CN1504573A - Confluent protein capable of self-cracking into polypeptide with antibiosis and reparation function - Google Patents

Abstract

The invention discloses an antibiotic peptide, blood coagulation apoenzyme slicing sequence and fusion protein sequence of phoroblast growth factor, wherein the antibiotic peptide can be bacteria resistant peptide, antimycotic peptide, or genetic engineering hybrid peptide for bacteria and fungus resistant simultaneously, while the phoroblast growth factor can be acidic phoroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF), the nucleic acid sequence for coding the fusion protein can be inserted into appropriate expression carrier and expressed in Pichia pastoris yeast.

Description

A kind of fusion rotein that can be cracked into the antibiotic and repairing effect polypeptide of tool voluntarily

Technical field:

The present invention relates to a kind of fusion rotein of forming by antibacterial peptide, zymoplasm albumen cutting sequence and fibroblast growth factor structure, reach and use.

Background technology:

Antibiotic discovery is a milestone on the medical history, but along with antibiotic a large amount of uses, also make the resistance of pathogenic bacterium improve greatly, even more serious is, the super bacterium that antibiotics resistance now also occurred, nearly all microbiotic does not all have effect to them.Antibacterial peptide then can address these problems.

Up to the present, the human discovery and synthetic a large amount of antibacterial peptide wherein is representative with the insect antimicrobial peptide.Experiment shows that these antibacterial peptides not only have the broad-spectrum antimicrobial ability to bacterium, fungi, and virus, protozoon and tumour cell are also had effect.In addition, because the mechanism of action uniqueness, antibacterial peptide does not have the sick effect of teratogenesis, no cumulative toxicity, the living resistance of difficult labour and to characteristics such as the higher animal normal cell are harmless, and antibacterial peptide may become antibiotic new source.

Insect antimicrobial peptide can be divided into antibacterium peptide, anti-fungus peptide and not only antibacterium but also antimycotic antibacterial peptide.

According to amino acid The Nomenclature Composition and Structure of Complexes feature, the antibacterium peptide of having studied is divided into four classes, promptly forms the antibacterium peptide class of the antibacterium peptide class of amphipathic molecule alpha-helix, the antibacterium peptide class that the intramolecular disulfide bridge is arranged, proline rich and the antibacterium polypeptide class that is rich in glycine.

1. forming the antibacterium peptide class of amphipathic molecule alpha-helix, is representative with cecropin class (Cecropins).In this class antibacterium peptide molecule amphiphilic α-Luo Xuanjiegou is arranged, but do not contain halfcystine, do not have a disulphide bridges.At present, in insect body, more than 20 kind of Cecropin and analogue thereof have been found.Foremost Boman in 1981 etc. at first isolate Cecropin A and B, and next year is isolated Cecropin D again.The antimicrobial spectrum of Cecropin comprises Gram-positive and negative bacterium.

2. the antibacterium peptide class of intramolecular disulfide bridge is arranged, similar to Cecropin, also form amphiphilic α-Luo Xuanjiegou in this class antibacterium peptide molecule, but in this peptide molecule, contain halfcystine, and form the intramolecular disulfide bridge.(insect defensins) is representative with insect defensin.At present, except dipteral insect, all found insect defensin in nearly all order.Insect defensin mainly works to gram-positive microorganism.

3. the antibacterium peptide class of proline rich is representative with apidaecins and abaecin.This is the novel insect antimicrobial peptide of just finding the end of the eighties of a class.Be made up of 15-34 amino-acid residues, wherein proline content is more than 25%.This class antibacterium peptide molecule only works to Gram-negative bacteria.

4. being rich in the antibacterium polypeptide class of glycine, is representative with attacins and sarcotoxins II.There are some researches show that attacins only works to a few species, in-growth Gram-negative bacteria.

Insect anti-fungus peptide: mainly be divided into AFP, holotricin3 and drosomycin.AFP is isolated a kind of anti-fungus peptide from sarcophagid, and rising has stronger sterilizing ability in distilled water and salts solution; Holotricin3 separates from the coleopteron larva and obtains, and is rich in glycine and Histidine; Drosomycin by the larva of fruit bat and adult through the bacteria-induction gained, the tool broad-spectrum antifungal activity.

