CN103333256B

CN103333256B - Human acid fibroblast growth factor fusion protein, and coding gene and applications thereof

Info

Publication number: CN103333256B
Application number: CN201310282455.2A
Authority: CN
Inventors: 刘德虎; 谭亚清; 李刚强; 王楠
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2013-07-05
Filing date: 2013-07-05
Publication date: 2014-11-05
Anticipated expiration: 2033-07-05
Also published as: CN103333256A

Abstract

The invention provides a fusion protein. The fusion protein comprises suppressors of gene silencing, 2A polypeptide and a human acid fibroblast growth factor protein. All or part of the human acid fibroblast growth factor protein is a natural human acid fibroblast growth factor protein; or the human acid fibroblast growth factor protein is a fusion protein formed by co-expression or splicing of the natural human acid fibroblast growth factor protein with another fibroblast factor protein, or with another protein which possesses completely different biological functions. The invention also provides the coding gene of the fusion protein, and a preparation method of genetically modified radix salviae miltiorrhizae which is used for high-efficiency expression of the human acid fibroblast factor. The genetically modified radix salviae miltiorrhizae can be used for effective treatment of operative incisions, nerve injuries, body mechanical injuries, diabetic foot, anabrosis, burned and scalded injuries, and the like, and is also applied in developing a new generation of recombinant human acidic fibroblast factor drugs.

Description

Human acid fibroblast growth factor fusion protein and encoding gene thereof and application

Technical field

The present invention relates to genetically engineered field, be specifically related to a kind of human acid fibroblast growth factor fusion protein and encoding gene thereof and application.

Background technology

Human fibroblastic growth factor belongs to a large protein families, up to the present, has found at least 23 members.Wherein, acid fibroblast growth factor (acidic fibroblast growth factor, aFGF) be by the people such as Thomas in 1984 from ox brain separation first obtain, because its iso-electric point is 5～7, be acid, therefore hence obtain one's name.

Human acid fibroblast growth factor (haFGF) is mainly distributed in kidney and cerebral tissue, is a kind of cytoplasmic protein, itself lacks N end signal peptide structure, mainly by autocrine and two kinds of modes of paracrine, peripheral cell is worked.HaFGF polypeptide is comprised of 154 amino-acid residues, and molecular weight is about 16kDa, is a full β-pleated sheet structure albumen that does not contain disulfide linkage, the cloverleaf structure that its secondary structure is comprised of 12 antiparallel β chains.This cloverleaf structure is not to work separately, at least there is functional unit separate in two structures (Chi Y H, Kumar T K, Chiu I M, Deng, Identification of rare partially unfolded states in equilibrium with native conformation in an all beta-barrel protein[J] .J Biol Chem, 2002,277 (38): 34941-34948), comprise Heparin-binding district, receptor binding domain and nuclear translocation district.

HaFGF has biologic activity widely, can be by realizing growth, differentiation and the function of the various kinds of cell in mesoderm and neuroderm source are exerted an influence with receptors bind.Its biologic activity is divided into two large types: 1) mitogenic activity, can promote histocyte division, propagation, and comprise fetal development, form generation, vasculogenesis and tissue injury reparation etc.; 2) non-mitogenic is active, comprises vasodilator, myocardial preservation, local asphyxia protection and neuroprotective etc.

HaFGF can participate in the forming process of new vessel directly.(the Zhang Bin such as Zhang Bin, plum is lifted, Zhang Baoren etc., the experimental study [J] of the short vascular endothelial cell proliferation effect of acid fibroblast growth factor. Wannan Medical College's journal, 2003,22 (1): 18-19) in the nutrient solution of endotheliocyte, add a certain amount of aFGF, found that, its growth to vascular endothelial cell has very significantly promoter action, and the propagation of vascular endothelial cell and differentiation are the important steps of angiogenesis.The aFGF angiogenesispromoting effect ischemic in a organized way of still needing, the factor of anoxic exists, in healthy tissues, aFGF is without the effect that promotes angiogenic growth, under the state of ischemic or anoxic, the up-regulated of tissue to aFGF, stimulate capillary endothelial cell to produce collagenase and scleroproein lytic enzyme, promote the hydrolysis of collagen and formation (the Ferrara N. of vascular endothelial cell tube chamber spline structure, Vascular endothelial growth factor and the regulation of angio-genesis[J] .Recent Prog Horm Res, 2000, 55:15-35).

The trauma repair of skin is a complicated dynamic biological procedures, aFGF plays an important role in this process: the chemotactic cell infiltration surface of a wound, promote vascular endothelial cell and inoblast division growth, accelerate the re-epithelialization of granulation tissue formation and the surface of a wound, thereby the generation of scar is avoided in the excessive formation of inhibition collegen filament.(Liu's Xin such as Liu's Xin, Ma Rong, Ma Ke etc., rh-aFGF is to the effect of wound healing [J]. China Medicine University's journal, 2008,39 (1): 87-91) by rat trauma model, observe the effect of rh-aFGF in wound healing.Research discovery, aFGF can promote granulation tissue growth in the early stage of wound healing, accelerates to hinder face healing; Later stage again can be direct or indirect the fibroblastic apoptosis of promotion, maintain the balance of Cell apoptosis and proliferation, avoid the formation of scar tissue.

AFGF can promote division growth the energy induction of vascular new life of the various kinds of cell such as vascular endothelial cell, smooth muscle cell and myofibroblast, so it also has provide protection to the ischemia injury of internal organ.Research is found to inject aFGF after Ischemia and reperfusion of intestine; after for some time, record hepatic and renal function index: alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and serum creatinine all have rising; illustrate that liver, renal function after aFGF is to Ischemia and reperfusion of intestine all have provide protection (Weng Lixin; pay dogface; Li Xiuxia etc., change configuration and wild-type aFGF on Ischemia and reperfusion of intestine after the impact of hepatic and renal function, Chinese critical illness emergency medicine; 2004,16 (1): 19-21).Transgenic mice Cardiac-specific is crossed expression mankind aFGF; the number showed increased of its arteriole and main coronary arterial tree; thereby volume of blood flow coronarius is increased, delay the expansion of myocardial infarction area, reduce the damage of cardiac muscle disappearance; protection cardiac muscle (Buehler A; Martire A, etc., Angiogenesis-independent cardioprotection in FGF-1transgenic mice[J] .Cardiovasc Res; 2002,55 (4): 768-777).Therefore, can, by aFGF simultaneously for promoting the reparation of skin histology damage and the reparation of internal organs ischemia damage, demonstrate the extremely great advantage of aFGF in clinical application.

In vitro under culture condition, aFGF can have neurotrophic effect as periphery neurones such as the central neurones such as hippocampus, hypothalamus, spinal cord and ciliary body, retinas to multiple neurone.AFGF can promote the growth of neuronic survival and aixs cylinder.In addition, aFGF can also promote reparation and the regeneration after nervus centralis and peripheral nerve injury, studies confirm that aFGF can promote adult rat to cut off functional recovery (the Lee L M of spinal nerve root, Huang M C, Deng, Acidic FGF enhances functional regeneration of adult dorsal roots[J] .Life Sci, 2004,74 (15): 1937-1943), this treatment for spinal nerve root damage provides new method.

Thereby aFGF promotes bone growth by affecting chondrocyte and osteoblastic activity.During aFGF cultivates in vitro, can promote the chondrocyte in differentiation that migration and colony formation occur, promote the in vitro differentiation of chondrocyte's antecedent, chondrocyte's propagation and maturation.To joint cavity injection rh-aFGF, can delay preferably development (the Wu H of rabbit knee osteoarthritis cartilage degeneration and prevention osteoarthritis, Yao P, Liu N, Deng, Prevention of osteoarthritis cartilage degeneration with intraarticular injection of recombinant human acidic fibroblast growth factor[J] .J Clin Rehab Tissue Eng Res, 2007,11 (32): 6394-6396).In addition adjusting, the radioprotective that, aFGF ingests in addition, affect human immune system, hormone regulating and controlling, the anti-apoptosis of cell, cell migration, relieve the pain and the several functions such as hypnosis.

