CN103319609A - Human basic fibroblast growth factor (hbFGF) fusion gene and application thereof - Google Patents
Human basic fibroblast growth factor (hbFGF) fusion gene and application thereof Download PDFInfo
- Publication number
- CN103319609A CN103319609A CN2013102827315A CN201310282731A CN103319609A CN 103319609 A CN103319609 A CN 103319609A CN 2013102827315 A CN2013102827315 A CN 2013102827315A CN 201310282731 A CN201310282731 A CN 201310282731A CN 103319609 A CN103319609 A CN 103319609A
- Authority
- CN
- China
- Prior art keywords
- gene
- hbfgf
- red sage
- sage root
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention provides a human basic fibroblast growth factor (hbFGF) fusion gene and application thereof. According to the hbFGF fusion gene and the application of the hbFGF fusion gene, a hbFGF gene is fused with a protein gene P19 of a tomato bushy stunt virus and a polypeptide coding sequence 2A of a foot-and-mouth disease virus by adopting an in-vitro gene recombination method so as to obtain a fusion gene, the fusion gene is used for constructing a plasmid expression vector of a plant, and red-rooted salvia is converted under the induction of agrobacterium, so as to obtain a transgenic plant. The integration and expression of an exogenous gene are proved by molecular biological detection; and activity detection shows that an obtained recombinant protein has bioactivity which is equivalent to that of a hbFGF standard. The hbFGF fusion gene and the application of the hbFGF fusion gene have the advantages that bFGFs can be produced efficiently, and thus, a new way is provided for the development of bFGF drugs.
Description
Technical field
The present invention relates to the genetically engineered field, specifically, relate to rh-bFGF fusion gene and application thereof.
Background technology
Fibroblast growth factor belongs to a large protein families, up to the present, has found at least 23 members.Wherein, Prostatropin (basic fibroblast growth factor, bFGF) is separated first from the ox pituitary gland in 1974 by people such as Gospodarowicz and obtains, and because its iso-electric point is 9.6, is alkalescence, therefore hence obtain one's name.
Rh-bFGF (hbFGF) extensively is present in the various tissues of human body, such as tissues such as brain, the heart, liver, bone, eye, suprarenal gland, placentas, and with content in brain and the hypophysis for the highest.It is a kind of mitogen of wide spectrum, has obvious promotion proliferation function to deriving from mesoderm and neuroectodermal cell, and its target cell has inoblast, vascular endothelial cell, chondrocyte and scleroblast etc.Its biological function mainly contains: 1) promote new vessel to form; 2) promote soft tissue, the reparation of cartilage, osseous tissue and neural tissue injury; 3) promote limb regeneration (Gospodarowicz D, Cheng J, Lui G M, etal., Isolation of brain fibroblast growth factor by heparin sepharose affinity chromatography:identity with pituitary fibroblast growth factor[J], Proc Natl Acad Sci USA, 1984,81:6963-6967); 4) participate in neurotization etc.
BFGF forms with the external new vessel that all can obviously promote in vivo, it all has promoter action to a plurality of links in the new vessel forming process, but the various types of cells of chemotactic tunica intima, and induce the required plasminogen activator of these cell expressing tissue reconstructions, collagenase, proteolytic ferment etc., these enzymes are by impelling the degraded of blood vessel endothelium basement membrane and irritating inner membrance various types of cells propagation and migration, the induction of vascular endothelial cell is grown into and is formed tube chamber in the collagen stroma, and promotes neurone and new vessel jointly to grow.
After the tissue injury, local bFGF expresses to be increased.BFGF not only can make monocyte, neutrophil leucocyte, scavenger cell, fibroblast etc. assemble to damage is local by chemotaxis, and can pass through the various approach such as cell fission propagation relevant with reconstruction in Angiogensis, promotion soft tissue and the osseous tissue, promote the generation of granulation tissue, the injury repairing of organizing is played vital effect.In addition, Exogenous bFGF for the poor wound healing person (as diabetes, chemicotherapy, and the patient such as immunodeficiency) wound have significant promotion Healing.
BFGF extensively is present in maincenter and the peripheral nervous system, no matter in vivo or external, and the effect that all multiple neurone is had surviving and promotes to grow, and can promote reparation and the regeneration of injured neurons.BFGF widely neurotrophic activity draws attention day by day, and the clinical fields such as ischemia central nervous system injury that cause in treatment nerve injury, nerve retrograde affection, cerebro-vascular diseases and cerebral trauma tentatively are applied.
Up to now, utilizing gene recombination technology to produce in the process of bFGF, the bFGF that gives expression to need to purify through a series of purification step, not only so that production stage is loaded down with trivial details, and can the loss product in purge process.In addition, at the expression phase of bFGF, express productive rate and still have much room for improvement.
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 the expression efficiency of green fluorescent protein foreign gene in vegetable cell take tobacco mosaic virus (TMV) (TMV) as carrier improve (Lindbo J A more than at least 100 times, High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors, BMCBiotechnology, 2007,7:52doi:10.1186/1472-6750-7-52).P19 albumen size approximately about 19kDa, in each P19 protein molecular, contains 4 or 5 αhelix and 4 β-pleated sheet structure sheets.Two P19 albumen interacts, forms a homodimer in the tail tail mode of linking to each other with sat linkage by the hydrogen bond that forms between intramolecular the 4th the β-pleated sheet structure sheet separately and the hydrophobic bond that forms between the α spiral of PROTEIN C end separately.Formed a groove that is formed by the β-pleated sheet structure sheet between this homodimer, can mutually identify and combination with the phosphoric acid of the double-stranded mixture of small molecules interference RNA-glycosyl skeleton, the impact that this combination is not arranged by the RNA nucleotide base can not be identified mutually with the double-stranded DNA mixture.The αhelix tryptophan residue that is arranged in two C-terminal of P19 dimer protein consists of one skeleton construction each other, can preferentially be combined with the double-stranded mixture of the small molecules interference RNA of 21～22 nucleotide base length, every greater than 22 or less than the double-stranded mixtures of 20 nucleotide base small molecules interference RNAs, greatly weaken with the binding ability of this dimer protein.Because this difference on aspect the 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.Disturb the double-stranded mixture can not depolymerization just because of the RNA small molecules, can not further be combined and make its degraded and silence with the mRNA that transcription of foreign genes forms, final result, the mRNA that foreign gene is transcribed out can keep active state and not by silence and fast degradation always.