Not only antibacterium but also antimycotic insect antimicrobial peptide are divided into metchnikowin and thanatin, are famous representative with dead plain (thanatin) wherein.Dead element is that thorn is takeed on the stinkbug adult through inducing a kind of antibacterial peptide of generation, and this antibacterial peptide all has very strong anti-microbial activity to gram-positive microorganism, negative bacterium and fungi.

The structure of antibacterial peptide and function are closely-related.Many antibacterial peptides that come from different plant species, its primary structure has many similarities.N end half point as peptide is rich in hydrophilic amino-acid residue, and the C end then contains more hydrophobic amino acid residue.The design of present novel heterozygous antibacterial peptide mainly concentrates on cecropin (Crocepin), melittin (Melittin) and Magainin (Magainin).There are some researches show that Cecropin A and Magainin all have killing effect to gram-positive microorganism and negative bacterium, normal eukaryotic cell (as people's normal cell) is not then had lethal effect.And melittin is a kind of albumen of sterilization efficiently, and is all better than the above two, but it is harmful to eukaryotic cell.So Song etc. design a kind of hybrid protein according to the primary structure of these several antibacterial peptides, both had strong sterilization effect, harmless to normal eukaryotic cell again, can also kill cancer cell.

At present, more clearly is the mechanism of action of insect antibacterium peptide, comprises that the cytolemma electromotive force relies on the formation of passage, suppresses cellular respiration, suppresses the proteic formation of synthesizing and suppressing cell walls of epicyte.Existing antibacterial peptide sterilization model is by following 3 steps: 1. the polymer of antibacterial peptide and cytolemma attract each other antibacterial peptide are attached on the film; 2. the hydrophobic C end of antibacterial peptide inserts in the film, and the N end of formation amphiphilic alpha-helix is stayed on the membrane interface, and 3. the amphiphilic alpha-helix inserts plasma membrane, forms bigger hole on plasma membrane, thereby makes bacterium death.

Utilize relatively difficulty of gene engineering method mass production insect antimicrobial peptide now, subject matter is: 1. antibacterial peptide extracts purification technique.This is the principal element of restriction antibacterial peptide production cost; 2. expression system carrier.Zoogenous antibacterial peptide is very limited on the source, must express antibacterial peptide by engineered method.Because most of antibacterial peptide all has the anti-effect of killing to expression vector, generally select yeast or make the formal representation of antibacterial peptide with fusion rotein, but such difficulty that has increased post-treatment again; 3. the stability of antibacterial peptide and immune response problem.

Fibroblast growth factor (Fibroblast growth factor, FGFs), have tens kinds, wherein mainly comprise acid fibroblast growth factor (acidic Fibroblast Growth Factor, aFGF) and Prostatropin (bovine FibroblastGrowth Factor, bFGF) two kinds is a class to deriving from mesoderm and neuroectodermal polytype cell has the cell growth factor of biologic activity widely.Wherein, aFGF is with a wide range of applications clinically, can be used for trauma repair, cardiovascular and cerebrovascular diseases (as myocardial ischemia, cerebral apoplexy etc.), nervous system disorders (as Parkinson's disease, alzheimer's disease etc.) treatment etc.

1.aFGF the major physiological function

(1) aFGF can participate in the formation of new vessel directly.AFGF has stronger chemotaxis and short proliferation function to vascular endothelial cell, and stimulate vascular endothelial cell to produce collagenase and plasminogen activator (t-PA), further the degraded basilar membrane induces capillary endothelial cell to form the tube chamber spline structure simultaneously;

(2) propagation of promotion epidermic cell.The vivo and vitro experiment shows, after burn wound is used aFGF, immunohistochemical staining and microscopic examination show, the epidermic cell of aFGF group and the dna content of full thick-layer skin histology are higher than the physiological saline control group, S phase cell count shows that also apparently higher than control group aFGF can promote the mitotic division of epidermic cell, thereby promote the propagation of epidermic cell, quicken the healing of the surface of a wound;

(3) aFGF has tangible hormesis to the differentiation of mesoderm and neuroderm cell.The division of neuroblast is subjected to the adjusting of aFGF, and the Disproportional segregation axon process grows that growing tip, neurotransmitter are synthetic, the neurone features such as transhipment of mediator vesicle.