The P19 albumen of tomato bushy stunt virus coding is an exogenous gene expression silencing suppressors (Baulcombe D C.and Molnar A, Crystal structure of p19 – a universal suppressor of RNA silencing, TRENDS in Biochemical Sciences, 2004, 29 (6): 279-281), it can make to take green fluorescent protein foreign gene that tobacco mosaic virus (TMV) (TMV) the is carrier expression efficiency in vegetable cell to improve more than at least 100 times (Lindbo J A, High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors, BMC Biotechnology, 2007, 7:52doi:10.1186/1472-6750-7-52).The about 19kDa of P19 albumen size left and right, in each P19 protein molecular, contains 4 or 5 αhelix and 4 β-pleated sheet structure sheets.Two P19 albumen is interacted, in the tail tail mode of being connected, forms a homodimer with sat linkage by the hydrogen bond that forms between intramolecular the 4th β-pleated sheet structure sheet separately and the hydrophobic bond that forms between the α spiral of PROTEIN C end separately.

Between this homodimer, formed a groove being formed by β-pleated sheet structure sheet, can mutually identify and combination with phosphoric acid-glycosyl skeleton of the double-stranded mixture of small molecules interference RNA, the impact that this combination is not arranged by RNA nucleotide base, can not identify mutually with double-stranded DNA mixture.The αhelix tryptophan residue that is arranged in two C-terminal of P19 dimer protein forms the skeleton construction of each other, can preferentially be combined with the double-stranded mixture of small molecules interference RNA of 21～22 nucleotide base length, everyly be greater than 22 or be less than the double-stranded mixtures of 20 nucleotide base small molecules interference RNAs, greatly weaken with the binding ability of this dimer protein.Due to this difference on aspect binding ability, make it stop RNA helicase and RNA small molecules to disturb double-stranded mixture to bring into play vital role aspect mutually combining.Just because of RNA small molecules, disturb the double-stranded mixture can not depolymerization, can not further be combined and make its degraded and silence with the mRNA of transcription of foreign genes formation, final result, makes the mRNA that foreign gene is transcribed out can keep active state always and not be silenced and fast degradation.

2A protein polypeptide is that a class length is 18～22 amino-acid residue micromolecule polypeptides (Szymczak-Workman A L; Vignali K M and Vignali D A.A.; Design and Construction of2A Peptide-Linked Multicistronic Vectors; Cold Spring Harb Protoc, 2012; Doi:10.1101/pdb.ip067876), although it is larger to be positioned at the amino-acid residue diversity ratio of N-terminal, the amino-acid residue that is positioned at C-terminal has higher sequence homology,, with---GlyPro ending, is all the cleavage site of proteolytic ferment.In eukaryotic cell, proteolytic ferment can carry out specific cutting between glycine (Gly) and proline(Pro) (Pro), and this cutting is not generally positioned at the impact of 2A polypeptide upstream and downstream aminoacid sequence.Just because of this feature, generally 2A polypeptide is placed between two foreign proteins, under the effect of fusion protein expression product lytic enzyme in born of the same parents, become two portions with high efficiency cutting rapidly, the albumen of upstream has retained most sequences of 2A micromolecule polypeptide, the albumen that is positioned at downstream has only retained a proline residue at N-terminal, can at utmost retain like this biological activity of downstream albumen.2A protein polypeptide is the earliest at foot and mouth disease virus (foot-and-mouth disease virus, FMDV) (the Ryan M D finding in, Drew J, Foot-and-mouth disease virus2A oligopeptide mediated cleavage of an artificial polyprotein.EMBO J1994,13:928-933), after, people find again successively in the gene encoding productions such as No. 1, the prompt Shen of horse Coryzavirus, pig virus and bright arteries and veins thosea siensis virus, and their cutting efficiency exists certain difference.

Although haFGF biologic activity extensively and have a good clinical value, but it is as the trace activity substance in human body, very wide but content is very micro-although distribute in human body, only depend on traditional method to extract from body fluid or tissue, cost is too high-leveled and difficult to meet the clinical demand of expanding day.From nineteen eighty-two recombinant human insulin, as since first gene engineering product comes out in the world, the Application and Development of modern medicine biological technique has had the progress of breakthrough, for the batch production of aFGF has brought hope, for the treatment of various diseases has brought Gospel.

1986, (Jaye M, Howk R, the Burgess W such as Jaye, Deng, Human endothelial cell growth factor:cloning, nucleotide sequence, and chromosome localization.Science, 1986,233 (4763): 541-545DOI:10.1126/science.3523756) from human brain, cloned first haFGF gene, and measured its nucleotide coding sequence, confirmed that aFGF lacks classical signal peptide sequence.The brave EcoR I site (Wang Haoyong that the gene of coding haFGF is inserted into plasmid pBV220 that waits of the Wang Hao of Military Medical Science Institute in 1991, king can be doubly, Huang Peitang etc., clone and the expression of human acid fibroblast growth factor gene in intestinal bacteria. journal of biological chemistry, 1993,7 (6): 713-718), obtained the engineering strain can high efficient expression with aFGF bioactive product, expression product accounts for 15% of bacterial protein, and mainly the form with inclusion body exists, need be through a renaturation process.Their research has solved the problem that obtains haFGF sterling in the world first, and solid foundation has been laid in this mechanism of action, physiological function and clinical application for further investigation aFGF.1992, the people such as Zazo also in intestinal bacteria successful expression the haFGF albumen of complete form, and obtained similar result of study (Zazo M, Lozano RM, Ortega S, Deng, High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions[J] .Gene, 1992,113 (2): 231-238).

In order further to improve the expression amount of haFGF, the source of the biological activity of the expression product of improvement and stability, expansion external source aFGF, Chinese scholars has successively carried out exploring trial to multiple different expression system, and through long-term and unremitting experimental study, success has realized expression in the expression systems such as insect cell, silkworm, yeast and Mammals.

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

Plant is a kind of variation, low cost and reproducible material resources, developing rapidly of biotechnology makes us further widen the use range of plant, the external method that adopts this " Plant molecular farming ", a lot of biological products have successfully been produced, comprise some high value pharmaceutical protein polypeptide, as erythropoietin, Interferon, rabbit, tethelin, monoclonal antibody with can be used as antigen protein that vaccine uses etc., some research institutions and company have started to obtain huge economic benefit from the production of these pharmaceutical proteins.

Yet up to now, the P19 albumen that also nobody utilizes tomato bushy stunt virus coding is as exogenous gene expression silencing suppressors, and by 2A polypeptide and a human acidic fibroblast factor fusion rotein of composition common and high efficient expression in the transgenosis red sage root.This fusion rotein can be cut and discharge by specificity bioactive human acidic fibroblast factor recombinant protein in transgenosis red sage root cell, and this recombinant protein can be applied to by topical administration mode the treatment of the diseases such as people's operative incision, nerve injury, body physical abuse, diabetic foot, ulcer and burn and scald, therefore, there is important clinical value.

Summary of the invention

For addressing the above problem, the object of this invention is to provide a kind of in the red sage root method of high efficient expression human acid fibroblast growth factor.

Technical scheme of the present invention is as follows:

The invention provides a kind of fusion rotein, described fusion rotein comprises RNA silencing suppressor, 2A polypeptide and human acidic fibroblast factor protein, and described human acidic fibroblast factor protein is all or part of natural human acidic fibroblast factor protein.

In described fusion rotein, described human acidic fibroblast factor protein also can be albumen that natural human acidic fibroblast factor protein and another one inoblast factor protein have different biological function as human basic fibroblast factor protein or another one as human epidermal growth factor's albumen coexpression or the formation fusion rotein that is stitched together.Its objective is in order conveniently to purify or to improve its biological activity.The fusion of albumen can be passed through protein translation post-treatment, covalently bound mode, or by DNA recombinant technology, gene is just stitched together before accurate translation, these two kinds of known technologies that technology is this area.

Preferably, described fusion rotein is P19 albumen-2A polypeptide-human acidic fibroblast factor fusion protein (P19-2A-haFGF fusion rotein), and wherein, P19 is tomato bushy stunt virus P19 albumen; 2A is foot and mouth disease virus 2A polypeptide; HaFGF is human acid fibroblast growth factor.

Further preferably, described fusion rotein P19-2A-haFGF is:

(1) protein that the aminoacid sequence shown in SEQ ID NO.2 forms;

(2) aminoacid sequence shown in SEQ ID NO.2 be substituted, lack or add one or several amino acid and have same function by (1) derivative protein.