The 2A 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, all with the Gly-Pro ending, is 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 generally is not positioned at the impact of 2A polypeptide upstream and downstream aminoacid sequence.Just because of this characteristics, generally the 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 kept most sequences of 2A micromolecule polypeptide, the albumen that is positioned at the downstream has only kept a proline residue at N-terminal, can at utmost keep like this biological activity of downstream albumen.2A protein polypeptide the earliest is at foot and mouth disease virus (foot-and-mouth disease virus, FMDV) find in, after, people find 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 again successively, and their cutting efficiency exists certain difference.
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, 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, such as erythropoietin, Interferon, rabbit, tethelin, monoclonal antibody with can be used as antigen protein that vaccine uses etc., some research institutions and company have begun to obtain huge economic benefit from the production of these pharmaceutical proteins.
Summary of the invention
In order to solve problems of the prior art, the invention provides a kind of can be in transgenic plant the method for high efficient expression rh-bFGF, and the target protein of expressing does not need to carry out purifying and reclaims, and can directly carry out processed and applied to transgenic plant.
In order to realize purpose of the present invention, the present invention at first provides a kind of fusion rotein, described fusion rotein comprises RNA silencing suppressor, 2A polypeptide and human basic fibroblast factor protein, and described human basic fibroblast factor protein is all or part of natural human basic fibroblast factor protein;
In described fusion rotein, described human basic fibroblast factor protein also can be natural human basic fibroblast factor protein and another one inoblast factor protein such as human acidic fibroblast factor protein or another one and has albumen such as human epidermal growth factor's albumen coexpression of different biological function 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, perhaps by the DNA recombinant technology gene just is stitched together before accurate translation, these two kinds of known technologies that technology is this area.
Preferably, described fusion rotein is P19-2A-hbFGF; Wherein, P19 is tomato bushy stunt virus P19 albumen; 2A is foot and mouth disease virus 2A polypeptide; HbFGF is rh-bFGF.
For improving the expression amount of rh-bFGF in vegetable cell, the gene of encoding human Prostatropin albumen need to tomato bushy stunt virus P19 protein gene coexpression so that make foreign gene in red sage root cell not by silence.In addition, for improving the expression amount of rh-bFGF recombinant protein in red sage root cell, the gene of encoding human Prostatropin fusion rotein can be optimized according to the codon of plant-preference, in order to make this gene more stable in vegetable cell, efficient is higher when being transcribed into mRNA and translating into albumen.
Further, the aminoacid sequence of described fusion rotein is shown in SEQ ID No.2.
The present invention also provides the fusion gene of the above-mentioned fusion rotein of encoding.
Further, described fusion gene be following (a) or (b) definition polynucleotide:
(a) polynucleotide of nucleotide sequence shown in SEQ ID No.1;
The polynucleotide of (b) under stringent condition, hybridizing with the DNA of nucleotide sequence shown in the SEQ ID No.1.
Wherein, described stringent condition is 65 ℃, hybridization and wash film in the solution of 0.1 * SSPE or 0.1 * SSC, 0.1%SDS.
The present invention also provides the carrier that contains above-mentioned fusion gene.
Preferably, described carrier is recombinant expression vector.Described recombinant expression vector comprises resistant gene or selection markers gene, also comprise and to handle foreign gene in the promotor of vegetable cell transcription, described promotor can obtain from plant or virus, such as 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 such as ribose bisphosphate carboxylase small subunit (rbcS) or adversity inducible gene such as ethanol dehydrogenase (Adhl) etc.
More preferably, described carrier is recombinant expression vector pBI-P19-2A-hbFGF, and its construction process comprises the steps:
(1) synthetic nucleotide sequence fusion gene shown in SEQ ID NO.1;
(2) fusion gene cloning that step (1) is synthetic obtains carrier pT-P19-2A-hbFGF to the pEASY-T1 carrier;
(3) respectively carrier pT-P19-2A-hbFGF and pBI121 are carried out double digestion with restriction enzyme XbaI and SacI, reclaim fusion gene fragment and pBI121 carrier segments, connect, obtain recombinant expression vector pBI-P19-2A-hbFGF.
The present invention also provides the host cell that contains above-mentioned carrier.
The present invention also provides a kind of method that makes up transgenosis red sage root plant: above-mentioned carrier is transformed in the red sage root explant, make the foreign gene stable integration enter red sage root chromogene group DNA, material after transforming is broken up-takes root through inducing-exercise and the transplanting of transgenic seedling, the screening transgenic rice plant.
The present invention selects the red sage root as the host plant of expressing rh-bFGF, is 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.The modern pharmacological research confirmation, the red sage root has vasodilation, improves the widely biological activity such as microcirculation and antisepsis and anti-inflammation.If realize the expression of rh-bFGF 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 rh-bFGF trauma repair and improve the 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.
Described method for transformation 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.
As preferably, the present invention adopts agrobacterium-mediated transformation to transform.Agrobacterium-mediated transformation is to utilize plasmid vector that specific dna fragmentation is integrated into the plant chromosome genomic dna.The most frequently used method is Ye Panfa, and it is applicable to any plant explants that is easy to the regeneration whole plant.Conversion method for agrobacterium is specially adapted to the conversion that dicotyledons comprises the red sage root.
Contain one section in the Ti-plasmids of Agrobacterium and be called transfer DNA (T-DNA), it can enter in the red sage root cell and be integrated at last in the karyomit(e) of the red sage root.The structure of transfer vector was divided into for two steps, at first be to make up the plasmid that can in intestinal bacteria, copy, this plasmid contains the dna sequence dna of coding for tomato bushy stunt virus P19 albumen-2A polypeptide-human basic fibroblast growth factor gene and handle the plant promoter composition that this dna sequence dna is expressed in the red sage root, the 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.Usually another one selected marker gene (such as anti-kalamycin resistance gene) also is comprised in the left and right sides 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 behind red sage root cells.The second step of vector construction is that plasmid is transferred to the Agrobacterium from intestinal bacteria.This can finish by mode or the direct lead-in mode of DNA 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 red sage root cell at T-DNA and play an important role.
The present invention also provides the rh-bFGF of expressing by in the above-mentioned transgenosis red sage root plant.
The application of the transgenosis red sage root in preparation recombination human basic fibroblast growth factor newtype drug that the present invention also provides aforementioned construction process structure to obtain.
The present invention also provides the application of the described transgenosis red sage root in preparation treatment people performs the operation the medicine of otch, nerve injury, body physical abuse, diabetic foot, ulcer and burn and scald disease.
Further, described be applied as transgenosis red sage root plant that structure is obtained through freezing, dry and grind after, form with pharmaceutically acceptable carrier, be made into preparation.
