworm Lumbrukinase, according to a preferential embodiment, the invention provides in vegetable cell and to produce the proteic method of one-component earthworm Lumbrukinase, and the known formation that can cause human body or other Mammals heart and brain and limb vessel thrombus under native state of this albumen is suppressed or thrombus is dissolved.In other words, the earthworm earthworm kinase single component of indication of the present invention has plasmin activity, has and the similar plasmin activity of natural Lumbrukinase that is derived from same composition in the earthworm.Perhaps more definite theory, Lumbrukinase albumen of the present invention is the same with this Lumbrukinase albumen that is derived from earthworm or other conventional expression system generation, all can cause that the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed or thrombus is dissolved by oral and mode enterally administering.According to a preferential embodiment, this Lumbrukinase recombinant protein is a kind of plasmin albumen, it can express generation in vegetable cell, and has and be derived from the fibrinolytic activity of earthworm or this Lumbrukinase protein similar that other conventional expression system produced.

One-component Lumbrukinase of the present invention can be expressed generation in plant, and the plant that express to produce this one-component Lumbrukinase have at least a part be can directly eat or pass through after the roughing edible, for example tomato, the red sage root and clover etc.The edible part of this kind of plant or plant should not have toxicity, and therefore, many common food plants all are included within the defined plant scope of the present invention.In addition, in some cases, as potato, also be considered to edible plants here, some part that let it be to the greatest extent is considered to deleterious (the potato ball stem portion is nontoxic, therefore belong within the claim scope of the present invention, but its fruit is deleterious), in this case, this kind of plant also should be included within the claim scope of the present invention.

Recombinant protein of the present invention can be the whole of this Lumbrukinase native protein.Yet in some specific embodiments, Lumbrukinase also may be the part of this native protein.Sometimes, this Lumbrukinase can be expressed simultaneously in same plant with another one Lumbrukinase component or be stitched together and be formed the fusion rotein co expression.Proteic fusion can be passed through protein translation post-treatment, covalently bound mode, perhaps by the DNA recombinant technology gene is just linked together before accurate translation, and these two kinds of technology are technology that the expert of the art knows already.

The height of contained Lumbrukinase recombinant protein composition in the transgenic plant, the height of the processing and utilization of these transgenic plant and the related products cost of producing after being directly connected to, according to a preferential scheme of implementing, for improving the expression amount of Lumbrukinase gene in vegetable cell, the gene of coding Lumbrukinase can be optimized according to the codon of plant-preference, so that make this gene more stable in vegetable cell, efficient is higher when being transcribed into mRNA and translating into albumen.

In other embodiments of the present invention, a kind of transgenic plant of expressing one-component earthworm Lumbrukinase are fabricated.For the present invention, a kind of transgenic plant should have been expressed the Lumbrukinase recombinant protein at least in the part cell of this plant.According to some preferential embodiment of the present invention, it is edible that transgenic plant of the present invention have a part at least, its expressed recombinant protein is the earthworm Lumbrukinase, the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed for it or thrombus is dissolved, just as Lumbrukinase that native protein or other expression system produced.

The present invention relates to a kind of composition problem of food, contained some edible part of transgenic plant in this food at least, the transgenic plant here should be able to be expressed one-component earthworm Lumbrukinase.For the present invention, certain part of plant or plant is considered to have some nutritive value, and it can provide metabolism required energy for people or other Mammals, VITAMIN and other cofactors.Although lettuce does not provide energy, protein, carbohydrate and fat in a large number, just, it is specially eaten by people because containing reorganization Lumbrukinase albumen, and lettuce also should be included in the claim scope of the present invention.The robust fibre of lettuce is favourable to mammiferous health, even Mammals can not digest the robust fibre of lettuce.

The present invention relates to the composition problem of a kind of capsule, electuary or other preparation, contained some edible part of transgenic plant in capsule, electuary or other preparation at least, the transgenic plant here should be able to be expressed earthworm Lumbrukinase recombinant protein.For the present invention, the Lumbrukinase recombinant protein that contains in capsule, electuary or other preparation should be able to accurate quantification, so that more effectively bring into play the thrombolytic effect of this plasmin.According to a preferential embodiment of the present invention, the recombinant protein that is contained in capsule of the present invention, electuary or other preparation is the earthworm Lumbrukinase of one-component, the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed for it or thrombus is dissolved, just the earthworm Lumbrukinase that produces as native protein or other expression system.

Therefore, according to a specific embodiment more, the food of indication of the present invention will comprise the part of transgenic plant at least, it has some nutritive value, and contain the earthworm Lumbrukinase recombinant protein of one-component or fusion rotein or the derivative that forms with other albumen, and this recombinant protein can enter into by people or mammiferous Digestive tract and brings into play thrombolytic effect in the blood.Under any circumstance, food of the present invention all comprises root, stem, leaf, fruit or the said plant seed of plant.

To the relevant item with other of the method used in the present invention structure of some plasmid vectors very importantly, they play an important role in the processed and applied of transgenic plant that obtain indication of the present invention and transgenic plant.The plasmid vector that is used for transforming plant is by the dna sequence dna of the earthworm Lumbrukinase gene or derivatives thereof of coding one-component and handle the plant promoter that this dna sequence dna expresses said plant and form.In some specific embodiments, plasmid vector also comprises a selectivity or marker gene, is mainly used in the detection of transgenic plant or cell.In some specific embodiments, plant promoter of the present invention is the cauliflower mosaic virus 35S promoter.As mentioned above, according to some specific embodiment, plasmid vector of the present invention is used to transform edible plants, its encoded protein is a kind of Lumbrukinase (plasmin), furtherly, the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed the coded recombinant protein of plasmid vector or thrombus is dissolved, as native protein be derived from this Lumbrukinase albumen that other expression system produces.Dna fragmentation provided by the invention can be used for particle bombardment and transforms plant.

The present invention also provides the method that obtains transgenic plant cells, it may further comprise the steps: make up a plasmid vector or section of DNA sequence 1., wherein all contain the earthworm Lumbrukinase protein gene or derivatives thereof of the one-component of encoding gene (comprise gene codon optimised and with other gene Fusion etc.), and this gene is placed under the manipulation of a plant promoter; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells.According to a preferential embodiment, also comprise the method that goes out complete transfer-gen plant from plant transformed cell regeneration.

The invention provides the method for transformation of various vegetable cells or plant.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, electricity merges.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 transformed plant cells or plant.

The present invention has only mentioned potato and tomato when introducing method for transformation in detail, yet as the methods for plant transformation that the expert of the art knew already, many kind of plant can utilize method provided by the invention to transform.Therefore, all these plants all should be included within the claim scope of the present invention, and these plants can be monocotyledons or dicotyledons.

Will describe in detail in following example, used plasmid vector is a Ti-plasmids system in the methods for plant transformation of the present invention.According to an embodiment, the plasmid that the present invention uses is a double base conformability carrier.According to a more preferential embodiment, plasmid used in the present invention is pBI121.

The invention provides a kind of production method of transgenosis food.It may further comprise the steps: make up a plasmid vector or section of DNA sequence 1., the gene (comprise gene codon optimised and with other gene Fusion etc.) that wherein all contains the earthworm Lumbrukinase protein gene or derivatives thereof of the one-component of encoding, and this gene is placed under a plant promoter handles; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells; 3. from transgenic plant cells, bear complete transgenic plant again; 4. gather in the crops the edible part of transgenic plant; 5. edible a certain amount of this transgenic plant can make the formation of human body or other Mammals heart and brain and limb vessel thrombus be suppressed or thrombus is dissolved.

The present invention also provides the production method that contains set amount Lumbrukinase protein capsule, electuary or other preparation, it may further comprise the steps: make up a plasmid vector or section of DNA sequence 1., the gene (comprise gene codon optimised and with other gene Fusion etc.) that wherein all contains the single component earthworm Lumbrukinase protein gene or derivatives thereof of encoding, and this gene is placed under a plant promoter handles; 2. with plasmid vector or above-mentioned dna fragmentation transformed plant cells; 3. from transgenic plant cells, bear complete transgenic plant again; 4. in transgenic plant cell or complete plant, extract Lumbrukinase albumen, be processed into capsule, electuary or other preparation then.According to a preferential embodiment, containing some edible part of the proteic complete transgenic plant of Lumbrukinase or its can directly carry out freezing, dry and pulverize, be processed into capsule, electuary or other preparation then, and definite this Lumbrukinase albumen quantity that wherein contains.

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

The clone of Fig. 1 Lumbrukinase gene F-II gene and the structure of bacterial expression vector pETFII thereof.

The clone of Fig. 2 Lumbrukinase gene F-III gene and the structure of bacterial expression vector pETFIII thereof.

The building process of Fig. 3 plant expression vector pBIFII or pBIFIII, it contains Lumbrukinase F-II or F-III gene.Wherein GUS represents β-Glucuronidase (glucuronidase) gene; F-II represents Lumbrukinase F-II gene, and F-III represents Lumbrukinase F-III gene.

2 structure gene that Fig. 4 plasmid pBIFII or pBIFIII are contained and regulating and expressing sequence thereof.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 not homophyletic is the Northern results of hybridization of Lumbrukinase F-II protein mRNA in the transgenic Rhizoma Solani tuber osi blade.1 positive contrast (is the pcr amplification product of template with plasmid pGEMFIIDNA), 2 is the non-transgenic potato plant, 3-6 is a transgenic potato plant.

Fig. 6 PCR detects the integration situation of Lumbrukinase F-III recombinant protein gene in different transgenic Fructus Lycopersici esculenti strains are.Wherein 1 is λ DNA (EcoRI+HindIII) marker; 2 positive contrasts (the pGEMIII plasmid is a template amplification); 3,4 is non-transgenic tomato thing pcr amplification product; 5-12 is a part transgenic Fructus Lycopersici esculenti pcr amplification product.