In described fusion rotein, coexpression tomato bushy stunt virus P19 albumen can improve the biological yield of human acidic fibroblast factor recombinant protein; Use 2A polypeptide can facilitate human acidic fibroblast factor recombinant protein in intracellular release and keep its biological activity.

The present invention also provides the gene of the above-mentioned fusion rotein of encoding.Preferably, the nucleotide sequence of described gene is as shown in SEQ ID NO.1.

In described nucleotide sequence, the nucleotide sequence of coding for tomato bushy stunt virus P19 albumen, is derived from GenBank NC_001554(accession number) in 3888～4403 sites, its DNA length is 516bp, 172 amino-acid residues of encoding; The nucleotide sequence of coding foot and mouth disease virus 2A polypeptide, is derived from GenBank AJ251476(accession number) 3472～3537 sites, its DNA length is at 66bp, 22 amino-acid residues of encoding; The nucleotide sequence of encoding human acidic fibroblast factor protein, for the human acidic fibroblast factor gene sequence of having announced (is shown in to GenBank accession number: X65778), do not changing under the prerequisite of its Argine Monohydrochloride sequence, the codon of having a preference for according to plant, its gene coded sequence is optimized and is obtained, full length gene 465bp after optimization, 154 amino-acid residues of encoding.Before 5 ' end ATG initiator codon, being restriction enzyme site XbaI and kozaK sequence (being conducive to the expression of antigen-4 fusion protein gene in the red sage root), is restriction enzyme site SacI after 3 ' end terminator codon TAA.Wherein, the AACAATGG in SEQ ID NO.1 is kozaK sequence.

For improving the expression amount of human acidic fibroblast factor recombinant protein in red sage root cell, the gene of encoding human acidic fibroblast factor fusion protein can be optimized according to the codon of plant-preference, to make this gene more stable in vegetable cell, when being transcribed into mRNA and translating into albumen, efficiency is higher.

The present invention also provides the carrier that contains described P19-2A-haFGF fusion rotein encoding gene.

Preferably, described carrier is recombinant expression vector.Described recombinant expression vector comprises resistant gene or selection markers gene, also comprise and can handle foreign gene in the promotor of vegetable cell transcription, described promotor can obtain from plant or virus, if cauliflower mosaic virus 35 S promoter (comprise through splicing, double and the improved 35S promoter that suddenlys change), oil body protein gene promotor, photoinduction gene promoter are if ribose bisphosphate carboxylase small subunit (rbcS) or adversity inducible gene are as ethanol dehydrogenase (Adhl) etc.

Further preferably, described recombinant expression vector is pBI-P19-2A-haFGF, and its construction process comprises the steps:

(1) the P19 albumen-2A polypeptide-human acidic fibroblast factor fusion protein encoding gene of synthetic nucleotide sequence as shown in SEQ ID NO.1;

(2) gene clone step (1) being obtained, to pEASY-T1 carrier, obtains carrier pT-P19-2A-haFGF;

(3) with restriction enzyme XbaI and SacI, respectively carrier pT-P19-2A-haFGF and pBI121 are carried out to double digestion, reclaim P19 albumen-2A polypeptide-human's acidic fibroblast factor fusion protein encoding gene fragment and pBI121 carrier segments, connect, obtain recombinant expression vector pBI-P19-2A-haFGF.

The present invention also provides the host cell that contains described P19-2A-haFGF fusion rotein encoding gene.

The present invention also provides the host cell containing the recombinant expression vector of described P19-2A-haFGF fusion rotein encoding gene.

Described host cell comprises recombinant bacterium, as intestinal bacteria, Agrobacterium etc.

The present invention also provides a kind of method of preparing the transgenosis red sage root, described method for will contain described fusion rotein (as P19-2A-haFGF fusion rotein) encoding gene (as described in fusion rotein encoding gene be placed in plant promoter handles under) or containing as described in the recombinant expression vector of gene transform red sage root cell, from transgenosis red sage root cell, bear again the complete transgenosis red sage root.

The present invention also provide a kind of in the red sage root method of high efficient expression human acid fibroblast growth factor, described method for will contain described fusion rotein (as P19-2A-haFGF) encoding gene or containing as described in the recombinant expression vector of gene transform red sage root cell, from transgenosis red sage root cell, bear again the complete transgenosis red sage root, the described transgenosis red sage root can be stablized, produce efficiently human acidic fibroblast factor recombinant protein, described human acidic fibroblast factor recombinant protein can cut down and have biological activity in principal and subordinate's fusion rotein, do not need purifying.

The main method that wherein described fusion rotein encoding gene stable integration is entered to red sage root chromogene group DNA comprises: 1) agrobacterium-mediated transformation; 2) DNA directly takes in method, comprises Protoplast fusion method, electrization, the microtubule injection of PEG mediation and uses particle gun that DNA is directly sent into vegetable cell and tissue etc.

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

Wherein, Agrobacterium Ti-plasmids system is selected.In the Ti-plasmids of Agrobacterium, contain one section and be called transfer DNA (T-DNA), it can enter in red sage root cell and finally be integrated in the karyomit(e) of the red sage root.The structure of transfer vector is divided into two steps, first be to build a plasmid that can copy in intestinal bacteria, the plant promoter that the DNA sequence dna that this plasmid contains coding for tomato bushy stunt virus P19 albumen-2A polypeptide-human acidic fibroblast factor gene and this DNA sequence dna of manipulation are expressed in the red sage root forms, T-DNA border sequence is contained at the two ends of this DNA sequence dna, and it is responsible for the insertion point of goal gene in red sage root genome.Conventionally another one selected marker gene (as anti-kalamycin resistance gene) is also comprised in the left and right border sequence of T-DNA, and this gene can provide a selective marker to confirm whether the red sage root or red sage root cell contain the T-DNA sequence of integration after red sage root cells.The second step of vector construction is that plasmid is transferred to Agrobacterium from intestinal bacteria.This can complete by mode or the direct lead-in mode of DNA of triparental mating.For the transfer of T-DNA, in agrobacterium strains used, also to contain a set of induced gene, they are transferred in red sage root cell and play an important role at T-DNA.

What the conversion of the red sage root was the most frequently used is c4 plant leaf discs conversion method, in some cases, also needs to add some helpers.Other method for transformation comprises protoplast transformation, then by protoplast regeneration, goes out red sage root cell and whole plant.

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

As various DNA already mentioned above, directly take in method for transformation, electrization is first protoplastis to be placed in and to be placed on a highfield, under galvanic action, foreign DNA is sent in protoplastis.Microtubule injection is with microtubule, DNA to be injected directly in cell.Particle bombardment is that DNA is first adsorbed on magnesium sulfate or tungsten particle, and these particles are accelerated to be injected in vegetable cell or tissue.

The present invention selects the red sage root (Salvia miltiorrhiza Bunge) as the host plant of expressing the human acidic fibroblast factor, that it is in use for a long time in China as representative Chinese medicine promoting blood circulation and removing blood stasis because the red sage root is the per nnial herb of Labiatae Salvia.Modern pharmacological research confirmation, the red sage root has vasodilation, improves the biological activity widely such as microcirculation and antisepsis and anti-inflammation.If realize the expression of the human acidic fibroblast factor in the red sage root, the effect that makes the promoting blood circulation and removing blood stasis of the active and red sage root of human acidic fibroblast factor trauma repair and improve microcirculation isoreactivity adds up mutually, not only save the cost of product separation purifying but also avoided the side effect of vivo medicine-feeding, increased again the medical functions of the two simultaneously.

In the transgenosis red sage root, can co expression tomato bushy stunt virus the fusion rotein that forms of P19 albumen, 2A polypeptide and the human acidic fibroblast factor of coding.This fusion rotein is stable and high efficient expression in the transgenosis red sage root, can significantly improve the biological yield of human acidic fibroblast factor recombinant protein, and the human acidic fibroblast factor recombinant protein that is derived from the transgenosis red sage root can cut down and have biological activity in principal and subordinate's fusion rotein, do not need purifying.

The present invention also provides the application of the described transgenosis red sage root in preparing recombination human acidic mechanocyte cytokine newtype drug.

The present invention also provides the application of the described transgenosis red sage root in the medicine of preparation treatment people operative incision, nerve injury, body physical abuse, diabetic foot, ulcer and burn and scald disease.