Described preparation can be pulvis, paste, sprays or other preparation.
At least some organ, tissue or its crude extract that contain the transgenosis red sage root in the described preparation.Can be complete plant, or the part of plant such as fruit, seed, leaf, stem, stem tuber and piece root, or a part of crude extract of plant or be derived from the human basic fibroblast factor recombinant protein of the transgenosis red sage root through Economical Purification and partial purification.Contained recombinant protein is rh-bFGF in the preparation of the present invention, have the biological activitys such as the generation of the people of promotion blood vessel, nerve and tissue injury reparation, the rh-bFGF that produces with native protein or other expression system has identical function.
The transgenosis salviae nutrient body that the present invention proposes to contain human basic fibroblast factor recombinant protein first divides the method that is processed into pulvis, paste, sprays or other preparation, since can carry out wherein contained human basic fibroblast factor recombinant protein composition strict quantitatively, thereby people's each use interval and usage quantity had clear and definite regulation.As approximately containing 2.4ug recombination basic fibroblast cytokine in every gram transgenosis red sage root pulvis, spray the dosage of 0.37mg dry powder (approximately containing 1ng rhbFGF) according to every square centimeter of wound, use every day 1 time, used continuously 7～10 days, just can reach the purpose for the treatment of people burn and scald mouth.
The present invention has following beneficial effect:
(1) height of contained human basic 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 basic fibroblast factor protein in red sage root cell, the present invention is gene and the tomato bushy stunt virus P19 protein gene coexpression of encoding human bFGF albumen, make foreign gene in red sage root cell not by silence.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 basic fibroblast factor gene of co expression greatly improve, and the biological yield that human basic fibroblast factor recombinant protein is accumulated in red sage root cell significantly increases.
(2) the present invention utilizes a 2A peptide sequence, tomato bushy stunt virus P19 albumen and human basic 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 the 2A polypeptide was cut open specifically by the proteolytic enzyme of self in cell, the P19 albumen that discharges and human basic fibroblast factor recombinant protein can keep respectively biological activity separately.It not only can not affect high efficient expression and the production of human basic fibroblast factor protein in the transgenosis red sage root, and the human basic fibroblast factor recombinant protein that discharges after cutting through the intracellular protein enzyme has the biological activity as native protein.
(3) the invention provides the method for utilizing exogenous gene expression silencing suppressors efficient and stably express human basic fibroblast factor recombinant protein in red sage root cell, under native state, have biological activitys such as the generation of promotion people blood vessel, nerve and tissue injury reparation and this recombinant protein is known.In other words, human basic fibroblast factor recombinant protein of the present invention has the biological activitys such as the generation of the people of promotion blood vessel, nerve and tissue injury reparation, has and biological activity like the natural basic fibroblast factor type that is derived from the people.Perhaps more definitely say, human basic fibroblast factor recombinant protein of the present invention is the same with this bFGF albumen that is derived from people or other conventional expression system generation, all has the biological activitys such as the generation of the people of promotion blood vessel, nerve and tissue injury reparation.
(4) the present invention expresses in the transgenosis red sage root and the human basic fibroblast factor recombinant protein produced does not need purifying, the transgenosis red sage root that contains human basic fibroblast factor recombinant protein is processed and applied directly, can directly be processed into pulvis, paste, sprays and other preparation etc., be applied to the perform the operation treatment of the diseases such as otch, nerve injury, body physical abuse, diabetic foot, ulcer and burn and scald of people by the topical administration mode, thereby can greatly reduce production costs, and be easy to use.Therefore, has important clinical value.
(5) adopt method of the present invention, Effective Raise the expression amount of human basic fibroblast factor recombinant protein, and the red sage root itself also has vasodilation, improves the widely biological activity 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 save the cost of human basic fibroblast factor recombinant protein separation and purification, and can bring into play again the cooperative effect of the two simultaneously.
(6) antigen-4 fusion protein gene of P19 albumen-2A-human basic fibroblast factor 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 the rat back scald wound, can promote the growth of surface of a wound granulation tissue and re-epithelialization, the restructuring hbFGF standard protein or the red sage root that significantly are better than bacterial origin are individually dosed.This research is laid a good foundation for the recombination human basic fibroblast cytokine newtype drug of Development of New Generation.
Description of drawings
Fig. 1 is contrast before and after inventor's bFGF gene codon is optimized;
Wherein: the gene order that the N representative is codon optimized and transformation is front; M represents codon optimized and improved gene order; Shaded letter is rear nucleotide base for a change;
Fig. 2 is structure and the composition of P19-2A-hbFGF antigen-4 fusion protein gene of the present invention;
Wherein, gene 5 ' end list underscore place is the XbaI restriction endonuclease sites; Dash area is the kozak sequence; The ripple underscore partly is tomato bushy stunt virus p19 albumen coded sequence; Two line place are foot and mouth disease virus 2A polypeptid coding sequence; There is not underscore partly to be people's bFGF encoding sequence; Gene 3 ' single underscore source is the SacI restriction enzyme site;
Fig. 3 is the clone of P19-2A-hbFGF antigen-4 fusion protein gene of the present invention and the structure of the middle plasmid vector pT-P19-2A-hbFGF of bacterium thereof;
Fig. 4 is the building process of plant expression vector pBI-P19-2A-hbFGF of the present invention; Wherein, GUS represents β-Glucuronidase(glucuronidase) gene; P19-2A-hbFGF representative bFGF antigen-4 fusion protein gene;
Fig. 5 is the contained structure gene of plasmid pBI-P19-2A-hbFGF of the present invention 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 cauliflower mosaic virus 35 S promoter; P19-2A-hbFGF representative bFGF antigen-4 fusion protein gene; RB representation DNA right border sequence; LB representation DNA left margin sequence;
Fig. 6 is that PCR of the present invention detects the gel electrophoresis figure of human basic fibroblast factor fusion protein gene in different transgenosis red sage root strains;
Wherein, M is DNA marker; 1 positive contrast (pcr amplification product take the pBI-P19-2A-hbFGF plasmid DNA as template); 2 are the pcr amplification product take non-transgenic red sage root genomic dna as template; 3～13 are the pcr amplification product take part transgenosis red sage root genomic dna as template;
Fig. 7 is that the Southern blot of the transgenosis red sage root of the present invention detects;
Wherein, 1 positive contrast (the Southern results of hybridization of the pcr amplification product take the pBI-P19-2A-hbFGF plasmid DNA as template); 2 is the Southern results of hybridization of non-transgenic red sage root genomic dna behind XbaI enzyme cutting; 3 are the Southern results of hybridization of non-transgenic red sage root genomic dna after the SacI enzyme is cut; 4 is the Southern results of hybridization of transgenosis red sage root genomic dna behind XbaI enzyme cutting; 5 are the Southern results of hybridization of transgenosis red sage root genomic dna after the SacI enzyme is cut.