AFGF and heparin and heparin class sulfuric acid protein-polysaccharide have very strong avidity, have another name called heparin binding growth factor 1.It is to exist with non-folding form under physiological temp under the condition that does not have heparin and heparin class sulfuric acid protein-polysaccharide to exist, and is very unstable.After experiment showed, heparin and heparin class sulfuric acid protein-polysaccharide and HAFGF combining, not only can stablize the molecular configuration of HAFGF, stop its degraded, reduce heat, acid, proteolytic ferment and oxygenant to its active influence, can also strengthen its biologic activity 20-100 doubly, prolong its biological action.

2.aFGF the mutation research of gene and active the detection

Three cysteine residues (Cys) are arranged in the aminoacid sequence of aFGF, lay respectively at the 31st, 98,132, wherein the 31st, 98 Cys is consistent in aFGF and bFGF, and the 132nd Cys is that aFGF is exclusive.Generally all come rock steady structure different with the extracellular protein of many Cys of containing, do not form disulfide linkage between the Cys residue of aFGF by forming disulfide linkage.Replace Cys with neutral amino acids as: Serine (Ser), L-Ala (Ala), not only very little to the influence of the molecular structure of aFGF and biologic activity, can also increase the stability of aFGF to heat, acid and some proteasome degradation.Harper JW etc. once modified some amino-acid residues of AFGF, the active variation of AFGF after research is modified, after finding that the 133rd Methionin (Lys) methylates, the avidity of AFGF and heparin and cell surface receptor, short mitotic activity all descend, show Lys-133 and structure on every side thereof, participate in the interaction of AFGF and heparin and transmembrane receptor.

The present invention can solve the fusion rotein separation and purification and obtain the method for a large amount of antibacterial peptides.Because antibacterial peptide is the small molecule peptide, separation and purification is extremely difficult, and fibroblast growth factor can reclaim by the heparin gel column chromatography purification, therefore, the present invention's (fusion rotein) can reclaim by the heparin gel column chromatography purification, thereby obtains the pure product of fusion rotein.It is to solve the problem that is difficult to obtain a large amount of antibacterial peptides that the present invention also has another advantage.Because antibacterial peptide to expression vector, has lethal effect as intestinal bacteria etc., it is extremely difficult to obtain antibacterial peptide in a large number.And prove that by experiment after antibacterial peptide and fibroblast growth factor merged, its fungicidal activity significantly reduced even disappears, therefore obtaining a large amount of antibacterial peptides by fusion rotein is a good way.In addition, the present invention does not need enzyme to cut or the chemical process crack fusion protein.Because contain the various zymoplasm factors in people and the mammiferous blood, therefore, the present invention's (fusion rotein) can be cut by zymoplasm in use, is cracked into the antibacterial peptide and the fibroblast growth factor of function automatically.

Summary of the invention:

Utilize round pcr to make up the antigen-4 fusion protein gene that coding contains antibacterial peptide, zymoplasm cutting sequence and fibroblast growth factor, be cloned among the pichia spp intermediate carrier pPICZ α A, and be converted into pichia spp (Pichiapastoris).Screening obtains high expression level amount recon from transformant, obtains pure fusion rotein by high density fermentation and heparin gel chromatography.This fusion rotein can be applied to the reparation of the mammiferous wound of treatment, burn, scald and prevent bacterium and fungi infestation.

Embodiment:

Embodiment one: cecropin (cecropin) and acid fibroblast growth factor (acidic fibroblast growth factor) Expression of Fusion Protein

Design and synthesize primers F 1 (SEQ ID NO:22), F2 (SEQ ID NO:23), R1 (SEQ ID NO:24) and R2 (SEQ IDNO:25).Wherein two primers of F1 and R1 contain different DNA restriction enzyme digestion sites, are respectively XhoI and XbaI.And F1 5 ' end has a signal sequence restriction enzyme digestion sites after, in order to guide fusion rotein in host cell secretion and after translation, excise.5 ' end of primers F 2 and R1 3 ' end contains the nucleotide sequence (SEQ ID NO:26) of zymoplasm albumen cleavage site.With two pairs of primers (F1R2 and F2R1) is that template is carried out the PCR reaction with the plasmid that contains cecropin gene and the acidic fibroblast factor (1-154a.a) gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 1 and R1.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/ml Zeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 21KD protein product.