The present invention also provides a kind of preparation containing human acidic fibroblast factor recombinant protein.Preferably, described preparation is pulvis, paste, sprays or other preparation.

The present invention also provides the preparation method of described preparation, described method for extracting human acidic fibroblast factor recombinant protein from the transgenosis red sage root, or directly use organ, tissue or the crude extract of the described transgenosis red sage root, be processed into described preparation, as pulvis, paste, sprays or other preparation.

The transgenosis salvia miltiorrhiza material of producing this pulvis, paste, sprays or other preparation etc. can have various ways, it can be complete plant, or a part for plant is as fruit, seed, leaf, stem, stem tuber and piece root, or a part of crude extract of plant or be derived from the human acidic fibroblast factor recombinant protein of the transgenosis red sage root through Economical Purification and partial purification.In fact in its each tissue and organ, all contain the human acidic fibroblast factor (haFGF) recombinant protein, comprise fruit, root, stem, blade and seed etc.The transgenosis red sage root is freezing, dry and grind after be made into pulvis, paste, sprays or other preparation.

For the present invention, the human acidic fibroblast factor recombinant protein containing in pulvis, paste, sprays or other preparation should be able to accurate quantification, so that more effectively performance promotes the biological activitys such as people's vasculogenesis, nerve and tissue injury reparation.In pulvis of the present invention, paste, sprays or other preparation, contained recombinant protein is the human acidic fibroblast factor, there is the biological activitys such as the people of promotion vasculogenesis, nerve and tissue injury reparation, just as the human acidic fibroblast factor of native protein or the generation of other expression system.

In a word, adopt which kind of mode to depend primarily on the biological activitys such as vasculogenesis that this albumen itself has and tissue injury reparation, the dosage of the required this recombinant protein that is derived from the transgenosis red sage root of the effects such as vasculogenesis and tissue injury reparation that produce and in this composition contained interference thrombolytic effect material number, as carbohydrate, pyrogen and toxin etc.

The homogenate of described transgenosis red sage root tissue can be treated by topical administration mode the diseases such as people's operative incision, nerve injury, body physical abuse, diabetic foot, ulcer and burn and scald.

The present invention proposes the transgenosis salviae nutrient body that contains human acidic fibroblast factor recombinant protein to divide the method that is processed into pulvis, paste, sprays or other preparation first, due to can carry out wherein contained human acidic fibroblast factor recombinant protein composition strict quantitatively, thereby there has been to clear and definite regulation at each use interval of people and usage quantity.As approximately contained 2.7ug human acid fibroblast growth factor recombinant protein in every gram of transgenosis red sage root pulvis, according to every square centimeter of wound, spray the dosage of 0.37mg dry powder (approximately containing 1ng rhaFGF), use every day 1 time, use continuously 7～10 days, just can reach the object for the treatment of people burn and scald mouth.

The present invention has following beneficial effect:

(1) height of contained human acidic fibroblast factor recombinant protein composition in the transgenosis red sage root, the height of the processing and utilization of this transgenosis red sage root and the related products cost of producing after being directly connected to, for improving the expression amount of human acidic fibroblast factor protein in red sage root cell, the present invention, by the gene of encoding human acidic fibroblast factor protein and tomato bushy stunt virus P19 protein gene coexpression, is not silenced foreign gene in red sage root cell.The present invention has confirmed that the P19 albumen of tomato bushy stunt virus coding has the effect of exogenous gene expression silencing suppressors in the red sage root, it can make the expression efficiency of the human acidic fibroblast factor gene of co expression greatly improve, and the biological yield that makes thus human acidic fibroblast factor recombinant protein accumulate in red sage root cell significantly increases.

(2) the present invention utilizes a 2A peptide sequence, tomato bushy stunt virus P19 albumen and human acidic fibroblast factor protein are together in series end to end, and the gene of this fusion rotein of encoding can be in the red sage root high efficient expression; After 2A polypeptide is cut open specifically by the proteolytic enzyme of self in cell, the P19 albumen discharging and human acidic fibroblast factor recombinant protein can keep respectively biological activity separately.It not only can not affect high efficient expression and the production of human acidic fibroblast factor protein in the transgenosis red sage root, and has the biological activity as native protein at the human acidic fibroblast factor recombinant protein discharging after the cutting of intracellular protein enzyme.

(3) the invention provides the method for utilizing exogenous gene expression silencing suppressors efficient and stably express human acidic fibroblast factor recombinant protein in red sage root cell, and this recombinant protein is known, under native state, there is the biological activitys such as promotion people vasculogenesis, nerve and tissue injury reparation.In other words, human acidic fibroblast factor recombinant protein of the present invention has biological activitys such as promoting people's vasculogenesis, nerve and tissue injury reparation, has and is derived from biological activity like people's natural acidic inoblast factor type.Or more definitely say, human acidic fibroblast factor recombinant protein of the present invention is the same with this acidic fibroblast factor protein that is derived from people or other conventional expression system generation, all has the biological activitys such as the people of promotion vasculogenesis, nerve and tissue injury reparation.

(4) the present invention expresses and the human acidic fibroblast factor recombinant protein produced does not need purifying in the transgenosis red sage root, containing the direct processed and applied of the transgenosis red sage root of human acidic fibroblast factor recombinant protein, can directly be processed into pulvis, paste, sprays and other preparation etc., by topical administration mode, be applied to the treatment of the diseases such as people's operative incision, nerve injury, body physical abuse, diabetic foot, ulcer and burn and scald, thereby can greatly reduce production costs, and be easy to application.Therefore, there is important clinical value.

(5) adopt method of the present invention, effectively improved the expression amount of human acidic fibroblast factor recombinant protein, and the red sage root itself also has vasodilation, improves the biological activity widely such as microcirculation and antisepsis and anti-inflammation.Directly use organ, tissue or the crude extract of this transgenosis red sage root, then be processed into certain pulvis, paste, sprays or other preparation etc., both saved the cost of human acidic fibroblast factor recombinant protein separation and purification, and can bring into play the cooperative effect of the two again simultaneously.

(6) P19 albumen-2A-human acidic fibroblast factor fusion protein encoding gene high efficient expression and accumulation in the transgenosis red sage root plant of regeneration, this transgenosis red sage root has plerosis function by the mode of topical administration to rat back scald wound, can promote the growth of surface of a wound granulation tissue and re-epithelialization, the haFGF standard protein or the red sage root that are significantly better than bacterial origin are individually dosed.The recombination human acidic mechanocyte cytokine newtype drug that this research is Development of New Generation is laid a good foundation.

Accompanying drawing explanation

Fig. 1 is the codon optimized front and back contrast of human acidic fibroblast factor gene, and N represents the gene order before codon optimized and transformation; M represents codon optimized and improved gene order; Shaded letter is the nucleotide base changing.

Fig. 2 is structure and the composition of P19-2A-haFGF antigen-4 fusion protein gene, and wherein, gene 5 ' end list underscore place is XbaI restriction endonuclease sites; Dash area is kozak sequence; Ripple underscore is partly tomato bushy stunt virus p19 albumen coded sequence; Two line place are foot and mouth disease virus 2A polypeptid coding sequence; There is no underscore is partly human acidic fibroblast factor encoding sequence; The single underscore of gene 3 ' source is SacI restriction enzyme site.

Fig. 3 is the clone of P19-2A-haFGF antigen-4 fusion protein gene and the structure of the middle plasmid vector pT-P19-2A-haFGF of bacterium thereof.

Fig. 4 is the building process of plant expression vector pBI-P19-2A-haFGF, and it contains human acidic fibroblast factor gene, and wherein, GUS represents β-Glucuronidase(glucuronidase) gene; P19-2A-haFGF representative acidic fibroblast factor fusion protein gene.

Fig. 5 is the contained structure gene of plasmid pBI-P19-2A-haFGF and regulating and expressing sequence thereof, and wherein, Nos-Pro represents rouge alkali synthetase promoter; Npt-II represents neomycin phosphotransferase gene; Nos-Ter represents rouge alkali synthetase terminator; 35S Pro represents cauliflower mosaic virus 35 S promoter; P19-2A-haFGF representative acidic fibroblast factor fusion protein gene; RB representation DNA right border sequence; LB representation DNA left margin sequence.