Fig. 8 is that the Western blot of the transgenosis red sage root of the present invention detects;
Wherein, M is the 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 the A transferring film; 1 is normal man's bFGF albumen (positive control); 2 is the non-transgenic red sage root; 3～5 is the transgenosis red sage root.
Fig. 9 is the result for the treatment of figure to the rat burn and scald of the transgenosis red sage root of the present invention;
Wherein, 0d, 3d, 7d, 14d and 21d represent respectively the time behind the burn and scald; Upper left is physiology saline control group; Upper right is rhbFGF standard control treatment group (positive control); The lower-left is non-transgenic Treated with Radix Salviae Miltiorrhizae group; The bottom right is transgenosis Treated with Radix Salviae Miltiorrhizae group of the present invention.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Synthetic and the clone of embodiment 1 antigen-4 fusion protein gene
1, the synthetic of P19-2A-hbFGF 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.Be to come from the plant virus genome because this gene is former, be suitable in vegetable cell, efficiently transcribing and translating, therefore, its codon is done 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.
The nucleotide sequence of encoding human Prostatropin, although the people with and plant all belong to eukaryote, but they are still existing certain difference aspect the gene codon preference, and this species diversity might have influence on stability and the high efficient expression of rh-bFGF gene in the transgenosis red sage root.For further improving its expression amount in the transgenosis red sage root, (see the GenBank accession number: E05102) according to the rh-bFGF gene order of having announced, under the prerequisite that does not change its Argine Monohydrochloride sequence, codon according to the plants preference is optimized its gene coded sequence.Full length gene 465bp after the optimization, 154 the amino-acid residue (see figure 1)s of encoding.Sequence optimisation relates to 97 codons, has changed altogether 116 nucleotide bases, but G+C content is by 52% before optimizing, and changes into 36% after the optimization.
Above-mentioned three encoding sequences are from beginning to end, consist of P19-2A-hbFGF antigen-4 fusion protein gene (see figure 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), behind 3 ' end terminator codon TAA of this antigen-4 fusion protein gene, increased restriction enzyme site SacI, synthetic the nucleotide sequence of encoding human Prostatropin antigen-4 fusion protein gene (shown in SEQ ID No.1 in the sequence table), said gene splicing and synthetic work by the luxuriant industry bio tech ltd of Beijing English on behalf of finishing.
2, the clone of P19-2A-hbFGF antigen-4 fusion protein gene
The dna fragmentation of above-mentioned synthetic is directly inserted and is connected in the T site in the pEASY-T1 plasmid (available from the Beijing Quanshijin Biotechnology Co., Ltd), the method that provides according to pEASY-T1 plasmid specification sheets, the a plurality of bacterium mono-clonals of random choose are identified, obtain containing the bacterial clone (see figure 3) of rh-bFGF antigen-4 fusion protein gene plasmid vector pT-P19-2A-hbFGF, then, by nucleotide sequence analysis, determine that coding P19-2A-hbFGF antigen-4 fusion protein gene is correct and complete (dna sequencing is by the luxuriant industry bio tech ltd of Beijing English).
The structure of embodiment 2 transgenosis red sage root plant
1, the structure of plant expression vector pBI-P19-2A-hbFGF
In embodiment 1 described bacterial clone, extract pT-P19-2A-hbFGF plasmid vector DNA, utilize restriction enzyme XbaI and SacI to carry out double digestion, separate target DNA fragment, its orientation is inserted in the former gus gene site of plasmid pBI121 (through XbaI and SacI double digestion), is built into double base plant expression vector pBI-P19-2A-hbFGF(and sees Fig. 4).
Plasmid pBIl21 is available 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 then between gus gene terminator codon and NOS terminator.Select plasmid pBIl21 to be because can be with former gus gene deletion with XbaI and SacI double digestion, and can insert subsequently the new foreign protein genes of another one, 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 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 pBI-P19-2A-hbFGF contains: 1) Liu Suanyan NEOMYCIN SULPHATE synthase gene II(nptII), it offers the vegetable cell kalamycin resistance; 2) P19-2A-hbFGF antigen-4 fusion protein gene and handle the cauliflower mosaic virus 35 S promoter of this gene; 3) T-DNA left and right sides border sequence, it can be transferred to nptII gene and P19-2A-hbFGF antigen-4 fusion protein gene in the red sage root body and be incorporated in the red sage root karyomit(e).The structure of pBI-P19-2A-hbFGF expression vector such as Fig. 5.
2, expression vector pBI-P19-2A-hbFGF is imported Agrobacterium (A.tumefaciens)
The plasmid vector pBI-P19-2A-hbFGF that contains the P19-2A-hbFGF antigen-4 fusion protein gene can transfer in the Agrobacterium LBA4404 bacterial strain by the mode of triparental mating, and this bacterial strain is available from U.S. Clonetech company.Bacterial strain LBA4404 now is widely used, and it is improved agrobacterium strains, contain complete Vir gene, but the T-DNA of self is deleted.The Vir gene can be trans adjusting T-DNA from the transfer of plasmid pBI-P19-2A-hbFGF in the vegetable cell.
Agrobacterium is at 50mL YEP(peptone 10 gram that contains the 25mg/L Streptomycin sulphate, yeast extract 10 grams, and the NaCl5 gram, adding distil water to 1 liter, pH7.0) shaking culture in the substratum is at OD
600nmValue reaches at 0.4～0.7 o'clock, and the centrifugal collection bacterial cell of 4000g does not contain in any antibiotic YEP substratum stand-by at 1mL the agrobatcerium cell Eddy diffusion.Contain the DH5 α (available from U.S. GIBCO company) of expression vector pBI-P19-2A-hbFGF and contain helper plasmid pRK2013(available from U.S. Clonetech company) HB101(available from U.S. Clonetech company) be seeded in respectively and contain shaking culture in the 50mg/L kantlex 50mL LB substratum, at OD
600nmValue reaches at 0.4～0.7 o'clock, and then the centrifugal collection bacterial cell of 4000g is suspended in cell respectively 1mL 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 simultaneously 25mg/L Streptomycin sulphate and 50mg/L kantlex in this substratum, cultivate after 2 days for 28 ℃, the Agrobacterium bacterium colony that contains the pBI-P19-2A-hbFGF plasmid begins to produce.