The stability of Fig. 7 Lumbrukinase F-II recombinant protein in water is measured.After wherein containing potato tuber (0.5g) that earthworm swashs the F-II recombinant protein and grinding in the 2ml phosphoric acid buffer, (15-20 ℃) placed after 1-7 days under the room temperature, measured OD after getting 100ul supernatant liquor integrated enzyme reaction ₄₀₅The nm absorbance value.

Fig. 8 Lumbrukinase F-II recombinant protein stability is at high temperature measured.After the potato tuber (0.1g) that wherein contains Lumbrukinase F-II recombinant protein grinds in the 400ul phosphoric acid buffer, placed 1 hour under the differing temps, measure OD after getting 100ul supernatant liquor integrated enzyme reaction then ₄₀₅Nm light light absorption value.

The Lumbrukinase F-II that Fig. 9 goes out through affinitive layer purification and the SDS-PAGE of F-III recombinant protein analyze.Wherein A1 contains BL21 (DE3) the bacterial strain expression product negative control (IPTG induces) of plasmid pET-28b (+); B1 is for containing BL21 (DE3) the bacterial strain expression product negative control (IPTG induces) of plasmid pET-28a (+); A2 contains BL21 (DE3) the bacterial strain expression product negative control (IPTG does not induce) of plasmid pETFII; B2 is BL21 (DE3) the bacterial strain expression product negative control (IPTG does not induce) that contains plasmid pETFIII; A3 is BL21 (DE3) the bacterial strain expression product (IPTG induces) that contains plasmid pETFII; B3 is BL21 (DE3) the bacterial strain expression product (IPTG induces) that contains plasmid pETFIII; A4 is bacterium reorganization Lumbrukinase F-II behind the His6 column purification; B4 is bacterium reorganization Lumbrukinase F-III behind the His6 column purification; A5 is the plant reorganization Lumbrukinase F-II behind affinitive layer purification; B5 is the plant reorganization Lumbrukinase F-III behind affinitive layer purification; A6 and B6 are standard molecular weight albumen (Pharmacia)

Figure 10 utilizes the fibrin plate method to measure the Lumbrukinase fibrinolytic of different sources.P1 and P2 are respectively the extracting solution (positive control) of two Eisenia foetidas; 1 is the F-III of purifying from transgenic Fructus Lycopersici esculenti; 2 is the F-II of purifying from transgenic Rhizoma Solani tuber osi; 3,5-6 is a transgenosis horse tomato leaf extracting solution; 4,7-8 is the transgenic Rhizoma Solani tuber osi leaf extract; N1 is PBS damping fluid (negative control); N2 is 100mMTris-HCl, pH8.0 damping fluid (negative control); N2 is a non-transgenic potato leaf extracting solution (negative control); N4 is a non-transgenic tomato leaf extracting solution (negative control).

The present invention includes following integral part: 1. utilize DNA reorganization skill wood to make up plasmid expression vector, it contains the earthworm Lumbrukinase protein gene or derivatives thereof (comprise gene codon optimised and with other gene Fusion etc.) of one-component, and the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed for people or other Mammals or thrombus is dissolved; 2. select suitable recipient plant to transform, the stable gene ground of coding Lumbrukinase is integrated in the karyomit(e) of recipient plant also can stably entails the offspring; 3. from the recipient plant cell that transforms, bear complete transgenic plant again, and the Lumbrukinase recombinant protein can be stablized, be produced efficiently to this plant; The directly edible this transgenic plant of people or Mammals or be main the active ingredient capsule, electuary or other preparation that process so that the mode of enterally administering is oral with the edible part of these transgenic plant or its extract after, the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed or thrombus is dissolved, thus the purpose that reaches diseases prevention and cure the disease.

The invention provides the construction process of expressing earthworm Lumbrukinase albumen or derivatives thereof transgenic plant, this transgenic plant can directly be eaten as food or after being processed into capsule, electuary or other preparation or enterally administering after the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed or thrombus is dissolved, thisly can fibrinolytic result make people or Mammals have certain resistivity to thrombus disease.This thrombolytic effect is the result who produces behind the earthworm Lumbrukinase or derivatives thereof owing to expressing in transgenic plant, and the generation of Lumbrukinase recombinant protein then is because this protein gene is integrated in the genome of plant and the result that can stably express under plant promoter is regulated and control.

1. utilize the plant production recombinant exogenous protein

Producing one of reorganization pharmaceutical protein polypeptide example the earliest in plant is the high level expression characteristic of utilizing the plant storage protein natural, produces neuropeptide-leucine enkephalin of people.It is a micromolecule polypeptide that contains 5 amino-acid residues, in rape, be produced with the albuminised form of seed storage protein 2S earlier, after trypsin hydrolyzing is cut down from reserve protein, reclaimed by HPLC again, can be used as pain killer or tranquilizer clinically and use.

After early stage research, the antibody of expressing humans and animals in plant becomes the focus that people pay close attention to again.Immunoglobulin (Ig) such as IgG, IgM, IgG/IgA chimeric antibody, Fab fragment, single-chain antibody and single domain antibody etc. have all been realized expression, and expression product content is up to more than 2% of plant soluble proteins.This antibody-like has biologic activity, useful as drug, diagnostic reagent and affinity agent etc. behind purifying.In addition, expressing antibodies also might strengthen anti-virus ability, the raising output of crop and improve other economical character etc. in plant.

An other approach of producing the reorganization pharmaceutical protein in plant is to use genetically engineered plants virus as expression vector, this class is cut out body can instantaneously efficiently express a large amount of foreign proteins, be that institutes such as Agrobacterium transformation system can not accomplish, existing tens kind of plant viruses are transformed into dissimilar exogenous protein expression carriers, comprise cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV) (TMV) and cowpea mosaic virus (CpMV) etc., wherein surpass protein polypeptide successful expression in the TMV carrier of kind more than 150 ^[29]Utilize the expressing fusion protein strategy to produce to contain the chimeric coat protein of malaria, animal foot and mouth disease virus, rhinovirus and human immunodeficiency virus (HIV) antigenic determinant, the mode of production of this class fusion rotein may be in the future to produce an important and cost-effective approach of vaccine.

The present invention not only allows in a kind of plant to produce a kind of earthworm Lumbrukinase albumen, and can be in a kind of plant earthworm Lumbrukinase recombinant protein of production various ingredients simultaneously.Gene constructedly on same expression vector, transform plant with it then with the earthworm Lumbrukinase of each component of coding is proteic, thereby in transgenic plant, can express multiple earthworm Lumbrukinase simultaneously.For example, the Lumbrukinase that contains 6 kinds of components such as F-I-1, F-II and F-III-1 in the positive earthworm of powder, on same plant expression vector, also controlled by different plant promoters if general's two Lumbrukinases wherein are gene constructed, import plant then simultaneously, thereby in transgenic plant, just can produce these two kinds of Lumbrukinases simultaneously; The gene of these two kinds of Lumbrukinases of also this can being encoded just merges before conversion mutually, but do not change frameshit framework separately, after importing plant, in plant materials, just can express a fusion rotein that forms by these two kinds of albumen, thus, just may further improve these two kinds of proteic thrombolysis activities.In addition, transgenic plant also can be by repeatedly transforming or containing the method acquisition that different Lumbrukinase expression carrier transform simultaneously with a plurality of simultaneously.

2. the selection of recipient plant

Utilize different foreign DNA transfer techniques, now transformed various plants, comprise many dicotyledonss and some monocotyledonss.Consider the complexity of genetic transformation operation, the present invention is more prone to use dicotyledons as recipient plant, although some monocotyledons such as wheat, corn and paddy rice may be more suitable for as people's food or be processed into capsule, electuary or other preparation.

Select recipient plant to carry out genetic transformation and will consider whether the Lumbrukinase gene can express in its edible part.Because, if after Lumbrukinase is expressed in certain part reality, leaf, stem, seed or the root of plant, the plant tissue of people or direct-edible this part of Mammals or after processing, make capsule, electuary or other preparation is oral for people.According to not preferential embodiment, Lumbrukinase also can be expressed in the plant that anorexia is used, and therefrom is purified into required Lumbrukinase recombinant protein then fully, is reprocessed into capsule, electuary and other preparation.

When selecting recipient plant, also to consider some other factor.The edible part of recipient plant preferably need not heat just edible, because heating can cause the Lumbrukinase sex change, thereby reduces its fibrinolytic ability.

The recipient plant that is suitable for the present invention's conversion comprises dicotyledons and the monocotyledons that all can be used as human or animal's food, part or all edible of plant, these plants comprise potato, tomato, the red sage root, clover, Radix Dauci Sativae, lettuce, cucumber, wheat, soybean, paddy rice, corn, apple, pears and banana etc., but are not limited only to this.

3. methods for plant transformation

There are many kinds of methods foreign gene can be imported monocotyledons and dicotyledons.The main method of the foreign gene stable integration being gone into the plant chromosome genomic dna comprises: 1) agrobacterium-mediated transformation; 2) dna direct is taken in method, comprises protoplasma fusion method, electrization, the microtubule injection of PEG mediation and uses particle gun that dna direct is sent into vegetable cell and tissue etc.

Agrobacterium-mediated transformation is to utilize plasmid vector that specific dna fragmentation is integrated into the plant chromosome genomic dna.The method for transformation of plant tissue is different because of plant and Agrobacterium system.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 of dicotyledons.