Fig. 6 is that PCR detects the integration of human acidic fibroblast factor fusion protein gene in different transgenosis red sage root strains, and wherein, M is DNA marker; 1 positive contrast (pcr amplification product that the pBI-P19-2A-haFGF plasmid DNA of take is template); 2 for take the pcr amplification product that non-transgenic red sage root genomic dna is template; 3～8 for take the pcr amplification product that part transgenosis red sage root genomic dna is template.

Fig. 7 is that the Southern blot that turns the haFGF gene red sage root detects, wherein, and 1 positive contrast (the Southern results of hybridization of the pcr amplification product that the pBI-P19-2A-haFGF plasmid DNA of take is template); 2 is the Southern results of hybridization of non-transgenic red sage root genomic dna after XbaI enzyme cutting; 3 is the Southern results of hybridization of non-transgenic red sage root genomic dna after SacI enzyme is cut; 4 is the Southern results of hybridization of transgenosis red sage root genomic dna after XbaI enzyme cutting; 5 is the Southern results of hybridization of transgenosis red sage root genomic dna after SacI enzyme is cut.

Fig. 8 is that the Western blot of the haFGF transgenosis red sage root detects, and wherein, M is standard protein molecular weight; A is the SDS-PAGE result of transgenosis and non-transgenic red sage root total protein; B is the Western blot detected result after A transferring film; 1 is standard human acidic fibroblast factor protein (positive control); 2～3 is the non-transgenic red sage root; 4～6 is the transgenosis red sage root.

Fig. 9 is the therapeutic action to rat burn and scald that turns the haFGF gene red sage root, and wherein, 0d, 3d, 7d, 14d and 21d represent respectively the time after burn and scald; Upper left is physiology saline control group; Upper right is rhaFGF standard control treatment group (positive control); Lower-left is non-transgenic Treated with Radix Salviae Miltiorrhizae group; Bottom right is haFGF transgenosis Treated with Radix Salviae Miltiorrhizae group.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.

If do not specialize, experiment material, reagent and instrument etc. used in the embodiment of the present invention are all commercially available, if specifically do not indicate, and the conventional means that in embodiment, technique means used is well known to the skilled person.

Embodiment 1

1, the synthetic of tomato bushy stunt virus P19 albumen-2A polypeptide-haFGF antigen-4 fusion protein gene (P19-2A-haFGF antigen-4 fusion protein gene)

The nucleotide sequence of coding for tomato bushy stunt virus P19 albumen, is derived from GenBank NC_001554(accession number) in 3888～4403 sites, its DNA length is 516bp, 172 amino-acid residues of encoding.Because this gene is former, be to come from plant virus genome, be suitable in vegetable cell, efficiently transcribing and translating, therefore, its codon is not done to further optimization.

The nucleotide sequence of coding foot and mouth disease virus 2A polypeptide is derived from GenBank AJ251476(accession number) 3472～3537 sites, its DNA length is at 66bp, 22 amino-acid residues of encoding.

Although people with and plant all belong to eukaryote, they are still existing certain difference aspect gene codon preference, this species diversity likely can have influence on stability and the high efficient expression of human acidic fibroblast factor gene in the transgenosis red sage root.For further improving its expression amount in the transgenosis red sage root, according to the human acidic fibroblast factor gene sequence of having announced, (see GenBank accession number: X65778), do not changing under the prerequisite of its Argine Monohydrochloride sequence, the codon of having a preference for according to plant, is optimized its gene coded sequence.Full length gene 465bp after optimization, 154 amino-acid residues of encoding.Sequence optimisation relates to 108 codons, has changed altogether 123 nucleotide bases, but does not change before and after the optimization of G+C content, is 52%(and sees Fig. 1).

Above-mentioned three encoding sequences are from beginning to end, just form a new P19-2A-haFGF antigen-4 fusion protein gene (gene structure and composition are shown in Fig. 2).For the ease of the structure of various plasmid vectors subsequently, in this antigen-4 fusion protein gene of synthetic, before 5 ' end ATG initiator codon of this gene, synthesize and added restriction enzyme site XbaI and kozaK sequence (being conducive to the expression of antigen-4 fusion protein gene in the red sage root), after 3 ' end terminator codon TAA of this antigen-4 fusion protein gene, increased restriction enzyme site SacI, synthetic the nucleotide sequence of encoding human acidic fibroblast factor fusion protein gene (said gene splicing and synthetic work by the luxuriant industry of Beijing English bio tech ltd on behalf of completing).The nucleotide sequence of described antigen-4 fusion protein gene is as shown in SEQ ID NO.1, and the aminoacid sequence of its proteins encoded is as shown in SEQ ID NO.2.2, the clone of P19-2A-haFGF antigen-4 fusion protein gene

The DNA fragmentation of above-mentioned synthetic is directly inserted and is connected in the T site in pEASY-T1 plasmid (sees Beijing Quanshijin Biotechnology Co., Ltd's product description), the method providing according to the said firm, a plurality of bacterium mono-clonals of random choose are identified, obtain the bacterial clone (see figure 3) that contains human acidic fibroblast factor fusion protein gene plasmid carrier pT-P19-2A-haFGF, then, by DNA sequencing, analyze, determine that coding P19-2A-haFGF antigen-4 fusion protein gene is correct and complete (DNA sequencing is by the luxuriant industry of Beijing English bio tech ltd).

Embodiment 2

1, the structure of plant expression vector pBI-P19-2A-haFGF

In above-mentioned bacterial clone, extract pT-P19-2A-haFGF plasmid vector, utilize restriction enzyme XbaI and SacI to carry out double digestion to carrier pT-P19-2A-haFGF and pBIl21, reclaim P19 albumen-2A polypeptide-haFGF fusion rotein encoding gene fragment and pBI121 carrier segments, connect, P19 albumen-2A polypeptide-haFGF fusion rotein encoding gene fragment orientation is inserted in the former gus gene site of plasmid pBI121 (through XbaI and SacI double digestion), is just built into double base plant expression vector pBI-P19-2A-haFGF(and sees Fig. 4).

Plasmid pBIl21 is purchased from U.S. Clonetech company, and its XbaI restriction enzyme site is between cauliflower mosaic virus 35 S promoter and gus gene initiation codon, and SacI is between gus gene terminator codon and NOS terminator.Select plasmid pBIl21 to be because former gus gene can be deleted with XbaI and SacI double digestion, and can insert subsequently the foreign protein genes that another one is new, new gene can be in vegetable cell high efficient expression.Plasmid pBI121 also contains neomycin phosphotransferase II gene (nptII), and the enzyme of its coding can be vegetable cell kalamycin resistance is provided, thereby the cell that contains T-DNA and tissue and unconverted vegetable cell or tissue division can be come.NptII gene has promotor and the poly gland acid tailing signal sequence of oneself, and they are all derived from nopaline (Nopaline) synthetic enzyme (NOS) gene.

Plasmid pBI-P19-2A-haFGF contains: 1) Liu Suanyan NEOMYCIN SULPHATE synthase gene II(nptII), it offers vegetable cell kalamycin resistance; 2) P19-2A-haFGF antigen-4 fusion protein gene and handle the cauliflower mosaic virus 35 S promoter of this gene; 3) T-DNA left and right border sequence, it can be transferred to nptII gene and P19-2A-haFGF antigen-4 fusion protein gene in red sage root body and be incorporated in red sage root karyomit(e).The structure of pBI-P19-2A-haFGF is as Fig. 5.

2, expression vector pBI-P19-2A-haFGF is imported to Agrobacterium (A.tumefaciens)

The plasmid vector pBI-P19-2A-haFGF that contains P19-2A-haFGF antigen-4 fusion protein gene can transfer in Agrobacterium LBA4404 bacterial strain by the mode of triparental mating, and this bacterial strain is purchased from U.S. Clonetech company.Bacterial strain LBA4404 is now widely used, and it is improved agrobacterium strains, contain complete Vir gene, but the T-DNA of self is deleted.Vir gene can be trans adjusting T-DNA from plasmid pBI-P19-2A-haFGF to the transfer in vegetable cell.