From Agrobacterium, extract plasmid DNA (Sambrook J and Rusell D W with alkaline lysis, Molecular Cloing:A Laboratory Manual, whether 2001, Cold Springer harbor Laboratory Press) also can detect the pBI-P19-2A-hbFGF plasmid DNA with digestion with restriction enzyme exists.
3, agriculture bacillus mediated Genetic Transformation in Higher Plants
The genetic transformation of the red sage root at first 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 the 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 will contain expression vector pBI-P19-2A-hbFGF 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, use again the MS Eddy diffusion to OD
600nm=0.2～0.4, for subsequent use.
The people's such as blade conversion Main Basis Horsch method (Horsch, etal., A Simple and General Method for Transferring Genes into Plants, Science, 1985,227:1229-1231).Salvia seeds is available from drug germchit station, northwest, Shangluo City, Shaanxi Province.Get full salvia seeds and soak 10min with washing composition, then repeatedly wash to remove washing composition with clear water, seed is placed Bechtop; To the ethanol that fills adding 75% in the seed-bearing bottle, outwell behind the 1min; Add the sterilized water contain 10% clorox, process 15min, during constantly rock, outwell clorox; Rinsed with sterile water 3 times, each 5min; Blot the moisture on the seed and be seeded on the MS solid medium; Put into illumination box and cultivate, 16h illumination/8h is dark, 25 ℃/23 ℃ of temperature.After 2～3 weeks of salvia seeds sowing, choose relatively more healthy and stronger aseptic seedling, with the young leaflet tablet edge that newly grows cut then be cut into 0.5cm * 0.5cm square as the explant that infects, place the culture dish that fills a small amount of sterilized water; Immerse in the above-mentioned Agrobacterium bacterium liquid for preparing shearing explant, infect 5～8min, constantly rock therebetween; Explant after infecting is used the rinsing of MS liquid nutrient medium once, and aseptic filter paper sucks residual liquid; At the common substratum of culture dish (MS minimum medium+2.0mg/L6 ?BA) surface tiling one deck aseptic filter paper, explant is placed on the filter paper, culture dish seals with sealed membrane, places that incubator is dark to be cultivated 3 days, and temperature is 25 ℃; Explant after secretly cultivating is transferred in the 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; Per 2 weeks are changed a division culture medium, until grow resistant buds; By the time during resistant buds length to the 1～2cm left and right sides, resistant buds is downcut, change in the 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 clean hardening in the vermiculite Nutrition Soil (1:1) that the root substratum moves into the bacterium of going out, after the stalwartness of growing, move into the field.
4, PCR detects the integration of P19-2A-hbFGF 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, etal., 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 the mortar liquid nitrogen grinding, in the powder dislocation 20mL centrifuge tube after grinding, then (100mMTris-HCl, pH8.0 contain 1.4M NaCl, 20mM EDTA to add 9mL CTAB extracting solution, the 10mL/L beta-mercaptoethanol, 20g/L CTAB), thermal agitation 1 minute makes it abundant mixing.Temperature was bathed 60 minutes in 65 ℃ of water-baths, during vibration in per 10 minutes once.From water-bath, take out centrifuge tube, placed under the room temperature 4～5 minutes, make it cooling, then add the saturated phenol of isopyknic Tris: chloroform (1:1), the concussion mixing is put into 4 ℃ of whizzer 10000rpm, centrifugal 15min; Collect supernatant liquor in another 20mL centrifuge tube, add again isopyknic chloroform and repeat extracting once, put into 4 ℃ of whizzer 10000rpm, centrifugal 15min; The careful supernatant of drawing does not suck organic phase, adds the Virahol of 0.6 times of volume in the supernatant liquor, the gentle mixing of putting upside down, and room temperature is placed 30min, has cotton-shaped genomic dna to occur in the visible pipe; Put into whizzer 10000rpm, centrifugal 10min carefully outwells supernatant liquor; Collecting precipitation contains 0.2M NaOAc and 2mL76% ethanol contains 10mM NH with 3ml76% ethanol
4OAc respectively washes precipitation once, puts into super clean bench or the ventilation dries up, and adds at last TE(pH8.0) the damping fluid dissolving DNA, 1 μ L RNase A places 1h in 37 ℃ of incubators; DNA concentration is transferred to 1ug/uL, be stored in-20 ℃ for subsequent use.
Get above-mentioned red sage root genomic dna 1ug approximately 1uL as masterplate, add primer P1:5 ＇-ATGGCTGCTGGATCTATTACTACTCTTCCAGC-3 ＇ (shown in SEQ ID NO.3) and each 1uL of primer P2:5 ＇-GAGCTCTTAAGACTTAGCAGACAT TGG-3 ＇ (shown in SEQ ID NO.4), 10xPCR damping fluid 3uL, each 2.5mM of dNTP() 3uL, Taq enzyme 0.5uL is 1.5U approximately, adds water to cumulative volume 30uL.
Response procedures is: 95 ℃ of denaturation 5min, 94 ℃ 30 seconds, 50 ℃ 45 seconds, 72 ℃ 1.5 minutes, carry out altogether 35 circulations, 72 ℃ are extended 10min.After reaction finishes, get 5uL amplification sample and carry out the detection of 1% agarose gel electrophoresis.Detected result as shown in Figure 6, the negative control red sage root (swimming lane 2) is not found specific band, then can amplify 1 approximately specific band of 500bp (swimming lane 3～13) in the swimming lane of transgenosis red sage root plant, this result shows that the rh-bFGF gene has been integrated in the red sage root genomic dna.
5, the Southern of the transgenosis red sage root detects
Utilize the rh-bFGF gene DNA fragment of digoxigenin labeled to make probe (the random primer labelling method is seen the test kit PCR DIG Labeling Mix of Roche company working instructions), the part kalamycin resistance red sage root genomic dna after the pBI-P19-2A-hbFGF carrier is transformed has carried out the Southern hybridization analysis.
The extracting method of transgenosis red sage root leaf DNA as mentioned above.Get 40uL(and contain 40 μ gDNA) red sage root genomic dna, then add respectively 40 μ L10 * buffer enzyme cutting buffering liquid, 10 μ L restriction enzyme XbaI or SacI and carry out single endonuclease digestion, add aseptic redistilled water to cumulative volume 400 μ L, reactant is mixed rear wink from making abundant mixing, whether 37 ℃ of enzymes are cut spend the night (at least more than the 12h), get 10 μ L enzymes and cut product and carry out the electrophoresis detection enzyme and cut complete.Enzyme is cut product to be concentrated, Xiang Guanzhong adds isopyknic chloroform/primary isoamyl alcohol and carries 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, with 75% washing with alcohol twice, put into super clean bench and dry up, add 40 μ L ultrapure waters with resolution of precipitate.