Embodiment two: cecropin and acid fibroblast growth factor disappearance modifier (19-154a.a) Expression of Fusion Protein

Design and synthesize primers F 3 (SEQ ID NO:27), 3 ' end contains the nucleotide sequence of zymoplasm albumen cleavage site for it.With two pairs of primers (F1R2 and F3R1) is that template is carried out the PCR reaction with the plasmid that contains cecropin gene and the acidic fibroblast factor (19-154a.a) gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 1 and R1.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/ml Zeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 19KD protein product.

Embodiment three: cecropin and acid fibroblast growth factor disappearance modifier (27-154a.a) Expression of Fusion Protein

Design and synthesize primers F 4 (SEQ ID NO:28), 3 ' end contains the nucleotide sequence of zymoplasm albumen cleavage site for it.With two pairs of primers (F1R2 and F4R1) is that template is carried out the PCR reaction with the plasmid that contains cecropin gene and the acidic fibroblast factor (27-154a.a) gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 1 and R1.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/ml Zeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 18KD protein product.

Embodiment four: cecropin and acid fibroblast growth factor mutant (aFGF-Ser-2-3) Expression of Fusion Protein

With two pairs of primers (F1R2 and F2R1) is that template is carried out the PCR reaction with the plasmid that contains cecropin gene and acidic fibroblast factor mutant (1-154a.a) gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 1 and R1.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/ml Zeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 18KD protein product.

Embodiment five: cecropin T1249 (Cecropin AD) and acid fibroblast growth factor (1-154a.a) Expression of Fusion Protein

Design and synthesize primer R3 (SEQ ID NO:29), 3 ' end contains the nucleotide sequence of zymoplasm albumen cleavage site for it.With two pairs of primers (F1R3 and F2R1) is that template is carried out the PCR reaction with the plasmid that contains cecropin T1249 gene and the acidic fibroblast factor (1-154a.a) gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 1 and R1.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/ml Zeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 21KD protein product.

Embodiment six: cecropin T1249 (Cecropin AD) and Prostatropin (base fibroblast growth factor) Expression of Fusion Protein

Design and synthesize primers F 5 (SEQ ID NO:29) and R4 (SEQ ID NO:30), wherein 5 of F5 ' end contains the nucleotide sequence of zymoplasm albumen cleavage site, and the 5 ' end of R4 then contains the XbaI enzyme cutting site.With two pairs of primers (F1R3 and F5R4) is that template is carried out the PCR reaction with the plasmid that contains cecropin T1249 gene and basic fibroblast factor gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 1 and R4.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/ml Zeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 22KD protein product.

Embodiment seven: Africa xenopus Magainin (magainin) and Prostatropin Expression of Fusion Protein

Design and synthesize primers F 6 (SEQ ID NO:31) and R5 (SEQ ID NO:32), wherein 5 of F6 ' end contains the XhoI restriction enzyme digestion sites, and the 5 ' end of R5 then contains the nucleotide sequence of zymoplasm albumen cleavage site.With two pairs of primers (F6R5 and F5R4) is that template is carried out the PCR reaction with the plasmid that contains Magainin gene and basic fibroblast factor gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 6 and R4.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/mlZeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 22KD protein product.

Embodiment eight: lopper worm alexin (defensin) and Prostatropin Expression of Fusion Protein

Design and synthesize primers F 7 (SEQ ID NO:33) and R6 (SEQ ID NO:34), wherein 5 of F7 ' end contains the XhoI restriction enzyme digestion sites, and the 5 ' end of R6 then contains the nucleotide sequence of zymoplasm albumen cleavage site.With two pairs of primers (F7R6 and F5R4) is that template is carried out the PCR reaction with the plasmid that contains Magainin gene and basic fibroblast factor gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 7 and R4.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/mlZeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 22KD protein product.

Embodiment nine: thorn shoulder stinkbug dead plain (thanatin) and Prostatropin Expression of Fusion Protein

Design and synthesize primers F 8 (SEQ ID NO:35) and R7 (SEQ ID NO:36), wherein 5 of F8 ' end contains the XhoI restriction enzyme digestion sites, and the 5 ' end of R7 then contains the nucleotide sequence of zymoplasm albumen cleavage site.With two pairs of primers (F8R7 and F5R4) is that template is carried out the PCR reaction with the plasmid that contains Magainin gene and basic fibroblast factor gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 8 and R4.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/mlZeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 22KD protein product.