Agrobacterium is 10 grams of the 50mL YEP(peptones that contains 25mg/L Streptomycin sulphate, 10 grams of yeast extracts, NaCl5 gram, adding distil water to 1 liter, pH7.0) shaking culture in substratum, in OD600nm value, reach at 0.4～0.7 o'clock, the centrifugal collection bacterial cell of 4000g, agrobatcerium cell Eddy diffusion is stand-by in 1mL does not contain any antibiotic YEP substratum.The DH5 α that contains expression vector pBI-P19-2A-haFGF (purchased from U.S. GIBCO company) and contain helper plasmid pRK2013(purchased from U.S. Clonetech company) HB101(purchased from U.S. Clonetech company) be seeded in respectively and contain shaking culture in 50mg/L kantlex 50mL LB substratum, in OD600nm value, reach at 0.4～0.7 o'clock, the centrifugal collection bacterial cell of 4000g, is then suspended in cell respectively 1mL not containing stand-by in any antibiotic LB substratum.Get above-mentioned three kinds of each 200uL of cell suspending liquid, be placed in same 1.5mL centrifuge tube, 28 ℃ of standing overnight incubation, get this culture 5～10uL, evenly spread upon on YEP solid medium flat board, in this substratum, contain 25mg/L Streptomycin sulphate and 50mg/L kantlex simultaneously, cultivate after 2 days for 28 ℃, the Agrobacterium bacterium colony that contains pBI-P19-2A-haFGF plasmid starts to produce.

With alkaline lysis, from Agrobacterium, extract plasmid DNA (Sambrook J and Rusell D W, Molecular Cloing:A Laboratory Manual, whether 2001, Cold Springer harbor Laboratory Press) also with digestion with restriction enzyme, can detect pBI-P19-2A-haFGF plasmid DNA exists.

3, agriculture bacillus mediated Genetic Transformation in Higher Plants

First the genetic transformation of the red sage root is that red sage root histoorgan or cell and Agrobacterium are cultivated altogether, after cultivating approximately 2 days, red sage root explant dislocation is selected in substratum accordingly.Red sage root explant can be protoplastis, callus or other organ-tissue, and selecting the organ with leaf is the most frequently used method.

The Agrobacterium LBA4404 that contains expression vector pBI-P19-2A-haFGF is seeded in 50mL YEP substratum (containing 50mg/mL kantlex and 25mg/mL Streptomycin sulphate) to 28 ℃ to be cultivated 2 days, 4000g collects thalline for centrifugal 5 minutes, with 25mL liquid MS nutrient solution (not containing microbiotic), washing once, use again MS Eddy diffusion to OD600nm=0.2～0.4, standby.

The people's such as blade conversion Main Basis Horsch method (Horsch, R.B., etc., A Simple and General Method for Transferring Genes into Plants, Science, 1985,227:1229-1231).Salvia seeds is purchased from drug germchit station, northwest, Shangluo City, Shaanxi Province.Get full salvia seeds and soak 10min with washing composition, then with clear water, repeatedly rinse to remove washing composition, seed is placed in to Bechtop; To filling the ethanol that adds 75 ℅ in seed-bearing bottle, after 1min, outwell; Add the sterilized water that contains 10 ℅ clorox, process 15min, during constantly rock, outwell clorox; Rinsed with sterile water 3 times, each 5min; Blot the moisture on seed and be seeded on MS solid medium; Put into illumination box and cultivate, 16h illumination/8h is dark, 25 ℃/23 ℃ of temperature.After salvia seeds sowing 2～3 weeks, choose more healthy and stronger aseptic seedling, the young leaflet tablet edge newly growing is cut be then cut into 0.5cm * 0.5cm square as the explant infecting, be placed in the culture dish that fills a small amount of sterilized water; By shearing explant, immerse in the above-mentioned Agrobacterium bacterium liquid preparing, infect 5～8min, constantly rock therebetween; With the rinsing of MS liquid nutrient medium once, aseptic filter paper sucks residual liquid to explant after infecting; Common substratum (MS minimum medium+2.0mg/L6-BA) surface tiling one deck aseptic filter paper at culture dish, is placed in explant on filter paper, and culture dish seals with sealed membrane, is placed in the dark cultivation of incubator 3 days, and temperature is 25 ℃; Explant after secretly cultivating is transferred in inductive differentiation medium (MS minimum medium+1.0mg/L6-BA+400mg/L Pyocianil (Carb)+50mg/L kantlex (Kan)), and culture condition is: 16h illumination/8h is dark, 25 ℃/23 ℃ of temperature; Within every 2 weeks, change a division culture medium, until grow resistant buds; When resistant buds grows to 1～2cm left and right by the time, resistant buds is cut, proceed in root media (1/2MS substratum+2% sucrose+30mg/L kantlex (Kan)) and take root, the concentration of Kan is down to 30mg/L by 50mg/L, is in order to increase the surviving rate of regeneration plant; When red sage root regrowth grows to the high and well developed root system of 5～6cm, it is taken out from substratum and clean root substratum and move into hardening in sterilized vermiculite Nutrition Soil (1:1), after robust growth, move into field.

2, PCR detects the integration of P19-2A-haFGF antigen-4 fusion protein gene in the red sage root

The extracting method of red sage root genomic dna substantially according to the method for Chee (Chee, P P, Drong, R F and Slightom J L, Plant Molecular Biology Manual, 1991, C3:1-28).Get the fresh blade of the 3g red sage root, put into mortar liquid nitrogen grinding, in powder dislocation 20mL centrifuge tube after grinding, then add 9mL CTAB extracting solution (100mM Tris-HCl, pH8.0 is containing 1.4M NaCl, 20mM EDTA, 10mL/L beta-mercaptoethanol, 20g/L CTAB), thermal agitation 1 minute, makes it fully to mix.In 65 ℃ of water-baths, temperature is bathed 60 minutes, during vibration in every 10 minutes once.From water-bath, take out centrifuge tube, under room temperature, place 4～5 minutes, make it cooling, then add the saturated phenol of isopyknic Tris: chloroform (1:1), concussion mixes, and puts into 4 ℃ of whizzer 10000rpm, centrifugal 15min; Collect supernatant liquor in another 20mL centrifuge tube, then add isopyknic chloroform to repeat extracting once, put into 4 ℃ of whizzer 10000rpm, centrifugal 15min; The careful supernatant of drawing, does not suck organic phase, and to the Virahol that adds 0.6 times of volume in supernatant liquor, gentle putting upside down mixes, and room temperature is placed 30min, in visible pipe, has cotton-shaped genomic dna to occur; Put into whizzer 10000rpm, centrifugal 10min, carefully outwells supernatant liquor; Collecting precipitation, with 3ml76% ethanol (containing 0.2M NaOAc) and 2mL76% ethanol (containing 10mM NH4OAc), respectively wash precipitation once, put into super clean bench or ventilation dries up, finally add TE(pH8.0) damping fluid dissolving DNA, 1 μ L RNase A, places 1h in 37 ℃ of incubators; DNA concentration is adjusted to 1ug/uL, be stored in-20 ℃ standby.

PCR reaction system: get the about 1uL of above-mentioned red sage root genomic dna 1ug as masterplate, add primer P1:5 ＇-GCTGAAG GAGAAATTACTACTTTTACTGC-3 ＇ (SEQ ID NO.3) and each 1uL of primer P2:5 ＇-GAGCTCTTAA TCAGAAGAAACTGGA AG TGG-3 ＇ (SEQ ID NO.4), 10 * PCR damping fluid 3uL, each 2.5mM of dNTP() 3uL, the about 1.5U of Taq enzyme 0.5uL, adds water to cumulative volume 30uL.

Response procedures is: 95 ℃ of denaturations 5 minutes; 94 ℃ 30 seconds, 50 ℃ 45 seconds, 72 ℃ 1.5 minutes, carry out altogether 35 circulations; 72 ℃ are extended 10 minutes.

After reaction finishes, get 5uL amplification sample and carry out 1% agarose gel electrophoresis detection.As shown in Figure 6, the negative control red sage root (swimming lane 2) is not found specific band, the specific band (swimming lane 3～8) that can amplify 1 about 500bp in the swimming lane of transgenosis red sage root plant, this result shows that human acidic fibroblast factor gene has been integrated in red sage root genomic dna.