Enzyme is cut product carry out 0.8% agarose gel electrophoresis, the 20V electrophoresis spends the night, stop electrophoresis when tetrabromophenol sulfonphthalein moves to the terminal 2cm of blob of viscose.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), 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, then hybridization solution [the 5XSSC that more renews, 0.1%(w/v) N-Lauroylsarcosine, 0.02%SDS, 1% encapsulant (providing in the test kit)], the dna probe 60ng that adds the digoxigenin labeled of sex change in the 5mL hybridization solution is hybridized, the long 465bp of probe, and it is as template take the pT-P19-2A-hbFGF plasmid DNA, adopt the arbitrarily primed PCR amplification method to prepare, comprised whole encoding sequences of rh-bFGF gene.68 ℃ of hybridization were taken out Hybond membrane after 24 hours in hybridization solution, in 200mL2XSSC, washed film under the room temperature 2 times in the 0.1%SDS damping fluid, then in 100mL0.1XSSC, washed film 2 times for 68 ℃ in the 0.1%SDS damping fluid.According to the method that provides in the test kit (the DIG Nucleic Acid Detection Kit of Roche company), nylon membrane is prior to elution buffer [0.1M Maleic acid, pH7.5 substantially for color reaction; 0.15M NaCl; 3%Tween20(w/v)] middle balance is 5 minutes, and then with 50mL1X sealing damping fluid sealing 30 minutes, DigiTAb to the concentration that adds alkali phosphatase enzyme mark was 75mU/mL, and reaction is 30 minutes under the room temperature, uses elution buffer 2 times, each 15 minutes.With film dislocation 20mL colour developing damping fluid (0.1M Tris-HCl, pH9.5; 0.1M NaCl; 50mM MgCl
2) in balance 5 minutes, the substrate buffer solution that more renews (200uL NBT/BCIP substrate adds 10mL colour developing damping fluid), colour developing is 16 hours under the shading condition, after desired band occurs, the water termination reaction.
The transgenosis red sage root that after plasmid pBI-P19-2A-hbFGF transforms, obtains and Southern results of hybridization such as Fig. 7 of negative control red sage root plant.In transgenosis red sage root swimming lane, two bands that after XbaI and SacI single endonuclease digestion genomic dna and probe are hybridized, obtained varying in size, proof P19-2A-hbFGF external source antigen-4 fusion protein gene successfully is incorporated in the red sage root genome, and the insertion copy number of this gene is two copies.And the genomic dna of the negative control red sage root is not observed any hybridization signal.
6, the Western of the transgenosis red sage root detects
Take by weighing 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/LKH
2PO4, 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 dissolving, 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/LDTT), boils 5min in the boiling water, place 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 the gel is transferred on the nitrocellulose filter of a homalographic.
Plate is put in the film taking-up, will be contacted one with gel phase and face up, add an amount of TBST damping fluid [TBS damping fluid (12.11g/L Tris, 8.775g/L NaCl)+0.05%(v/v) Tween-20] with the film submergence, place on the 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), place and seal 2h on the shaking table.Outwell the sealing damping fluid, use TBST buffer solution for cleaning 3 times, each 10min; Outwell and wash the film damping fluid, with dilution buffer liquid (TBST damping fluid+0.1%BSA) 200 times of hbFGF antibody (available from Antigenix America company) dilutions are added in the plate jog 1h on the shaking table; With TBST buffer solution for cleaning 3 times, each 10min; Outwell the damping fluid of washing film, with dilution buffer liquid with Streptarvidin-HRP(available from Antigenix America company) 2000 times of dilutions add in the plates jog 30min on the shaking table, TBST buffer solution for cleaning 3 times, each 10min; Outwell the damping fluid of washing film, then add DAB nitrite ion (available from Antigenix America company), lucifuge leaves standstill colour developing, after colour developing fully, uses distilled water flushing, termination reaction.Found that, single dark-brown band appears in transgenosis red sage root swimming lane, size is consistent with the hbFGF positive control, approximately 16kD(sees Fig. 8), show that the hbFGF gene expresses in the transgenosis red sage root, but the expression amount of hbFGF recombinant protein exists certain difference because plant is different, and is consistent with the result that following ELISA detects.
7, the mensuration of human fibroblastic growth factor recombinant protein content in the transgenosis red sage root
Measure the content (concrete operation method is seen the Human FGF-basic ELISA Construction Kit of Antigenix America company working instructions) of human fibroblastic growth factor recombinant protein in the transgenosis red sage root blade by enzyme linked immunosorbent assay (ELISA).1) gets approximately 0.02g of the positive 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 Na2HPO4 12H2O, 0.24g/L KH2PO4, pH7.4)+0.1%BSA] grind 12, the centrifugal 5min of 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, then room temperature reaction 2h dries liquid in the hole, and enzyme plate washes 5～7 times with the PBST damping fluid, each 3min; 4) with dilution buffer liquid Biotin Tracer(two is resisted) dilute 200 times, add 100 μ L in every hole, room temperature is placed 30min, then dries liquid in the hole, and enzyme plate washes 5～7 times with the PBST damping fluid, at every turn 3min; 5) Streptarvidin-HRP is carried out 2000 times dilution with dilution buffer liquid, add 100 μ L in every hole, room temperature is placed 30min, dries liquid in the hole, and enzyme plate is with PBST damping fluid flushing 5～7 times, each 3min; 6) with TMB chromogenic substrate A liquid and B liquid balanced mix, every hole adds 100 μ L, room temperature lucifuge reaction 1h; 7) the 650nm wavelength reads absorption photometric value (OD650), or absorbance value (OD450) is read after adding 100 μ L stop buffer (2mol/L H2SO4) termination reactions in every hole under 450nm.
With dilution buffer liquid the hbFGF standard substance that provide in the test kit are diluted 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, the reference standard curve can calculate rh-bFGF recombinant protein content in the testing sample.Survey discovery by the enzyme joint inspection, do not find to contain rh-bFGF recombinant protein (rhbFGF) in the negative control plant, then can detect the Prostatropin recombinant protein in part transgenosis red sage root plant, just content is different.The reference standard curve finds that the high-content of the rhbFGF albumen of the red sage root is about 24ng/mL, is equivalent to every gram fresh weight red sage root blade and contains rhbFGF and be about 240ng.
The effect experiment of the embodiment 3 transgenosis reds sage root
1, the modeling of mouse burn and scald
5 of male SD rats (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) are bought 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 scissors the hair of back part of animal is cut short, and lost hair or feathers with 8% sodium sulphite rat anesthesia with ether, area is 9cm * 8cm, and with the povidone iodine sterilization, physiological saline cleans.Experiment fasting on the same day, etherization fill up a gauze in the belly below of rat and make the back smooth, are placed on the anchor.The desk-top super temperature control scald apparatus of YLS-5Q is connected the 220V power supply, temperature regulation to 80 ℃, pressure transfers to 0, as the pressure of causing injury, scalds time 8s with the own wt of perming (0.5kg), select perming of area 2.5cm2, at 1.5cm place, the middle backbone both sides of distance rat back, each scalds 2 circular surface of a wound up and down after the sterilization, and degree of injury is that dark II degree is (through histopathological examination, injure skin corium, only the residual deep appendages of skin).
2, the healing of mouse burn and scald
4 surface of a wound of 5 rats of modeling success are done following processing: 1) rhbFGF positive controls; 2) hbFGF 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 each administration 100 μ L.
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 for subsequent use; Take 100 μ L physiological saline as blank; With 10mL physiological saline 0.1mg bFGF standard substance (nomenclature of drug: hold up Ji multiple, the two aigret pharmaceutcal corporation, Ltds in Beijing) are diluted to 10ug/mL as positive control.Namely began medication the same day after each group was scalded, and changes dressings every other day once later on 21d after extremely hindering.Open gauze when changing dressings at every turn, use the disinfectant with hydrogen peroxide wound, physiological saline cleans, wound circumference carries out disinfection with Iodophor, with aseptic cotton carrier excessive moisture is blotted, and sample is added in corresponding surface of a wound surface and smoothens with the liquid-transfering gun device, effect 5min, paper using immobilization with adhesive tape after the 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 the transparent pan paper, cut off unnecessary trimming, to weigh, calculate surface of a wound area by following formula, surface of a wound area=7.5cm * 7.5cm * scraps of paper are heavy/after 0.1604g) record is hindered 3,7,14,21d surface of a wound area.All data resultss all represent with ± SD, carry out monofactorial variance analysis with SPSS17.0 statistical analysis software system, significance level P＜0.1 or P＜0.5.
As can be seen from Figure 9, prolong with post burn, respectively form the face area and dwindle gradually, interior all surface of a wound of 3d all have a small amount of exudate after hindering, no significant difference between each group of postoperative l～7d; After surgery 14d and 21d, each treatment group is compared with the physiological saline control group, and the surface of a wound all obviously diminishes, but the effect that hbFGF transgenosis Treated with Radix Salviae Miltiorrhizae group and positive controls are accelerated wound healing more obviously (P＜0.5).In four treatment group, take the result for the treatment of that applies hbFGF transgenosis red sage root blade homogenate supernatant liquor treatment group as best (approximately using the rhbFGF2ng/ wound), its action intensity with apply the effect of rhbFGF standard substance (positive controls is approximately used rhbFGF standard substance 100ng/ wound) close (P〉0.5) (the active ratio of short injury repairing is 1:50).What be worth proposition is that non-transgenic Treated with Radix Salviae Miltiorrhizae group also has certain promoter action to the rat wound healing, but compares with hbFGF transgenosis Treated with Radix Salviae Miltiorrhizae group with positive controls, still exists significant difference (P＜0.1) (seeing Table 1).
Table 1 is respectively organized rat and is hindered rear different time points surface of a wound Area comparison
Annotate: n is the rat number, compares * p＜0.5, #p＜0.1 with the physiological saline group; Compare ▲ p＜0.1 with non-transgenic red sage root group.
Salvia miltiorrhiza is the piece root with part, but in fact all contains the hbFGF recombinant protein in its each tissue and the organ, comprises root, stem, leaf, flower and seed etc.This transgenosis red sage root piece root or all nourishing body through cryodrying or air-dry at a slow speed after, can wear at low temperatures 200～1000 purpose dry powder, after measured, the rh-bFGF recombinant protein that approximately contains 2ug 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 hbFGF recombinant protein can enter human body by wound, reaches thus the generation of promotion people wound site blood vessel and the purpose 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 more is applicable to be applied in the treatment of the wounds such as people's burn and scald, physical abuse or diabetes brothers than pulvis.
Also can add the transdermal agent of trace in the transgenosis red sage root dry powder, 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 the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, 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 (10)
1. fusion rotein, it is characterized in that, described fusion rotein comprises RNA silencing suppressor, 2A polypeptide and human basic fibroblast factor protein, and described human basic fibroblast factor protein is all or part of natural human basic fibroblast factor protein;
Perhaps, described human basic fibroblast factor protein is that natural human basic fibroblast factor protein and another one inoblast factor protein or another one have the albumen coexpression of different biological function or the formation fusion rotein that is stitched together.
2. fusion rotein as claimed in claim 1 is characterized in that, described fusion rotein is P19-2A-hbFGF;
Wherein, P19 is tomato bushy stunt virus P19 albumen; 2A is foot and mouth disease virus 2A polypeptide; HbFGF is rh-bFGF.
3. fusion rotein according to claim 2 is characterized in that, described fusion rotein albumen is:
(1) protein of the composition of the aminoacid sequence shown in the SEQ ID No.2;
(2) aminoacid sequence shown in the SEQ ID No.2 be substituted, lack or add one or several amino acid and have same function by (1) derivative protein.
4. the fusion gene of each described fusion rotein of coding claim 1-3; Preferably, the nucleotide sequence of described gene is shown in SEQ ID NO.1.
5. the carrier that contains the described fusion gene of claim 4.
6. carrier as claimed in claim 5 is characterized in that, described carrier is recombinant expression vector pBI-P19-2A-hbFGF, and its construction process comprises the steps:
(1) synthetic nucleotide sequence fusion gene shown in SEQ ID NO.1;
(2) fusion gene cloning that step (1) is synthetic obtains carrier pT-P19-2A-hbFGF to the pEASY-T1 carrier;
(3) respectively carrier pT-P19-2A-hbFGF and pBI121 are carried out double digestion with restriction enzyme XbaI and SacI, reclaim fusion gene fragment and pBI121 carrier segments, connect, obtain recombinant expression vector pBI-P19-2A-hbFGF.
7. contain the described gene of claim 4 or contain claim 5 or the host cell of 6 described recombinant expression vectors.
8. a method that makes up transgenosis red sage root plant is characterized in that, described method is with the described gene of claim 4 or claim 5 or 6 described recombinant expression vectors conversion red sage root cells, bears the complete transgenosis red sage root from transgenosis red sage root cell again.
9. preparation that contains human basic fibroblast factor recombinant protein, it is characterized in that, described preparation is that Accessory Right requirement 8 described methods make up extraction human basic fibroblast factor recombinant protein in the transgenosis red sage root that obtains, perhaps directly right to use requires 8 described methods to make up organ, tissue or the crude extract of the transgenosis red sage root that obtains, and is processed into described preparation.
10. preparation as claimed in claim 9 is characterized in that, described preparation is pulvis, paste or sprays.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102827315A CN103319609A (en) | 2013-07-05 | 2013-07-05 | Human basic fibroblast growth factor (hbFGF) fusion gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102827315A CN103319609A (en) | 2013-07-05 | 2013-07-05 | Human basic fibroblast growth factor (hbFGF) fusion gene and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103319609A true CN103319609A (en) | 2013-09-25 |
Family
ID=49188690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013102827315A Pending CN103319609A (en) | 2013-07-05 | 2013-07-05 | Human basic fibroblast growth factor (hbFGF) fusion gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103319609A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946656A (en) * | 2015-06-08 | 2015-09-30 | 吉林省农业科学院 | Human-derived bFGF (Basic Fibroblast Growth Factor), tobacco chloroplast expression vector and production method |
| CN111357627A (en) * | 2020-03-16 | 2020-07-03 | 山西省农业科学院棉花研究所 | Method for preparing salvia miltiorrhiza aseptic seedlings |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1448510A (en) * | 2002-04-04 | 2003-10-15 | 北京三元基因工程有限公司 | High-efficiency expression of recombination human basic fibroblast growth factor |
| WO2007135480A1 (en) * | 2006-05-22 | 2007-11-29 | Plant Bioscience Limited | Bipartite system, method and composition for the constitutive and inducible expression of high levels of foreign proteins in plants |
| CN101323859A (en) * | 2007-06-13 | 2008-12-17 | 吉林农大生物反应器工程有限公司 | Construction and transform expression method of human alkaline fibroblast growth factor plant expression vector |
-
2013
- 2013-07-05 CN CN2013102827315A patent/CN103319609A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1448510A (en) * | 2002-04-04 | 2003-10-15 | 北京三元基因工程有限公司 | High-efficiency expression of recombination human basic fibroblast growth factor |
| WO2007135480A1 (en) * | 2006-05-22 | 2007-11-29 | Plant Bioscience Limited | Bipartite system, method and composition for the constitutive and inducible expression of high levels of foreign proteins in plants |
| CN101323859A (en) * | 2007-06-13 | 2008-12-17 | 吉林农大生物反应器工程有限公司 | Construction and transform expression method of human alkaline fibroblast growth factor plant expression vector |
Non-Patent Citations (2)
| Title |
|---|
| 刘德虎: "利用转基因植物生产药用蛋白", 《生物技术通报》 * |
| 王献兵: "植物病毒编码 RNA 沉默抑制子的研究进展", 《中国农业大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946656A (en) * | 2015-06-08 | 2015-09-30 | 吉林省农业科学院 | Human-derived bFGF (Basic Fibroblast Growth Factor), tobacco chloroplast expression vector and production method |
| CN104946656B (en) * | 2015-06-08 | 2018-08-17 | 吉林省农业科学院 | A kind of people source basic fibroblast growth factor, tobacco chloroplast expression vector and production method |
| CN111357627A (en) * | 2020-03-16 | 2020-07-03 | 山西省农业科学院棉花研究所 | Method for preparing salvia miltiorrhiza aseptic seedlings |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107022551B (en) | A kind of regulation arabidopsis seedling stage trophosome is big, early blossoming and the increased corn gene of grain weightZmGRAS37And its application | |
| CN107435047A (en) | In a kind of plant phosphorus signal network Tolerant to low P key gene GmPHR25 and its with application | |
| CN106868023B (en) | Cucumber CsERF004 gene and coding protein and application thereof | |
| CN104488945B (en) | The purposes of insecticidal proteins | |
| CN110117320A (en) | Cotton GhCAL-D07 gene is promoting the application in flowering of plant | |
| CN110283824A (en) | A method of using CsXTH04 gene silencing to improve citrus to canker resistance | |
| CN105063085B (en) | The application of cabbage type rape gene BnMPK3 and its anti-sclerotinia sclerotiorum | |
| CN102851306B (en) | For the production of the transgenic aloe plants of albumen and relevant method thereof | |
| CN101628941B (en) | Bovine lactoferrin antibacterial peptide fusion protein, coding gene and application thereof | |
| CN106480163A (en) | A kind of joint Fructus Mali pumilae callus cell culture and the method for genetic transformation identification Fructus Mali pumilae disease-resistant gene | |
| WO2014089950A1 (en) | Method for producing recombinant human basic fibroblast growth factor from rice seeds | |
| CN103319609A (en) | Human basic fibroblast growth factor (hbFGF) fusion gene and application thereof | |
| CN105296492B (en) | A kind of javanese root knot nematode effector Mj-1-1, GAP-associated protein GAP and its application | |
| CN101768597A (en) | Fusion gene fragment rolB-FGFs and application thereof | |
| CN103911384B (en) | A kind of gene and application thereof of controlling sclerotinia rot of colza | |
| WO2022089340A1 (en) | Method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by using genetically engineered rice | |
| CN103333256B (en) | Human acid fibroblast growth factor fusion protein, and coding gene and applications thereof | |
| CN114015716A (en) | Method for verifying quinoa gene function mediated by virus-induced gene silencing | |
| CN102321665B (en) | Method for expressing human humanin protein in peanut seed | |
| CN107641155A (en) | A kind of method of the recombinant Human Serum Albumin Expression in plant | |
| CN102731646B (en) | Pteromalus puparum venom Kazal serine proteinase inhibitor polypeptide and its application | |
| CN102321163B (en) | Sea island cotton lipid transfer protein and application in fiber improvement thereof | |
| CN113214404A (en) | Epidermal growth factor-lysozyme fusion protein and application thereof | |
| CN103484442B (en) | Nod factor lytic enzyme, its encoding gene and application | |
| Liu et al. | Application of oleosin-flanked keratinocyte growth factor-2 expressed from Arabidopsis thaliana promotes hair follicle growth in mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130925 |