Embodiment ten: fruit bat anti-fungus peptide (anti-fungal peptide) and Prostatropin Expression of Fusion Protein

Design and synthesize primers F 9 (SEQ ID NO:37) and R8 (SEQ ID NO:38), wherein 5 of F9 ' end contains the XhoI restriction enzyme digestion sites, and the 5 ' end of R8 then contains the nucleotide sequence of zymoplasm albumen cleavage site.With two pairs of primers (F9R8 and F5R4) is that template is carried out the PCR reaction with the plasmid that contains Magainin gene and basic fibroblast factor gene respectively, and the product of acquisition is re-used as template, carries out the PCR reaction with primers F 9 and R4.With final PCR product cloning and order-checking.With XhoI and XbaI double digestion and be connected to simultaneously on the pPICZ α A carrier that produces with XhoI and XbaI digestion.Be transformed in the pichia spp cell by electroporation, in the YPD substratum that contains 100 μ l/mlZeocin resistances, select subsequently.Thereby obtain the clone of stably express fusion rotein.Confirm protein expression level by the Western hybridization assays of aFGF antibody and with SDS-PAGE, it shows about 22KD protein product.

Embodiment 11: the purifying of fusion rotein and cytoactive are measured

The supernatant of fermentation is at first crossed CM-Sepherose FF ion exchange column, carry out gradient elution, collect elute protein solution with the phosphate buffer soln that contains 0.01M-1M sodium-chlor.After the heparin affinity chromatography post, carry out gradient elution with phosphoric acid salt (10Mm-50mM) buffered soln that contains 0.06M-3M sodium-chlor, collect elution peak solution, promptly get the pure product of fusion rotein.People 3T3 tumor cell culture to the logarithmic growth stage, is linked on the 96 porocyte culture plates in the ratio of every hole 7000-8000 cell, adds 1640 substratum that contain 10% calf serum, cultivated 24 hours for 37 ℃.Remove substratum, change to and contain 0.4% 1640 substratum and continue to cultivate 24 hours.Remove substratum, add and contain the different types of deletion mutantion aFGF of different concns albumen, make positive control with the wild-type sample of purifying simultaneously, add PBS solution as blank.Cultivate that every hole adds 20 μ l MTT solution (5mg/ml) after 24-48 hour, continue to cultivate 4-6 hour.Remove substratum, add 100 μ l dimethyl sulfoxide (DMSO) (DMSO) solution, room temperature was placed 0.5 hour, put into microplate reader and measured with A570.

Embodiment 12: the bacteriostatic experiment of fusion rotein

Contain the LB substratum of 1.5% agar powder as base at plate upper berth one deck.After treating that base solidifies, preparation contains the LB substratum of 0.7% agar powder, and equitemperature is reduced to 45 and added the e. coli k12 D31 that is in logarithmic phase when spending, and fully mixing pours in the plate rapidly.After waiting to solidify, in solid medium, punch with punch tool.In the hole, add an amount of fusion rotein then.37 spend night cultivates, with antibacterial peptide, human acid fibroblast growth factor and the sterilized water of same concentrations in contrast.Found that, antibiotic Toplink suppresses the growth of peripheral bacterium, and fusion rotein, human acid fibroblast growth factor and sterilized water all can not suppress the growth of bacterium, this illustrates that this fusion rotein can not suppress the growth of bacterium, can utilize yeast even intestinal bacteria to enlarge production as expression vector.

Embodiment 13: carry out inhibition zone and short cell fission activity experiment with the zymoplasm cleavage of fusion proteins and with cleaved products

(5mM CaCl2 pH7.5), adds the fusion rotein of 20 micrograms for 0.05MHepes, 0.25M NaCl, 37 degree water-baths under buffer condition.Took a sample at 0.5,1.0,6,12,24 hour respectively, and carry out the SDS-PAGE electrophoresis.Surplus sample is urged the active and inhibition zone experiment of cell fission according to method in embodiment 11 and 12.The SDS-PAGE electrophoresis finds that fusion rotein begins after 0.5 hour to decompose in water-bath, and along with the prolongation of time, branch solves obvious more.Inhibition zone experiment showed, that the antibacterial peptide after the decomposition has bacteriostatic activity, but does not have the height of wild-type.Short cell fission activity does not then have significant difference with the human acid fibroblast growth factor of wild-type.