3, the Southern of the transgenosis red sage root detects

Utilize the human acidic fibroblast factor gene DNA fragmentation of digoxigenin labeled to make probe (random primer labelling method is shown in the test kit PCR DIG Labeling Mix of Roche company), the part kalamycin resistance red sage root genomic dna after pBI-P19-2A-haFGF carrier is transformed has carried out Southern hybridization analysis.

The extracting method of transgenosis red sage root leaf DNA as mentioned above.Get 40uL(containing 40 μ g DNA) red sage root genomic dna, then add respectively 40 μ L10 * buffer enzyme cutting buffering liquids, 10 μ L restriction enzyme XbaI or SacI to carry out single endonuclease digestion, add aseptic redistilled water to cumulative volume 400 μ L, after reactant is mixed, wink fully mixes from making, whether 37 ℃ of enzymes are cut spend the night (at least more than 12h), get 10 μ L enzymes and cut product and carry out electrophoresis detection enzyme and cut complete.Enzyme is cut to product to be concentrated, Xiang Guanzhong adds isopyknic chloroform/primary isoamyl alcohol to carry out extracting, centrifugal absorption supernatant adds the NaAc of 1/10 volume and the dehydrated alcohol of 2.5 times of volumes, room temperature is placed 30min, centrifugal acquisition DNA precipitation, by 75% washing with alcohol twice, put into super clean bench and dry up, add 40 μ L ultrapure waters by resolution of precipitate.

Enzyme is cut to product and carry out 0.8% agarose gel electrophoresis, 20V electrophoresis spends the night, and tetrabromophenol sulfonphthalein stops electrophoresis while moving to blob of viscose end 2cm.It is upper that nucleic acid is transferred to nylon membrane (Hybond N+) by hair pipette method, and damping fluid used is 20XSSC, pH7.2(3M sodium-chlor, 0.3M Trisodium Citrate), be 16 hours transfer time.Nucleic acid by uviolizing after by commissure on nylon membrane, film is placed in to prehybridization solution (5XSSC, 0.1%(w/v) N-Lauroylsarcosine, 0.02%SDS, 100ug/mL salmon sperm dna), 68 ℃ of prehybridizations 3 hours, then hybridization solution (the 5XSSC more renewing, 0.1%(w/v) N-Lauroylsarcosine, 0.02%SDS, 1% encapsulant (providing in test kit (the DIG Nucleic Acid Detection Kit of Roche company))), in 5mL hybridization solution, add the DNA probe 60ng of the digoxigenin labeled of sex change to hybridize, the long 465bp of probe, it is to take pT-P19-2A-haFGF plasmid DNA as template, adopt arbitrarily primed PCR amplification method to prepare, the whole encoding sequences that comprised human acidic fibroblast factor gene.In hybridization solution, 68 ℃ of hybridization, after 24 hours, are taken out Hybond membrane, in 200mL2XSSC, in 0.1%SDS damping fluid, wash film 2 times under room temperature, then in 100mL0.1XSSC, in 0.1%SDS damping fluid, wash film 2 times for 68 ℃.Color reaction is substantially according to the method providing in test kit (the DIG Nucleic Acid Detection Kit of Roche company), and nylon membrane is prior to elution buffer (0.1M Maleic acid, pH7.5; 0.15M NaCl; 3%Tween20(w/v)), balance is 5 minutes, and then, with 50mL1X sealing damping fluid sealing 30 minutes, adding DigiTAb to the concentration of alkali phosphatase enzyme mark is 75mU/mL, reacts 30 minutes under room temperature, uses elution buffer 2 times, each 15 minutes.By film dislocation 20mL colour developing damping fluid (0.1M Tris-HCl, pH9.5; 0.1M NaCl; 50mM MgCl ₂) in balance 5 minutes, the substrate buffer solution more renewing (200uL NBT/BCIP substrate adds 10mL colour developing damping fluid), develops the color under shading condition 16 hours, after desired band occurs, water termination reaction.

The transgenosis red sage root obtaining after plasmid pBI-P19-2A-haFGF transforms and the Southern results of hybridization of negative control red sage root plant are as Fig. 7.In transgenosis red sage root swimming lane, after hybridizing, XbaI and SacI single endonuclease digestion genomic dna and probe obtained the single band varying in size, proof P19-2A-haFGF external source antigen-4 fusion protein gene is successfully incorporated in red sage root genome, and the insertion copy number of this gene is single copy.And the genomic dna of the negative control red sage root is not observed any hybridization signal.

4, the Western of the transgenosis red sage root detects

Take the tender red sage root blade of 1g children, add 500 μ L PBST damping fluid (PBS damping fluid (8.0g/L NaCl, 0.2g/L KCl, 2.98g/L Na ₂hPO ₄12H ₂o, 0.24g/L KH ₂pO ₄, pH7.4)+0.05%Tween-20 (v/v)) fully grind, 10,000rpm10min, collects supernatant liquor, after cryogenic temperature freezing drying is concentrated, adds 50uL PBS to dissolve, and 4 ℃ save backup.

Get the above-mentioned protein extract after concentrated of 20 μ L, add equal-volume 2 * sample-loading buffer (50mmol/L Tris-HCl, 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine, 100mmol/L DTT), in boiling water, boil 5min, be placed at once 2min on ice.Utilize 15% poly-propionic acid amide denaturant gel (SDS-PAGE) to carry out the separation that electrophoresis is realized different molecular weight protein, loading 30 μ L in each sample cell.After electrophoresis finishes, utilize semi dry electrophoresis to shift instrument the albumen equipotential on gel is transferred on the nitrocellulose filter of a homalographic.

Film is taken out and puts into plate, will contact one with gel phase and face up, add appropriate TBST damping fluid (TBS damping fluid (12.11g/L Tris, 8.775g/L NaCl)+0.05%(v/v) Tween-20), by film submergence, be placed on shaking table, room temperature is washed at a slow speed film, repeat each 10min 3 times; Outwell the damping fluid of washing film, add sealing damping fluid (TBST damping fluid+3%BSA), be placed in and on shaking table, seal 2h.Outwell sealing damping fluid, use TBST buffer solution for cleaning 3 times, each 10min; Outwell and wash film damping fluid, with dilution buffer liquid (TBST damping fluid+0.1%BSA), 200 times of haFGF antibody (purchased from Antigenix America company) dilutions are added in plate to jog 1h on shaking table; By TBST buffer solution for cleaning 3 times, each 10min; Outwell the damping fluid of washing film, with dilution buffer liquid by Streptarvidin-HRP(purchased from Antigenix America company) 2000 times of dilutions add in plate, jog 30min on shaking table, TBST buffer solution for cleaning 3 times, each 10min; Outwell the damping fluid of washing film, then add DAB nitrite ion (purchased from Antigenix America company), the standing colour developing of lucifuge, after colour developing completely, with distilled water flushing, termination reaction.Found that, in transgenosis red sage root swimming lane, there is single dark-brown band, size is consistent with haFGF positive control, about 16kD(is shown in Fig. 8), show that haFGF gene expresses in the transgenosis red sage root, but the expression amount of haFGF recombinant protein exists certain difference because plant is different, consistent with the result that ELISA detects below.

5, the mensuration of human fibroblasts's factor recombinant protein content in the transgenosis red sage root

By enzyme linked immunosorbent assay (ELISA), measure the content (concrete operation method is shown in the Human FGF-acidic ELISA Construction Kit of Antigenix America company working instructions) of human fibroblasts's factor recombinant protein in transgenosis red sage root blade.

1) get the positive about 0.02g of transgenosis red sage root blade of PCR, add 200 μ L dilution buffer liquid (PBST damping fluid (8.0g/L NaCl, 0.2g/L KCl, 2.98g/L Na ₂hPO ₄12H ₂o, 0.24g/L KH ₂pO ₄, pH7.4)+0.1%BSA) grind, the centrifugal 5min of 12,000rpm, draws supernatant as testing sample; 2) process equally the negative contrast of non-transgenic red sage root blade; 3) get each 100 μ L of above-mentioned sample supernatant liquor, add respectively in each aperture of enzyme-linked reaction plate after primary antibodie is coated with, room temperature reaction 2h, then dries liquid in hole, and enzyme plate rinses 5～7 times with PBST damping fluid, each 3min; 4) with dilution buffer liquid, Biotin Tracer(bis-is anti-) dilute 200 times, in every hole, add 100 μ L, room temperature is placed 30min, then dries liquid in hole, and enzyme plate rinses 5～7 times with PBST damping fluid, each 3min; 5) with dilution buffer liquid, Streptarvidin-HRP is carried out the dilution of 2000 times, add 100 μ L in every hole, room temperature is placed 30min, dries liquid in hole, and enzyme plate rinses 5～7 times with PBST damping fluid, each 3min; 6) by TMB chromogenic substrate A liquid and B liquid balanced mix, every hole adds 100 μ L, room temperature lucifuge reaction 1h; 7) 650nm wavelength reads absorption photometric value (OD ₆₅₀), or every hole adds 100 μ L stop buffer (2mol/L H ₂sO ₄) under 450nm, read absorbance value (OD after termination reaction ₄₅₀).