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 vegetable cell and be integrated in the karyomit(e) of plant.The structure of transfer vector was divided into for two steps, at first was to make up the plasmid that can duplicate in intestinal bacteria, and this plasmid contains the gene of the earthworm Lumbrukinase or derivatives thereof of one-component; The T-DNA border sequence is contained at the two ends of this goal gene, and it is responsible for the insertion site of goal gene in Plant Genome.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 plant or vegetable cell contain the T-DNA sequence of integration after this gene is expressed in vegetable cell.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 vegetable cell at T-DNA and play an important role.

The people who is familiar with the plant genetic 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 plant.Equally, people also will be appreciated that except agrobacterium tumefaciens in some cases, to also have other Agrobacterium such as root of hair type Agrobacterium to be more suitable for the conversion of some plants.

The method for transformation of plant tissue is different because of plant and agriculture bacillus mediated system.The most frequently used is the c4 plant leaf discs conversion method, and it is suitable for the multiple regenerated plant explants that is easy to.In some cases, also need to add some helpers.Other method for transformation comprises protoplast transformation, goes out vegetable cell and whole plant by protoplast regeneration then.

The present invention is not limited only to use Agrobacterium Ti-plasmids conversion system, should comprise that also the means of other physical property directly import foreign gene method and other method with exogenous gene transfered plant cell of vegetable cell or protoplastis.

4. genetic expression promotor

After recipient plant and method for transformation are determined, a composing type, grow the selected so that foreign gene of expression promotor adjustment type or the organizing specific type and can in plant, 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); Photoinduction gene promoter such as ribose bisphosphate carboxylase small subunit (rbcS); Adversity inducible gene such as ethanol dehydrogenase (Adhl) or maize actin gene promoter etc., but be not limited only to this.The present invention also can utilize known promotor such as the potato plastogene promotor that efficiently expresses at the plant edible portion.

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

Earthworm Lumbrukinase with transgenic Rhizoma Solani tuber osi and tomato production one-component is an example below, describe some preferential embodiments, but application of the present invention is not limited only to this.

Selecting the earthworm Lumbrukinase to express in transgenic plant is because heart and brain and limb vessel embolism are a kind of very common people or mammalian diseases, although present employed thrombolytic drug is a lot, all exists deficiencies such as price is high, side effect is big.Selecting potato and tomato is to illustrate for example in the different piece of plant to express earthworm Lumbrukinase recombinant protein as recipient plant, in addition, for the modification that shows gene with to transform improving the expression efficiency of Lumbrukinase in plant be helpful, when transforming potato, used the Lumbrukinase gene after codon optimized.

Embodiment 1

1. the synthetic of Lumbrukinase F-II gene

Although earthworm and plant all belong to eukaryote, they are still existing certain difference aspect the gene codon preference, and this species diversity might have influence on the stability of Lumbrukinase gene in transgenic plant and efficiently express.For improving the expression amount of Lumbrukinase in transgenic plant, aminoacid sequence according to the Lumbrukinase component F-II that had announced already (is seen GenBank, accession number: AF109648), under the prerequisite that does not change this Argine Monohydrochloride sequence, the codon of having a preference for according to plant ^[30], manually design and synthesized the full gene DNA nucleotide sequence (seeing sequence table 1 and 2) of Lumbrukinase component F-II.For the ease of the structure of various plasmid vectors from now on, in synthetic Lumbrukinase F-II gene DNA nucleotide sequence, synthetic and added restriction enzyme site NcoI before 5 ' end ATG initiator codon of this gene, after 3 ' end of this gene is ended codon TAG, increased restriction enzyme site XhoI (said gene splice and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing).

2. the clone of Lumbrukinase F-II gene

The gene DNA fragment of above-mentioned synthetic is directly inserted and is connected in the T site in the pGEM-T plasmid (seeing Promega company product description), according to the method that the said firm provided, by the white screening system of indigo plant, obtain containing the bacterial clone pGEMFII (see figure 1) of Lumbrukinase F-II gene, then, by nucleotide sequence analysis, the gene of determining coding Lumbrukinase F-II is correct and complete (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

3. the clone of Lumbrukinase F-III gene

Lumbrukinase F-III does not take the method for synthetic, but clones from earthworm by the method for RT-PCR.RNA extraction agent box (the total RNA extraction agent of Flash UNIQ-10 pillar box) is given birth to worker bio-engineering corporation available from Shanghai, according to the method that is provided in the test kit, from Eisenia foetida (Eisenia foetida) (this earthworm is provided by the Yang Zhenji researcher of China Agriculture Academe Fertilizer Institute), extract the template of its total RNA as the RT-PCR amplification.DNA nucleotide coding sequence according to the earthworm Lumbrukinase component F-III that has announced (is seen GenBank, accession number: AB045719), synthesized a pair of Auele Specific Primer (giving birth to worker bio-engineering corporation by Shanghai synthesizes), wherein upstream primer P1 (5 ' end primer) is: 5 '-AA CATATGGAACTTCCTCCCGGAACAAAAATTGTCGG-3 ' wherein contains NdeI restriction enzyme site (line place as follows); Downstream primer P2 (3 ' end primer) is: 5 '-AA GAGCTCTTAGTTGTTGGTGATTGTGTCGGTGATCCATC-3 ' wherein contains SacI restriction enzyme site (line place as follows).RT-PCR test kit (TaKaRa RNA LA PCR ^TMKit) available from the precious biotechnology in Dalian company limited, according to the method that is provided in this test kit, utilize P1 and P2 primer, with the above-mentioned total RNA of earthworm that extracts is template, obtain a length by RT-PCR amplification and be about dna fragmentation (wherein reverse transcription condition: reaction system 20 μ l about 750bp, with P2 is the synthetic cDNA chain of primer, and 42 ℃ of water-bath 60min carry out reverse transcription; Handle 5min deactivation ThermoScript II for 95 ℃.The PCR condition: reaction system 100 μ l are amplimer with P1 and P2, at first, and 94 ℃ of pre-sex change 4min; According to 94 ℃ of sex change 30sec, 55 ℃ of annealing 45sec, 72 ℃ of extension 90sec, carry out 35 circulations then; At last, 72 ℃ are extended 10min).This dna fragmentation is inserted directly in the T site in the pGEM-T plasmid (seeing Promega company product description), the method that provides according to the said firm, by the white screening system of indigo plant, obtain containing the bacterial clone pGEMFIII (see figure 2) of this gene, then, by nucleotide sequence analysis, the gene of determining Lumbrukinase F-III is correct and complete (Shanghai is given birth to worker bio-engineering corporation and finished).

4. the structure of bacterial expression vector pETFII and pETFIII

Because when synthetic Lumbrukinase F-II gene, in its encoding sequence 5 ' end and 3 ' end, NcoI and XhoI restriction enzyme site have been introduced respectively, so the pGEMFII plasmid DNA is behind NcoI and XhoI double digestion, Lumbrukinase F-II gene can be scaled off, pass through agarose gel electrophoresis, this dna fragmentation of separable acquisition is inserted into (NcoI and XhoI double digestion) among the plasmid pET-28b (+) subsequently, just is built into bacillary expression vector pETFII (see figure 1).

When from earthworm, directly cloning Lumbrukinase F-III gene, because in employed 5 ' end primer P1, introduced the NdeI site, and in 3 ' end primer P2, introduced the SacI site, (the F-III gene order contains the NcoI site so the pGEMFIII plasmid DNA is behind NdeI and SacI double digestion, so can not use NcoI and XhoI double digestion, and can only utilize NdeI and SacI double digestion, be inserted into then among the pET-28a (+), cause the 6 histidine-tagged N ends that are positioned at fusion rotein thus), the gene fragment of coding Lumbrukinase F-III can be scaled off, separate this dna fragmentation of acquisition by agarose gel electrophoresis, be inserted into subsequently (NdeI and SacI double digestion) among the plasmid pET-28a (+), just be built into bacillary expression vector pETFIII (see figure 2).

Plasmid pET-28a (+) and pET-28b (+) are all available from U.S. Novagen company, and they all are bacillary efficiently expression vectors, and all are subjected to the IPTG abduction delivering, the difference of the two be that change has taken place single open reading frame behind restriction enzyme site BamHI.The albumen label of 6 Histidines is all contained at the two ends that these two plasmid multienzyme are cut the site respectively, foreign gene is inserted into after suitable multienzyme cuts the site, under the situation that does not change the frameshit framework, can with the 6 histidine-tagged formation fusion roteins that are positioned at N end or C end, be convenient to the purification of external source recombinant protein.In pETFII, because use is that insert in NcoI and XhoI site, the 6 histidine-tagged proteic C of Lumbrukinase F-II that are positioned at hold, and in pETFIII, are NdeI and the insertion of SacI site owing to what use, the 6 histidine-tagged proteic N ends of Lumbrukinase F-III that are positioned at.

5. Lumbrukinase F-II and F-III albumen efficiently expressing in bacterium

Utilize plasmid vector pETFII and pETFIII transformed into escherichia coli BL21 (DE3) bacterial strain (available from Novagen company) respectively, (10g peptone, 5g yeast extract, 10g sodium-chlor from LB solid plate substratum, adding distil water to 1 liter, pH7.0 also contains kantlex 50mg and 15g agar).Picking transforms the single bacterium colony of bacterium that the back forms, and is inoculated on the LB liquid nutrient medium (to contain kantlex 50mg/L), and shaking culture is spent the night; Next day, get the part nutrient solution and be added in the fresh LB liquid nutrient medium (containing Kan50mg/L) in 1: 100 ratio, continue to be cultured to OD ₆₀₀Reach at 0.6 o'clock, add IPTG (available from Sigma company), making its final concentration is 1mmol/L, behind the inducing culture 3 hours, centrifugal, collect thalline, be suspended in 1 * SDS sample-loading buffer (50mmol/L Tris-HCL, pH6.8,100mmol/L DTT, 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), in 100 ℃ of water-baths, boil the broken thalline of 3min, centrifugal, get supernatant liquor and carry out the SDS-PAGE electrophoresis, whether have recombinant protein produce, the BL21 bacterial strain (not adding IPTG induces) that BL21 bacterial strain (IPTG induces) that transforms respectively with pET28a (+) and pET28b (+) respectively in the experiment and pETFII and pETFIII transform is respectively seen Fig. 9 A and 9B if analyzing.

6. the purifying of Lumbrukinase recombinant protein

A large amount of cultivations contain the BL21 bacterial cell of pETFII and pETFIII plasmid respectively, after inducing through IPTG, collect the BL21 thalline, subsequently thalline is suspended in bacterium lysis buffer (0.05mol/L Tris-HCl damping fluid, pH8.0), the ultrasonic disruption thalline, the centrifugal 15min of 12000rpm keeps precipitation (inclusion body).Adding solubilization of inclusion bodies liquid (0.05mol/L Tris-HCl damping fluid, 1mmol/L PMSF, 8mol/L urea, 10mmol/L DTT, pH8.0), room temperature treatment 4 hours; 12000rpm, centrifugal 15min gets supernatant.

Because the proteic C end of reorganization Lumbrukinase F-II contains the label of 6 Histidines, and the N end of reorganization Lumbrukinase F-III contains the label of 6 Histidines, so their purifying can utilize the method for Histidine affinity column to carry out respectively.His6 fusion rotein purification kit ProBond ^TMPurification System is available from American I nvitrongen company, and purge process is carried out according to the method that test kit provided fully.Compile the protein liquid behind the wash-out, through lyophilize, be dissolved in then in the 0.05mol/L Tris-HCl damping fluid (contain 10mmol/L EDTA, pH7.0), to final concentration be 1mg/ml.

7. sero-fast preparation

Available from the Department Of Medicine, Peking University, all is male for examination small white mouse (BALB/c), and body weight is about 20g.Get above-mentioned reorganization Lumbrukinase F-II and F_III albumen after His6 post affinity chromatography, each mouse peritoneal is injected into 0.1ml (mouse of injection reorganization Lumbrukinase F-II and F-III is separately to raise) respectively, every injection in 10 days 1 time, injected the back the 7th day at the 6th time, get blood from the mouse eye, collect in (mouse resisting anteserum of Lumbrukinase F-II and F-III is collected respectively) in the 10ml centrifuge tube, placed 1 hour for 37 ℃, place 4 ℃ to spend the night then, next day, the centrifugal 15min of 5000rpm collects top serum, after the packing, place the medium-term and long-term preservation of-20 ℃ of refrigerators and standby.

Embodiment 2

1. the structure of plant expression vector pBIFII and pBIFIII

In order to make up pBIFII high-efficiency plant expression vector, according to the Lumbrukinase F-II nucleotide sequence of synthetic, synthesized a pair of primer (giving birth to worker bio-engineering corporation by Shanghai synthesizes), wherein upstream primer P3 (5 ' end primer) is: 5 '-AAA TCTAGAAACAATGGTTATTGGAGGTACTAACGCTAGCCCAGGAG-3 ' wherein contains XbaI site (seeing underscore place in the primer).Downstream primer P4 (3 ' end primer) is: 5 '-AAA GAGCTCCTAACGAGAGTTGTCTCCAATCCAACCCAAG-3 ' wherein contains SacI site (seeing underscore place in the primer).And be template with the DNA of plasmid pGEMFII, through PCR (reaction system 100 μ l are primer with P3 and P4, at first, 94 ℃ of pre-sex change 4min; According to 94 ℃ of sex change 30sec, 55 ℃ of annealing 45sec, 72 ℃ of extension 90sec, carry out 35 circulations then; At last, 72 ℃ are extended 10min), amplification obtains Lumbrukinase F-II gene, because of in 5 ' end and 3 ' primer, having introduced XbaI and SacI site respectively, so amplified production with above-mentioned two enzyme direct enzyme cutting after, directly orientation is inserted in the former gus gene site of plasmid pBI121 (through XbaI and SacI double digestion), just is built into double base plant expression vector pBIFII (see figure 3).

In order to make up pBIFIII high-efficiency plant expression vector, according to known Lumbrukinase F-III nucleotide sequence, synthesized one 5 ' upstream primer P5 (giving birth to worker bio-engineering corporation by Shanghai synthesizes), its sequence is: 5 '-AAA TCTAGAAACAATGGAACTTCCTCCCGGAACAAAAATTGTCGG-3 ' wherein contains XbaI site (seeing underscore place in the primer).DNA with plasmid pGEMFIII is a template, makes primer with P5 and P2 (see embodiment 1 3), through PCR (reaction system 100 μ l are primer with P5 and P2, at first, 94 ℃ of pre-sex change 4min; According to 94 ℃ of sex change 30sec, 55 ℃ of annealing 45sec, 72 ℃ of extension 90sec, carry out 35 circulations then; At last, 72 ℃ are extended 10min), amplification obtains Lumbrukinase F-III gene, because of in 5 ' end and 3 ' primer, having introduced XbaI and SacI site respectively, so amplified production with above-mentioned two enzyme direct enzyme cutting after, also directly orientation is inserted in the former gus gene site of plasmid pBI121 (through XbaI and SacI double digestion), just is built into double base plant expression vector pBIFIII (see figure 3) thus.

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 former gus gene can be deleted with XbaI and SacI double digestion, and can insert the new foreign protein genes of another one subsequently, new 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 tailing signal sequence of oneself, and they all are derived from nopaline (Nopaline) synthetic enzyme (NOS) gene.

Plasmid pBIFII or pBIFIII contain: 1) Xin Meisu synthase gene II (nptII), and it offers the vegetable cell kalamycin resistance; 2) Lumbrukinase gene F-II or F-III gene and handle the cauliflower mosaic virus 35S promoter of this gene; 3) T-DNA left and right sides border sequence, it can be with nptII gene and Lumbrukinase transgenosis in plant materials and be incorporated in the plant chromosome.The structure of pBIFII and pBIFIII such as Fig. 4.

2. expression vector pBIFII and pBIFIII are imported Agrobacterium (A.tumefaciens) respectively

The plasmid vector pBIFIII that contains the plasmid vector pBIFII of Lumbrukinase F-II gene or contain Lumbrukinase F-III gene can transfer in the Agrobacterium LBA4404 bacterial strain by the mode of triparental mating respectively, 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 the T-DNA of self is deleted.The Vir gene can trans adjusting T-DNA transfer in vegetable cell from plasmid pBIFII or pBIFIII.

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 ₆₀₀When the nm 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 pBIFII or pBIFIII 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 ₆₀₀When the nm 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 pBIFII or pBIFIII plasmid begins to produce.

From Agrobacterium, extract plasmid DNA with alkaline lysis ^[31]And can detect pBIFII or whether the pBIFIII plasmid DNA exists with digestion with restriction enzyme.

3. agriculture bacillus mediated plant genetic transforms

The genetic transformation of plant at first is that plant tissue organ or cell and Agrobacterium are cultivated altogether, after cultivating about 2 days, the plant explants dislocation is selected in the substratum accordingly.Plant explants can be protoplastis, callus or other organ-tissue, because of plant different.Selecting the organ of band leaf for use is the most frequently used method.

Blade transforms main method according to people such as Horsch ^[32]Potato aseptic seedling (No. 3, middle potato, available from Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science) be kept in 22 ℃ of illumination boxs, give medium tenacity illumination in 16 hours, every 3 week clip top young shoot, again cuttage is not in containing any antibiotic MS solid medium, and the blade in 2 weeks of growing is suitable for transforming most.Tomato (middle vegetables No. 5, seed is available from Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science) then transforms with the cotyledon or the hypocotyl aseptic explant of 7-10 age in days.

No matter potato still is a tomato all is converted into best with blade.The Agrobacterium LBA4404 that will contain expression vector pBIFII or pBIFIII is seeded in the 50ml YEP substratum (containing 50mg/ml kantlex and 25mg/ml Streptomycin sulphate) 28 ℃ and cultivated 2 days, 4000g collected thalline in centrifugal 5 minutes, with 25ml liquid MS nutrient solution (not containing microbiotic) washing once, be suspended into OD again with MS again ₆₀₀Nm=0.2-0.4.To there be in the Agrobacterium nutrient solution after the blade of wound immerses dilution several minutes, blot bacterium liquid unnecessary on the blade, and then the blade dislocation not contained on any antibiotic regeneration culture medium 23-25 ℃ and cultivated altogether 2 days with aseptic filter paper.Cultivate on the selection substratum that the blade dislocation is fresh, contain 100ug/ml kantlex and 500ug/ml Pyocianil in this substratum, later per two weeks are changed and once select substratum.When blade wound callus has young shoot to form and grows to when enough big, young shoot cutting-out (usually in 6-10 week after the conversion) is transferred on the root media and grown.After root occurs, will grow under the regeneration plant dislocation aseptic condition or transfer in the soil, the growth down of the temperature that suits, illumination and nutritional condition.

4. the screening of express recombinant Lumbrukinase F-II or F-III transgenic engineering plant

After transforming about four months (tomato produces fruit and needs 10 months).Transfer-gen plant be can detect and reorganization Lumbrukinase F-II or F-III albumen whether contained.

1) biological chemistry and immunology detection

Blade, stem, stem tuber or fruit collected specimens from transgenic plant.And sample (as the PBS phosphoric acid buffer, contained 0.2g KH at water or damping fluid among the 1L ₂PO ₄, 2.9g Na ₂HPO ₄12H ₂O, 8.0gNaCl, 0.2g KCl, after grinding in pH7.4), homogenate centrifugal 10 minutes at 10000g is got supernatant liquor, and except that staying sub-fraction to be used for the Lowry standard measure soluble proteins, all the other are used for Lumbrukinase F-II or F-III recombinant protein Determination on content.Can measure the content of Lumbrukinase F-II in the plant or F-III recombinant protein by euzymelinked immunosorbent assay (ELISA).

2) detection of Lumbrukinase F-II or F-III gene

The integration situation of Lumbrukinase F-II or F-III gene can detect by pcr amplification purpose fragment gene or the reaction of Southern hybridization trace in the transgenic plant, in transgenic plant and control plant, extract its genomic dna respectively, with this as masterplate, Auele Specific Primer (seeing embodiment 1) with Lumbrukinase F-II or F-III gene increases, or with behind XbaI or the SacI single endonuclease digestion, agarose electrophoresis DNA isolation fragment, and then after transferring on the nylon membrane, with the Lumbrukinase F-II of digoxin (Digoxigenin) mark or F-III gene coded sequence as probe (marking method is seen Roche company test kit).In addition, can collect the transfer-gen plant selfed seed, the foreign gene of sprouting experiment or its offspring of pcr analysis by the kalamycin resistance situation of isozygotying.

5. express the breeding and the cultivation of Lumbrukinase F-II or F-III protein transgene plant

Behind the transgenic Rhizoma Solani tuber osi or tomato plant that filter out efficient and stably express Lumbrukinase F-II or F-III recombinant protein, must a large amount of these transfer-gen plants of breeding for producing this Lumbrukinase recombinant protein.Therefore potato, can just can obtain a large amount of seedling and micro potato at short notice by the method for cuttage, and needn't worry to cause owing to syngenesis offspring's genetic recombination problem by nourishing and generating.Tomato or other rely on the transgenic plant of seminal propagation will obtain the offspring of stably express, then need the long period, because seminal propagation has shortcoming, its offspring lacks homogeneity, and foreign gene is passed to the offspring according to mendelian inheritance.Basically, be different in each seed heredity, have its hereditary property.Therefore, transgenic plant preferably will be through the several generations screening, and after obtaining pure lines, foreign gene could stably be passed to the offspring.

6. contain the preparation of Lumbrukinase F-II or F-III recombinant protein genetically modified food

Lumbrukinase F-II or F-III recombinant protein can be produced by a large amount of breeding transgene plants, and the plant edible food part is reprocessed into capsule, electuary and other preparation in direct-edible after the results or direct processing or after part is purified or purified fully.Though potato tuber can not be eaten something rare, after lyophilize, wear into fine powder and be processed into capsule, electuary or other preparation.Tamato fruit then can be processed into bottled tomato juice conveniently to be drunk.Within a certain period of time, people are by oral a certain amount of capsule, electuary or tomato juice or directly edible tomato (one or many in for some time), the formation of human body or other Mammals heart and brain and limb vessel thrombus is suppressed for Lumbrukinase F-II that wherein contains or F-III recombinant protein or thrombus is dissolved, thereby reaches the purpose of control thrombus disease.

Embodiment 3

1. the conversion of potato

Under agriculture bacillus mediated, utilize leaf dish method to transform potato, then by on selecting substratum, [adding plant hormone (Sigma company) 6-BA1.5mg/L and NAA0.1mg/L among the MS in the basic ingredient, and contain 200ug/ml kantlex and 500ug/ml carboxylic Bian penicillin] cultured continuously and screening, complete kalamycin resistance potato plant is reproduced out.

According to people's such as Horsch method, utilize leaf dish method to transform potato.The potato aseptic seedling is cut blade from the petiole position that links to each other with stem with scissors after cuttage grew for 2 weeks in the MS solid medium again.The agrobacterium strains LBA4404 that contains expression vector pBIFII 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 in the Agrobacterium of cutting after blade immerses dilution 5 minutes, takes out then, blots with filter paper, and the blade face places on the MS solid medium up, cultivates altogether 2 days for 28 ℃.On then that the blade dislocation is the fresh selection substratum, 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 the 100ug/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. the RNA of transgenic Rhizoma Solani tuber osi analyzes

Utilize the Lumbrukinase F-II gene DNA fragment of digoxigenin labeled to make probe (marking method is seen Roche company test kit), the part kalamycin resistance potato plant RNA after the pBIFII expression vector is transformed has carried out hybridization analysis.

The total RNA of the blade of transgenic potato plant is extracted, the method document that sees reference ^[33]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 741bp of probe, it is from the NcoI of plasmid pGEMFII and the endonuclease bamhi of XhoI, has comprised whole encoding sequences of Lumbrukinase F-II 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 100ml 0.1XSSC, 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 company), 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 (0.1MTris-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 pBIFII as shown 5.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 RNA that transcribes has 800bp long approximately, with pre-estimate consistent.The RNA of negative control plant does not observe hybridization signal.Stable, the great expression of mRNA also can show that the stability of mRNA can not become the problem that Lumbrukinase F-II gene is expressed in potato with the result of the proteic DNA nucleic acid probe of coding Lumbrukinase F-II specific hybrid.

3. the mensuration of different transgenic potato plant Lumbrukinase F-II protein contents

Get different transgenic potato plant blade 0.1g, each adds the 1ml deionized water, grinds with glass homogenizer under 4 ℃.Centrifugal 5 minutes of 4 ℃ of homogenate 10000g utilize Lowry method (protein determination kit is available from Sigma company, and article No. is P5656), get the part supernatant liquor and measure soluble protein content.

Measure the content of Lumbrukinase F-II recombinant protein in the potato leaf by enzyme linked immunosorbent assay (ELISA), get blade 0.2g, be cushioned in the liquid at bag with the ratio of 1: 3 (W/V) and grind (1.59gNa ₂CO ₃, 2.93gNaHCO ₃, adding distil water to 1 liter), centrifugal 5 minutes of 4 ℃ of homogenate 10000g get in the aperture that supernatant liquor 200ul adds enzyme-linked reaction plate, each sample is established two repetitions, then elisa plate is placed 4 ℃ down bag spent the night, next day is with PBST damping fluid (8gNaCl, 0.2gKH ₂PO ₄, 2.9gNa ₂HPO ₄12H ₂O, 0.2gKCl, 0.5mlTween 20, adding distil water to 1 liter) wash 3 times, each 3 minutes, after the drying, add 200ul mouse anti Lumbrukinase F-II protein antiserum (seeing embodiment 1) in each aperture, antiserum(antisera) before use through PBST by 1/1000 times of dilution.37 ℃ were reacted 2 hours, wash 3 times with PBST, each 3 minutes, after the drying, the anti-mouse IgG (available from Sigma company) that respectively adds the 200ul alkali phosphatase enzyme mark in each aperture, enzyme labelled antibody press 1/5000 times of dilution through PBST before use, and 37 ℃ were reacted 1 hour, wash 4 times with PBST, each 3 minutes, after the drying, respectively add chromogenic substrate damping fluid [9.7ml diethanolamine in the aperture, adding distil water 80ml, transfer pH to 9.8 with HCl, add substrate p-oil of mirbane (Sigma company product) then to final concentration 0.6mg/ml], colour developing is 1 hour under the room temperature, each aperture respectively adds 50ul 3M NaOH stopped reaction then, reads absorbance value under the 405nm.

The positive contrast of Lumbrukinase F-II recombinant protein (seeing embodiment 1) of the standard content that is purified into the His6 affinity column, positive control is become different protein levels (2-512ng) by doubling dilution, behind color reaction, read the 405nm absorbance value, prepare a standard absorption curves.Survey discovery by the enzyme joint inspection, do not find to contain Lumbrukinase F-II recombinant protein in the negative control plant, then can detect Lumbrukinase F-II recombinant protein in the part transgenic potato plant, just content is different, generally is about plant soluble proteins 0.01-0.1%.Between the different transgenic Rhizoma Solani tuber osi strains system big this of diversity ratio may be since copy number that gene inserts and insertion the site is different causes.

4. Lumbrukinase F-II recombinant protein Determination on content in the transgenic Rhizoma Solani tuber osi different tissues organ

Get transgenic Rhizoma Solani tuber osi blade, stem and stem tuber and organize each 0.2g, processing and measuring method are the same.Listed the content of Lumbrukinase F-II recombinant protein in the transgenic Rhizoma Solani tuber osi different tissues organ in the table 1.

The content of Lumbrukinase F-II recombinant protein in table 1. potato leaf, stem and the stem tuber

Organ	Ng/mg soluble proteins (%)	The ng/g fresh weight
			Leaf stem stem tuber	290(0.03％) 110(0.01％) 1250(0.12％)	1500 140 4300

The highest 0.03% of the soluble proteins that is about of Lumbrukinase F-II expression amount in the potato leaf is the 1500ng/g fresh weight.F-II expression amount in the stem accounts for 0.01% of plant soluble proteins, and its every g fresh weight contains recombinant protein and is about 1/10th of blade.F-II recombinant protein content in the potato tuber is the highest, is about the 4300ng/g fresh weight, and this just provides great convenience for processing and utilizing potato tuber to produce the antithrombotic new drug.

5. the breeding of Lumbrukinase F-II transgenic Rhizoma Solani tuber osi

The breeding of transgenic Rhizoma Solani tuber osi is described in embodiment 2.

Embodiment 4

1. the conversion of tomato

The method for transformation of tomato (Lycopersicom esculentum) as embodiment 31 described in, also be under Agrobacterium LBA4404 mediation, to utilize Ye Panfa to carry out genetic transformation.Different is that the plasmid vector that contains in the Agrobacterium LBA4404 bacterial strain of use is pBIFIII.Cotyledon or hypocotyl aseptic explant after Agrobacterium is infected and grew 7-10 days, this explant does not need pre-cultivation, but after cutting-out, immersed immediately in the Agrobacterium bacterium liquid 5-10 minute, take out the back and blot unnecessary bacterium liquid with filter paper, back dislocation Z1 substratum (adding the 1mg/L zeatin in the MS minimum medium) was cultivated 2 days under 25 ℃ of shading conditions altogether.Then explant being transferred to SM substratum (adding 1mg/L zeatin, 100mg/L kantlex and 500mg/L Pyocianil in the MS minimum medium) evoked callus forms and the seedling differentiation ^[34]The seedling of downcutting is in RM substratum (adding 0.2mg/L NAA, 100mg/L kantlex and 500mg/L Pyocianil in the 1/2MS minimum medium) root induction, and certain altitude to be grown to is transferred in the sterile soil then, the condition incubation growth that suits.

2.PCR detect the integration situation of Lumbrukinase F-III gene in tomato

The extracting method of tomato dna group DNA is substantially according to the method for Chee ^[35]Get the fresh blade of 3g tomato, put into the mortar liquid nitrogen grinding, in the powder dislocation 20ml centrifuge tube after grinding, add then 9ml CTAB extracting solution (100mM Tris-HCl, pH 7.5 contains 5M NaCl, 10mM EDTA, 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 the about 1ul of above-mentioned tomato dna group DNA 1ug as masterplate, add P5 and each 1ul of P2 primer (seeing embodiment 1 and 2), 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 6, (swimming lane 3-4) do not find specific band in the negative control plant, then can amplify the specific band (swimming lane 5-12) of 1 about 760bp in part transgenic Fructus Lycopersici esculenti plant, this result shows that Lumbrukinase F-III gene has been integrated among the tomato dna group DNA.

2. Lumbrukinase F-III recombinant protein Determination on content in leaf and the fruit

With mortar plant tissue is ground, in attrition process, add liquid nitrogen, add 10 times of volume sterilization deionized waters in the plant tissue powder and dilute.Centrifugal 5 minutes of 4 ℃ of plant homogenate 10000g, (Sigma company, the method that p5656) provides are got the part supernatant liquor and are measured soluble protein content according to test kit.Lumbrukinase F-III recombinant protein content detecting method is with 3 among the embodiment 3 in the transfer-gen plant.

Listed the content of Lumbrukinase F-III recombinant protein in transgenic Fructus Lycopersici esculenti leaf and the fruit in the table 2.The greenery and the erythrocarpus that are taken at the transgenic Fructus Lycopersici esculenti of growing in the greenhouse carry out soluble proteins and Lumbrukinase F-III recombinant protein Determination on content, as above described.Detect less than Lumbrukinase F-III albumen in non-transgenic plant leaf and the fruit.

The content of the Lumbrukinase F-III recombinant protein in table 2 tomato leaf and the fruit

Organ	Ng/mg soluble proteins (%)	The ng/g fresh weight
			Leaf fruit (red)	42(0.004％) 37(0.003％)	210 95

From table 1 and table 2 relatively, Lumbrukinase F-III recombinant protein content in the tomato leaf is well below potato leaf (210/1500), expression amount only is 0.004% of a blade soluble proteins, trace it to its cause, may be because the gene of Lumbrukinase F-III is not modified according to the codon that plant is had a preference for, therefore influence this gene efficiently expressing in tomato, however, this expression amount has also reached the 210ng/g fresh weight, should be still than higher.The content of Lumbrukinase F-III recombinant protein accounts for 0.003% of soluble proteins in mature fruit, or is the 95ng/g fresh weight.Because the density of fruit is than higher, the proteic expression amount of Lumbrukinase F-III in the tamato fruit has been concentrated the major part of tomato plant Lumbrukinase F-III recombinant protein, and the tamato fruit of heavy 100 grams contains about 10ug Lumbrukinase F-III recombinant protein.

Embodiment 5

1. the stability of reorganization Lumbrukinase albumen F-II in plant tissue is measured

Reorganization Lumbrukinase albumen F-II can preserve in complete vegetable cell for a long time.As in the mode of potato tuber at 4 ℃ of following cryopreservation, preservation period reaches half a year, Lumbrukinase F-II does not have any loss yet.But after plant cell structures was destroyed, the various proteolytic enzyme in the organoid will be released, contained external source recombinant protein in their energy degrading plant juice.Therefore, detect the stability of reorganization Lumbrukinase albumen in water, most important for guaranteeing as far as possible in the course of processing that from now on Lumbrukinase albumen is not damaged.As can be seen from Figure 7, the time that the Lumbrukinase F-II albumen of reorganization is placed in water is long more, and proteic loss will be big more.The Lumbrukinase albumen of at room temperature placing after 1 day is little with the Lumbrukinase protein content difference in the new water of grinding, but from the 2nd day, the content of Lumbrukinase can sharply reduce, and after the 7th day, has detected basically less than the Lumbrukinase recombinant protein.This result shows, in the transgenic Rhizoma Solani tuber osi stem tuber course of processing, should it be dehydrated, and can prevent effectively that just the proteasome degradation that the Lumbrukinase recombinant protein can not discharged in the cell from destroying.

Dehydrate although high temperature can quicken the transgenic Rhizoma Solani tuber osi stem tuber, reorganization Lumbrukinase albumen is limited to the pyritous suffertibility.As can be seen from Figure 8, when temperature surpassed 60 ℃, the treatment time reached 1 hour, can the proteic quantity of detected Lumbrukinase sharply descend in the water, and this shows that the endurable limit high temperature of this Lumbrukinase albumen is 60 ℃.Surpass this temperature, Lumbrukinase albumen can be degraded rapidly by proteolytic enzyme.

Comprehensively above-mentioned, can draw as drawing a conclusion, dry potato tuber can effectively protect reorganization Lumbrukinase albumen not suffer a loss with low temperature, the most suitable fast with this understanding.

2. contain the capsular preparation of Lumbrukinase recombinant protein

Potato tuber is not easy to eat raw, can consider to be processed into capsule.1000g fresh weight potato tuber can obtain the about 140g of dry-matter after lyophilize, this dry-matter and then worn into 150-200 purpose dry powder at low temperatures, after measured, contain 2.8ugF-II reorganization Lumbrukinase albumen in the dry powder that this transgenic Rhizoma Solani tuber osi stem tuber of every 100mg is made approximately.This dry powder can directly pour in the capsule shell of enteric solubility or gastric solubility.As the case may be, each capsule can pour into the dry powder that 100-500mg transgenic Rhizoma Solani tuber osi stem tuber is made, and for example, the capsule of a 300mg contains the reorganization Lumbrukinase F-II albumen of 8.6ug approximately.The adult is each only need to take 1, just might reach the purpose of prevention or control thrombotic diseases.

3. contain the preparation of Lumbrukinase recombinant protein electuary

Capsular volumetric ratio is less, if directly pour into the dry powder that the transgenic Rhizoma Solani tuber osi stem tuber is made, the Lumbrukinase recombinant protein quantity that then every capsules contained is fewer, the patient may take the purpose that many capsules can reach diseases prevention and cure the disease at every turn when being in a bad way, therefore, the dosage that increases disposable oral administration may be essential in some cases.At this moment, can adopt the electuary packaged form, the dry powder that the 1-2g that packs in every bag is made by the transgenic Rhizoma Solani tuber osi stem tuber.Be packaged as example with 1g, contain the about 28ug of Lumbrukinase F-II recombinant protein.The patient is each only need to take 1 bag, just might reach and suppress thrombosis or thrombolytic purpose.When taking, cause protein denaturation for avoiding high temperature, can accompany by warm boiling water dilution back clothes down.

4. other preparation that contains the Lumbrukinase recombinant protein

There are some problems in the transgenic Fructus Lycopersici esculenti fruit that contains the Lumbrukinase recombinant protein when directly eating, because can not measure the Lumbrukinase protein content in each tomato, thereby just can't determine that also the at every turn edible several tamato fruits of people are comparatively suitable.In this case, can consider tamato fruit is processed into fruit tea or other beverage mode, only need every batch of product is carried out sampling Detection, just can be used as the guidance foundation of people's taking dose.Certainly, the mode that potato tuber or other transgenic plant also can be processed into beverage is convenient to take, its prerequisite is to guarantee that reorganization Lumbrukinase albumen can not degraded by the proteolytic enzyme in the vegetable cell under the liquid situation, at ambient temperature can standing storage.

The Lumbrukinase albumen of plant origin can also have other application mode, for example, the Lumbrukinase recombinant protein can be used as foodstuff additive through behind complete purifying or the partial purification, mixes in food such as milk powder, bean powder, fruit juice and sour milk and the beverage and is prepared into various specific functionality food.

Embodiment 6

1. utilize the affinity chromatography reorganization Lumbrukinase albumen of from transgenic Rhizoma Solani tuber osi or tomato, purifying

Embodiment 1 is seen in the preparation of Lumbrukinase F-II or F-III mouse resisting anteserum, after getting this mouse resisting anteserum of 2ml and the 8mlPBS damping fluid mixing, place the 50ml small beaker, slowly add equivalent saturated ammonium sulphate solution under the continuously stirring, stirred 1 hour under the room temperature, centrifugal 15 minutes of 10000rpm, get precipitation, be suspended in again in the 10mlPBS damping fluid, slowly add 5ml saturated ammonium sulphate solution under the continuously stirring, stirred 1 hour under the room temperature, centrifugal 15 minutes of 10000rpm gets precipitation, is suspended in again in the 2ml PBS damping fluid, place dialysis tubing that the PBS damping fluid of 2L is carried out dialysed overnight then, change the PBS damping fluid during this time 2 times, after dialysis is finished, with IgG antibody sucking-off in dialysis tubing, measure the 280nm light absorption value, then IgG concentration is transferred to 1mg/ml.According to the method that provides in the product description, this mouse antibodies is fixed on CNBr activatory Sepharose 4B (Pharmacia Biotech company product).

Get 100 gram transgenic Rhizoma Solani tuber osi stem tuber or 1000g tomato leafs, add equivalent PBS damping fluid and (contain 0.1%Triton X-100; 0.5mM PMSF), 4 ℃ of following homogenate.Homogenate is through 40, and 000g is centrifugal, and supernatant liquor is collected in the back.Supernatant liquor is dissolved in 20ml TSA damping fluid (20mMTris-HCl, pH8.0,140mM NaCl, 0.025%NaN again after lyophilize ₃) in, then with above-mentioned activatory antibody-gel composite 4 ℃ of following thorough mixing 16 hours.Behind the reactant upper prop, Tris damping fluid (50mM Tris-HCl with 5 times of column volume pH8.0 and pH9.0,0.1%Triton X-100,500mM NaCl) washes post respectively, then with 10ml trolamine damping fluid (the 50mM trolamine, pH 11.5,0.1%Triton X-100,150mMNaCl) carry out wash-out, elutriant is collected in respectively in 10 1.5ml centrifuge tubes.Detect having or not of Lumbrukinase recombinant protein in each centrifuge tube by the absorption reaction of enzyme connection.To have immunoreactive elutriant to pool together, and carry out 4 ℃ of dialysed overnight in the dialysis tubing of packing into, dialysis buffer liquid is 2 liters of PBS.The dialysis finish after from dialysis tubing the sucking-off dialysate, after lyophilize, be dissolved in again in the 1ml PBS damping fluid, therefrom take out 20ul then and carry out the SDS-PAGE electrophoresis.The SDS-PAGE electrophoresis detection finds that the proteic molecular weight of reorganization Lumbrukinase F-II that obtains in the plant is about 25KD, and the molecular weight of F-III be about 26KD, all with from earthworm isolated natural F-II and F-III Lumbrukinase protein seemingly ^[4,5], see Fig. 9 A and 9B.

2. the mensuration of reorganization Lumbrukinase fibrinolytic

The detection of Lumbrukinase fibrinolytic is fully according to people such as Deogny institute reported method ^[36], after the agar for the treatment of fibrinogen (Sigma company product) and zymoplasm (Sigma company product) solidifies fully, getting the circular hole of several diameters about 6mm on the flat board with the punch tool after the sterilization, be evenly distributed on the culture dish flat board around.Get transgenic Rhizoma Solani tuber osi blade or transgenic Fructus Lycopersici esculenti blade 0.1g (seeing embodiment 3 and 4), mixing the back with the 100mMTris-HCl damping fluid (pH8.0) of 300ul fully grinds with glass homogenizer, homogenate is centrifugal 5 minutes of 10000rpm at room temperature, gets supernatant liquor 80ul and joins in the above-mentioned circular hole of accomplishing fluently.Processing as the non-transgenic plant of negative control is the same.Get two of Eisenia foetidas, mix the back respectively fully grinds with glass homogenizer with the 100mM Tris-HCl damping fluid (pH8.0) of 1ml, homogenate is centrifugal 5 minutes of 10000rpm at room temperature, gets supernatant liquor 80ul and also joins in the above-mentioned circular hole of accomplishing fluently as positive control.Other gets above-mentioned reorganization Lumbrukinase F-II and each 40ul of F-III through affinitive layer purification, join respectively in the above-mentioned circular hole of having accomplished fluently, place after 18 hours down for 37 ℃, found that, except that the Eisenia foetida extracting solution as positive control can form (scleroproein is dissolved) the transparent circle, the reorganization Lumbrukinase F-II and the F-III that are purified into from transgenic plant also can form transparent circle.The extracting solution of part transfer-gen plant (comprising potato and tomato) blade also can form transparent circle, and The above results shows that all reorganization Lumbrukinase expressed in transgenic plant has plasmin activity.Negative control all can not form transparent circle, in addition, also has the part transfer-gen plant also to fail to form transparent circle, may be relatively lower relevant with the intravital reorganization Lumbrukinase of this plant expressing quantity, see Figure 10.

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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〉Liu Dehu

＜120〉production method and the product of expression vermis kinase tr-gene plant

<160>4

<210>1

<211>741

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(4)...(732)

<400>1

acc atg gtt att gga ggt act aac gct agc cca gga gag ttc cct tgg caa ctt tct 57

Met Val Ile Gly Gly Thr Asn Ala Ser Pro Gly Glu Phe Pro Trp Gln Leu Ser

1 5 10 15

cag caa aga caa agc ggt tct tgg tcg cac agc tgc gga gca tcg ctt ctt agc tca 114

Gln Gln Arg Gln Ser Gly Ser Trp Ser His Ser Cys Gly Ala Ser Leu Leu Ser Ser

20 25 30 35

act tcc gct ctc agc gca tct cac tgc gtt gat gga gtt ctt cca aac aac att aga 171

Thr Ser Ala Leu Ser Ala Ser His Cys Val Asp Gly Val Leu Pro Asn Asn Ile Arg

40 45 50 55

gtt att gct ggt ctt tgg caa cag tct gac aca agc ggc act cag acc gct aac gtc 228

Val Ile Ala Gly Leu Trp Gln Gln Ser Asp Thr Ser Gly Thr Gln Thr Ala Asn Val

60 65 70 75

gat agc tac acc atg cat gag aac tat ggt gct ggt act gct tct tac tct aat gac 285

Asp Ser Tyr Thr Met His Glu Asn Tyr Gly Ala Gly Thr Ala Ser Tyr Ser Asn Asp

80 85 90

att gct att ctt cac ctt gct act tct att agc ctt gga gga aac att cag gct gct 342

Ile Ala Ile Leu His Leu Ala Thr Ser Ile Ser Leu Gly Gly Asn Ile Gln Ala Ala

95 100 105 110

gtc ctt ccc gct aac aac aac aac gac tac gct gga acc aca tgc gtc att tct gga 399

Val Leu Pro Ala Asn Asn Asn Asn Asp Tyr Ala Gly Thr Thr Cys Val Ile Ser Gly

115 120 125 130

tgg ggt cgc aca gat gga act aac aat ctt ccg gac att ctt cag aag tct tct att 456

Trp Gly Arg Thr Asp Gly Thr Asn Asn Leu Pro Asp Ile Leu Gln Lys Ser Ser Ile

135 140 145 150

ccg gtc att act acc gct caa tgc acc gct gct atg gtt gga gtc ggt gga gct aac 513

Pro Val Ile Thr Thr Ala Gln Cys Thr Ala Ala Met Val Gly Val Gly Gly Ala Asn

155 160 165 170

att tgg gat aat cac att tgc gtc caa gat cca gct ggc aac acc gga gct tgc aat 570

Ile Trp Asp Asn His Ile Cys Val Gln Asp Pro Ala Gly Asn Thr Gly Ala Cys Asn

175 180 185

ggc gat agc ggt ggc cca ctt agc tgc cca gac ggc gga act aga gtg gtt ggt gtt 627

Gly Asp Ser Gly Gly Pro Leu Ser Cys Pro Asp Gly Gly Thr Arg Val Val Gly Val

190 195 200 205

act tct tgg gtt gta tct agt ggc ctt ggt gca tgt ctt ccg gac tac cct tct gtc 684

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

210 215 220 225

tac acc cgc gtc agc gcc tac ttg ggt tgg att gga gac aac tct cgt tag ctcgag 741

Tyr Thr Arg Val Ser Ala Tyr Leu Gly Trp Ile Gly Asp Asn Ser Arg *

230 235 240

<210>2

<211>243

<212>PRT

＜213〉the positive earthworm of two chests (Lumbricus bimastus) Lumbrukinase F-II

<400>2

Met Val Ile Gly Gly Thr Asn Ala Ser Pro Gly Glu Phe Pro Trp Gln

1 5 10 15

Leu Ser Gln Gln Arg Gln Ser Gly Ser Trp Ser His Ser Cys Gly Ala

20 25 30

Ser Leu Leu Ser Ser Thr Ser Ala Leu Ser Ala Ser His Cys Val Asp

35 40 45

Gly Val Leu Pro Asn Asn Ile Arg Val Ile Ala Gly Leu Trp Gln Gln

50 55 60

Ser Asp Thr Ser Gly Thr Gln Thr Ala Asn Val Asp Ser Tyr Thr Met

65 70 75 80

His Glu Asn Tyr Gly Ala Gly Thr Ala Ser Tyr Ser Asn Asp Ile Ala

85 90 95

Ile Leu His Leu Ala Thr Ser Ile Ser Leu Gly Gly Asn Ile Gln Ala

100 105 110

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

115 120 125

Val Ile Ser Gly Trp Gly Arg Thr Asp Gly Thr Asn Asn Leu Pro Asp

130 135 140

Ile Leu Gln Lys Ser Ser Ile Pro Val Ile Thr Thr Ala Gln Cys Thr

145 150 155 160

Ala Ala Met Val Gly Val Gly Gly Ala Asn Ile Trp Asp Asn His Ile

165 170 175

Cys Val Gln Asp Pro Ala Gly Asn Thr Gly Ala Cys Asn Gly Asp Ser

180 185 190

Gly Gly Pro Leu Ser Cys Pro Asp Gly Gly Thr Arg Val Val Gly Val

195 200 205

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

210 215 220

Pro Ser Val Tyr Thr Arg Val Ser Ala Tyr Leu Gly Trp Ile Gly Asp

225 230 235 240

Asn Ser Arg

<210>3

<211>741

<212>DNA

＜213〉Eisenia foetida (Eisenia foetida) Lumbrukinase F-III

<220>

<221>CDS

<222>(1)...(738)

<400>3

atg gaa ctt cct ccc gga aca aaa att gtc gga gga att gaa gcc aga cca tac gag 57

Met Glu Leu Pro Pro Gly Thr Lys Ile Val Gly Gly Ile Glu Ala Arg Pro Tyr Glu

1 5 10 15

ttc cca tgg cag gtg tcc gtc cga agg aag tct acc gat tcc cat ttc tgc gga ggt 114

Phe Pro Trp Gln Val Ser Val Arg Arg Lys Ser Thr Asp Ser His Phe Cys Gly Gly

20 25 30 35

agc atc ata aac gat cgt tgg gtt gtc tgc gct gct cac tgc atg cag gga gag agc 171

Ser Ile Ile Asn Asp Arg Trp Val Val Cys Ala Ala His Cys Met Gln Gly Glu Ser

40 45 50 55

cct gcc ctg gtt tca ttg gtc gtc ggc gag cac gat agc agc gct gcg agt aca gtt 228

Pro Ala Leu Val Ser Leu Val Val Gly Glu His Asp Ser Ser Ala Ala Ser Thr Val

60 65 70 75

cgt cag act cat gat gtt gat agc atc ttc gtc aac gaa aac tac aat ccc cgt aca 285

Arg Gln Thr His Asp Val Asp Ser Ile Phe Val Asn Glu Asn Tyr Asn Pro Arg Thr

80 85 90 95

ctt gaa aac gac gtt tct gtc atc aag aca gct atc gct atc acc ttc gac att aac 342

Leu Glu Asn Asp Val Ser Val Ile Lys Thr Ala Ile Ala Ile Thr Phe Asp Ile Asn

100 105 110

gtt ggg cca atc tgt gct cca gat ccg gct aac gac tac gtc tac cgt aag agc cag 399

Val Gly Pro Ile Cys Ala Pro Asp Pro Ala Asn Asp Tyr Val Tyr Arg Lys Ser Gln

115 120 125 130

tgc tct gga tgg gga tct ata aac tca ggt gga atc tgc tgt ccc gca gtt ttg cga 456

Cys Ser Gly Trp Gly Ser Ile Asn Ser Gly Gly Ile Cys Cys Pro Ala Val Leu Arg

135 140 145 150

tat gtt aca ctg aac atc acg acc aac gcc ttc tgc gac gcc gtc tac aca tcg gat 513

Tyr Val Thr Leu Asn Ile Thr Thr Asn Ala Phe Cys Asp Ala Val Tyr Thr Ser Asp

155 160 165 170

acc atc tac gac gat atg atc tgc gcc acg gac aac act ggg atg acc gac aga gac 570

Thr Ile Tyr Asp Asp Met Ile Cys Ala Thr Asp Asn Thr Gly Met Thr Asp Arg Asp

175 180 185 190

tca tgc cag ggt gac tcc ggc ggc cct ctg agc gtc aag gat ggc agc gga atc ttc 627

Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser Val Lys Asp Gly Ser Gly Ile Phe

195 200 205

agt ctg gtt ggc att gtg tct tgg gga att ggt tgc gct tcc ggc tat cca gga gtc 684

Ser Leu Val Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Ser Gly Tyr Pro Gly Val

210 215 220 225

tac tcc cga gtc gga ttt cat gct gga tgg atc acc gac aca atc acc aac aac taa 741

Tyr Ser Arg Val Gly Phe His Ala Gly Trp Ile Thr Asp Thr Ile Thr Asn Asn *

230 235 240 245

<210>4

<211>246

<212>PRT

＜213〉Eisenia foetida (Eisenia foetida) Lumbrukinase F-III

<400>4

Met Glu Leu Pro Pro Gly Thr Lys Ile Val Gly Gly Ile Glu Ala Arg

1 5 10 15

Pro Tyr Glu Phe Pro Trp Gln Val Ser Val Arg Arg Lys Ser Thr Asp

20 25 30

Ser His Phe Cys Gly Gly Ser Ile Ile Asn Asp Arg Trp Val Val Cys

35 40 45

Ala Ala His Cys Met Gln Gly Glu Ser Pro Ala Leu Val Ser Leu Val

50 55 60

Val Gly Glu His Asp Ser Ser Ala Ala Ser Thr Val Arg Gln Thr His

65 70 75 80

Asp Val Asp Ser Ile Phe Val Asn Glu Asn Tyr Asn Pro Arg Thr Leu

85 90 95

Glu Asn Asp Val Ser Val Ile Lys Thr Ala Ile Ala Ile Thr Phe Asp

100 105 110

Ile Asn Val Gly Pro Ile Cys Ala Pro Asp Pro Ala Asn Asp Tyr Val

115 120 125

Tyr Arg Lys Ser Gln Cys Ser Gly Trp Gly Ser Ile Asn Ser Gly Gly

130 135 140

Ile Cys Cys Pro Ala Val Leu Arg Tyr Val Thr Leu Asn Ile Thr Thr

145 150 155 160

Asn Ala Phe Cys Asp Ala Val Tyr Thr Ser Asp Thr Ile Tyr Asp Asp

165 170 175

Met Ile Cys Ala Thr Asp Asn Thr Gly Met Thr Asp Arg Asp Ser Cys

180 185 190

Gln Gly Asp Ser Gly Gly Pro Leu Ser Val Lys Asp Gly Ser Gly Ile

195 200 205

Phe Ser Leu Val Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Ser Gly

210 215 220

Tyr Pro Gly Val Tyr Ser Arg Val Gly Phe His Ala Gly Trp Ile Thr

225 230 235 240

Asp Thr Ile Thr Asn Asn

245

Claims (18)

1. method of producing earthworm Lumbrukinase or derivatives thereof by transgenic plant.

2. as the method in the claim 1, it is edible that wherein said plant has a part at least.

3. one kind causes that people or Mammals heart and brain and limb vessel thrombosis are suppressed or thromboclastic method, and this method comprises uses effective and definite dosage, as to derive from the transgenic plant that can produce this Lumbrukinase recombinant protein or derivatives thereof vegetable material or its processing extract that people or Mammals are carried out oral or enterally administering.

4. as the method in the claim 3, it is edible that wherein said plant has a part at least.

5. a food is made up of certain part of transgenic plant at least, it is eaten is because it has certain nutrient value, wherein said plant is a kind of plant that can express the Lumbrukinase or derivatives thereof, and this albumen has the effect of solution fibrin or plasminogen activator under native state.

6. as any food in the claim 5, wherein said plant edible food partly comprises fruit, leaf, stem, root or said plant seed.

7. a plasmid vector that is used to transform plant comprises:

The dna sequence dna of one section coding earthworm Lumbrukinase or derivatives thereof (comprise gene codon optimised or with other gene Fusion etc.), the albumen of this sequence encoding has the effect of solution fibrin or plasminogen activator under native state.

A plant promoter that is connected with said dna sequence dna, this promotor is handled said gene and is expressed in said plant.

8. as the plasmid vector in the claim 7, it contains a selectivity or marker gene, is edibility and wherein said plant has a part at least.

9. one section dna sequence dna that is used for particle bombardment conversion plant comprises:

The dna sequence dna of one section coding earthworm Lumbrukinase or derivatives thereof (comprise gene codon optimised or with other gene Fusion etc.), the albumen of this sequence encoding has the effect of solution fibrin or plasminogen activator under native state.

A plant promoter that is connected with said dna sequence dna, this promotor is handled said gene and is expressed in said plant.

10. as the dna sequence dna in the claim 9, it contains a selectivity or marker gene, is edibility and wherein said plant has a part at least.

11. a method that makes up transgenic plant cells comprises:

Make up a plasmid vector or section of DNA sequence, wherein all contain the gene (comprise gene codon optimised or with other gene Fusion etc.) of one section coding earthworm Lumbrukinase or derivatives thereof, the albumen of this genes encoding has the effect of solution fibrin or plasminogen activator under native state.

A plant promoter that is connected with said dna sequence dna, this promotor is handled said gene and is expressed in said plant.

With this plasmid vector or this dna sequence dna transformed plant cells.

12. as the method in the claim 11, it comprises bears complete transfer-gen plant again from said transgenic plant cells.

13. the method for a kind of capsule, electuary or other preparation of producing comprises:

Make up a plasmid vector or section of DNA sequence, wherein all contain the gene (comprise gene codon optimised or with other gene Fusion etc.) of one section coding earthworm Lumbrukinase or derivatives thereof and can handle the plant promoter that this gene is expressed in said plant, the albumen of this genes encoding has the effect of solution fibrin or plasminogen activator under native state.

With this plasmid vector or this dna sequence dna transformed plant cells.

With the edible part of transgenic plant freezing, dry and grind after be made into capsule, electuary or other preparation.

14. as the method for claim 13, wherein also be included in and utilize transgenic plant as food and be processed into before capsule, electuary or other preparation, should from said transgenic plant cells, bear complete transfer-gen plant again.

15., wherein also comprise from said vegetable cell partial purification or be purified into the active integral part of said earthworm Lumbrukinase recombinant protein fully as capsule, electuary or other preparation as the method in the claim 13.

16. as the method in the claim 13, wherein said vegetable cell transforms by Agrobacterium Ti-plasmids system.

17. as the method in the claim 13, wherein said plant is tomato, potato, the red sage root, clover, lettuce, Radix Dauci Sativae, cucumber, wheat and paddy rice or other edible dicotyledonous or monocotyledons.

18. as the method in the claim 13, wherein said vegetable cell is to transform as methods such as particle bombardment, microtubule injection, PEG absorption process and electro fusion methods by other method for transformation except that Agrobacterium transformation system.

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