Embodiment 14: fusion rotein is repaired injured mammal skin and antibacterial experiment

Adopt conventional method to make the white rabbit or the white mouse skin damage of normal health,, splash into a certain amount of Pseudomonas aeruginosa, golden staphylococcus or intestinal bacteria etc. as scratch, wound etc., the fusion rotein of Dropwise 5 00～2000ng again, raising 1-10 days, is contrast to add sterilized water, the observation experiment result.Found that the skin that adds fusion rotein began to tie cangue at second day, did not have the sign of infectation of bacteria, control group then has the phenomenon of infectation of bacteria.After adding fusion rotein, the skin that animal is injured was fully recovered in 4-7 days, and control group then needs 6-14 days.

Claims (10)

1. express a kind of fusion rotein, it comprises: (a) antimicrobial peptide protein sequence; (b) zymoplasm albumen cleavage site; (c) fibroblast growth factor protein sequence.

2. the fusion rotein described in the claim 1, wherein said antimicrobial peptide protein sequence, zymoplasm albumen cleavage site and fibroblast growth factor protein sequence are held C end coding successively from the N of fusion rotein.

3. the fusion rotein described in the claim 1, wherein said antimicrobial peptide protein sequence can be antibacterium peptide, anti-fungus peptide or not only antibacterium but also antimycotic antibacterial peptide and their varient.

4. the fusion rotein described in the claim 1, wherein said zymoplasm albumen cleavage site refers to shear the preferential aminoacid sequence of shearing of agent by proteolytic enzyme or other albumen, and useful proteolytic enzyme cutting site comprises the aminoacid sequence shown in SEQ ID NO:1 and the SEQ ID NO:41.

5. the fusion rotein described in the claim 1, wherein said fibroblast growth factor can be acid fibroblast growth factor (comprising the aminoacid sequence shown in the SEQ ID NO:2) or Prostatropin (comprising the aminoacid sequence shown in the SEQ ID NO:3) and their varient (comprising the aminoacid sequence shown in SEQ ID NO:4～20).

6. cutting can be cracked into two kinds of albumen to the fusion rotein described in the claim 1 through different zymoplasm albumen, and two kinds of proteic N ends or C end contain the amino-acid residue of the composition zymoplasm albumen cutting sequence of different quantities respectively.When wherein proteolytic enzyme cut sequence 1 (SEQ IDNO:1), the C of the antimicrobial peptide protein of generation end contained 3 amino acid (KLV), and the proteic N end of fibroblast growth factor also contains 3 amino acid (PRS); And during proteolytic enzyme cutting sequence II (SEQ IDNO:41), the C end of the antimicrobial peptide protein of generation contains 4 amino acid (Ile-Glu/Asp-Gly-Arg), and the proteic N end of fibroblast growth factor does not then contain any amino acid.

7. the term " varient " that is adopted in claim 3 and the claim 5 not only refers to full-length proteins, but also refers to specific varient and allelic variant respectively, and the varient of other natural generation or non-natural generation.For example by the gene engineering method of routine produce amino acid mutation body, indivedual chemistry of amino acids modify mutant and have wild-type protein bioactive fragment etc.They have at least 60% similarly or 50% identical with the protein sequence of antibacterial peptide disclosed herein or the natural generation of fibroblast growth factor, and preferred at least 75% is similar or 55% identical and most preferred 80% similar or 60% identical.

8. the recovery protein process can reclaim whole fusion rotein by heparin sepharose post or molecular sieve; Also can manually add zymoplasm (Mammals or people) cleavage of fusion proteins, thereby obtain to have the antibacterial peptide of anti-microbial activity and the fibroblast growth factor of skin repair function, adopt conventional purification process to reclaim two kinds of albumen respectively at last, for example adopt heparin sepharose post or molecular sieve etc.

9. the present invention's (fusion rotein) composition gives Mammals or people by any suitable administering mode, in order to diseases such as treatment wound, burn, scalds, and can prevent and treat bacterium or fungi infestation.

10. the administering mode described in the claim 11 is for directly giving body surface local organization position; Described composition is meant and comprises the liquid suspension or the solution that can mate on portion water or the physiology.