With dilution buffer liquid, the haFGF standard substance that provide in test kit are diluted to 200 times, 400 times, 800 times, 1600 times, 3200 times and 6400 times successively, working concentration is 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 3.125ng/mL, prepare a standard absorption curves, reference standard curve can calculate human acidic fibroblast factor recombinant protein content in testing sample.By enzyme joint inspection, survey and find, in negative control plant, do not find containing human acidic fibroblast factor recombinant protein (rhaFGF), in part transgenosis red sage root plant, acidic fibroblast factor recombinant protein can be detected, just content is different.Reference standard curve, finds that the high-content of the rhaFGF albumen of the red sage root is about 27ng/mL, is equivalent to every gram of fresh weight red sage root blade and is about 270ng containing rhaFGF.

Embodiment 3

1, the modeling of mouse burn and scald

5 of male SD rats (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), buy in each experiment the last week, freely drink water and ingest (experiment repeats 3 times, needs altogether 15 rats).Test the day before yesterday, with ether, by rat anesthesia, with scissors, the hair of back part of animal is cut short, and lost hair or feathers with 8% sodium sulphite, area is 9cm * 8cm, and with povidone iodine sterilization, physiological saline cleans.Experiment fasting on the same day, etherization, pad a gauze in the belly below of rat and make back smooth, is placed on anchor.The desk-top super temperature control scald apparatus of YLS-5Q is connected to 220V power supply, and temperature is adjusted to 80 ℃, and pressure is adjusted to 0, usings and perms own wt (0.5kg) as the pressure of causing injury, and scalds time 8s, selects area 2.5cm ²perm, after sterilization apart from 1.5cm place, backbone both sides in the middle of rat back, each boiling hot 2 circular surface of a wound up and down, degree of injury is dark II degree (through histopathological examination, injuring skin corium, only the residual deep appendages of skin).

2, the healing of mouse burn and scald

4 surface of a wound of successful 5 rats of modeling are done to following processing: 1) rhaFGF positive controls; 2) haFGF transgenosis red sage root experimental group; 3) non-transgenic red sage root control group; 4) physiological saline blank group.5 i.e. 5 repetitions of rat, each surface of a wound (2.5cm ²) each administration 100 μ L at every turn.

Get the 0.02g transgenosis red sage root and non-transgenic red sage root blade and add 200 μ L physiological saline, centrifugal after grinding is broken, get supernatant standby; The 100 μ L physiological saline of take are blank; With 10mL physiological saline, 0.1mg aFGF standard substance (nomenclature of drug: Ai Fujifu, Wanxing Biological Pharmaceutical Co., Ltd., Shanghai) are diluted to 10ug/mL as positive control.Each group is scalded and was started medication the rear same day, changes dressings every other day once, extremely 21d after wound later.While changing dressings, open gauze at every turn, use disinfectant with hydrogen peroxide wound, physiological saline cleans, wound circumference carries out disinfection with Iodophor, with aseptic cotton carrier, excessive moisture is blotted, and with liquid-transfering gun device, sample is added in to corresponding surface of a wound surface and smoothens, effect 5min, paper using immobilization with adhesive tape after sterile gauze wrapping, single cage is fed, and freely drinks water and ingests.

Postoperative with the surface of a wound take pictures, transparent film is traced weighing method (with transparent pan paper: 7.5cm * 7.5cm, weighted average is about 0.1604g, cover the rat back surface of a wound, surface of a wound edge is depicted on transparent pan paper, cut off unnecessary trimming, weigh, by formula below, calculate surface of a wound area, surface of a wound area=7.5cm * 7.5cm * scraps of paper weight/0.1604g) record wound is rear 3,7,14,21d surface of a wound area.All data resultss all with ± SD represents, with SPSS17.0 statistical analysis software system, carries out monofactorial variance analysis, significance level P < 0.1 or P < 0.5.

As can be seen from Figure 9, with post burn, extend, respectively form face area and dwindle gradually, after wound, in 3d, all surface of a wound all have a small amount of exudate, no significant difference between each group of postoperative l～7d; 14d and 21d after surgery, each treatment group is compared with physiological saline control group, and the surface of a wound all obviously diminishes, but the effect that haFGF transgenosis Treated with Radix Salviae Miltiorrhizae group and positive controls are accelerated wound healing more obviously (P<0.5).In four treatment group, the result for the treatment of that applies haFGF transgenosis red sage root blade homogenate supernatant liquor treatment group of take is best (approximately using rhaFGF2ng/ wound), its action intensity and the effect close (P>0.5) (the active ratio of short injury repairing is 1:50) that applies rhaFGF standard substance (positive controls is approximately used rhaFGF standard substance 100ng/ wound).What be worth proposition is, non-transgenic Treated with Radix Salviae Miltiorrhizae group also has certain promoter action to rat wound healing, but compare with haFGF transgenosis Treated with Radix Salviae Miltiorrhizae group with positive controls, still exist significant difference (P<0.1) (in Table 1, wherein, the number that in table 1, n is rat).

Table 1 is respectively organized the rear different time points surface of a wound Area comparison (cm of rat wound ², )

Note: with the comparison of physiological saline group, * p<0.5, #p<0.1; With the comparison of non-transgenic red sage root group, ▲ p<0.1.

3, contain the preparation of rhaFGF transgenosis red sage root pulvis, paste, sprays or other preparation

Transgenosis Salvia miltiorrhiza is piece root by part, but in fact in its each tissue and organ, all contains haFGF recombinant protein, comprises root, stem, blade and seed etc.This transgenosis red sage root piece root or all nourishing body through cryodrying or air-dry at a slow speed after, under low temperature or room temperature, can wear into 200～1000 object dry powder, after measured, the human acidic fibroblast factor recombinant protein that approximately contains 2.5ug in the dry powder that this transgenosis red sage root of every 1g piece root is made.This dry powder can regularly directly spray in wound surface such as people's burn and scald, physical abuse or diabetes brothers, and its contained haFGF recombinant protein can enter human body by wound, reaches thus and promotes the generation of people's wound site blood vessel and the object of accelerating wound healing.

Above-mentioned transgenosis red sage root dry powder also can be mixed and made into paste with wetting Agent for Printing Inkss such as adding glycerine, agar gel, xanthan gum, dextrin or Vaseline, also can in this paste, add mineral substance or protein transdermal agent etc., in some cases, such paste is more suitable for the treatment of wounds such as being applied in people's burn and scald, physical abuse or diabetes brothers than pulvis.

In transgenosis red sage root dry powder, also can add micro-transdermal agent, then be mixed and made into sprays with aqueous protein stabiliser, in some cases, the more convenient application of this sprays, the field of clinical application is wider.

Although, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims

1. containing a preparation for human acidic fibroblast factor recombinant protein, described preparation is pulvis, paste or sprays;

The preparation method of described preparation is for to extract human acidic fibroblast factor recombinant protein from the transgenosis red sage root, or directly uses organ, tissue or the crude extract of the described transgenosis red sage root, is processed into described preparation;

The preparation method of the described transgenosis red sage root is: the gene of encoding fusion protein P19-2A-haFGF or the recombinant expression vector that contains this gene are transformed to red sage root cell, bear the complete transgenosis red sage root from transgenosis red sage root cell again; Wherein, the aminoacid sequence of described fusion rotein P19-2A-haFGF is as shown in SEQ ID NO.2.

2. preparation according to claim 1, is characterized in that, described recombinant expression vector is pBI-P19-2A-haFGF, and its construction process comprises the steps: