CN102399293A - Recombinant protein specifically bound with fibrin and application thereof - Google Patents
Recombinant protein specifically bound with fibrin and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant protein specifically bound with fibrin and the application thereof. The protein is a fusion protein which is obtained by fusing the following polypeptides a) or b) or c) or d) as fusion partners to an amino terminal of a target protein: a) a polypeptide prepared from an amino acid sequence as shown in the 22-107 positions from an amino terminal of sequence 2 in the sequence table; b) the polypeptide prepared from the amino acid sequence as shown in the 22-109 positions from the amino terminal of sequence 4 in sequence table; c) a polypeptide which is derived from a) and is prepared after replacement and/or deletion and/or addition of one or more amino acids from the amino acid sequence restricted by a) and can be specifically bound with fibrin; d) a polypeptide which is derived from b) and is prepared after replacement and/or deletion and/or addition of one or more amino acids from the amino acid sequence restricted by b) and can be specifically bound with fibrin. The recombinant protein specifically bound with fibrin of the invention can be used for wound restoration and active tissue induction regeneration.
Description
The application is that application number is 200810114495.5, the applying date the dividing an application for the recombinant protein and the application thereof of scleroproein specific combination " a kind of with " that be on 06 03rd, 2008, invention and created name.
Technical field
The present invention relates to a kind of and recombinant protein and application thereof the scleroproein specific combination.
Background technology
Prostatropin (basic fibroblast growth factor; BFGF) be a very strong mitogen; Can promote the cell in multiple mesoderm source to comprise inoblast, vascular endothelial cell and smooth muscle cell proliferation, therefore be expected to be used for carry out therapeutic revascularization and injury repairing.Endogenic bFGF often is not enough to promote fast angiogenesis and injury repairing usually in damage and ischemic process.The injection recombinant bfgf can improve damage and limbs and myocardial ischemia process medium vessels new life and blood circulation; Yet one of restricted reason of use of recombinant bfgf is recombinant bfgf rapid diffusion in vivo at present; Cause the recombinant bfgf accumulation of therapentic part low; Medicine is difficult to continuous action (Rinsch; C., et al. (2001) .Delivery of FGF-2but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia.Gene therapy 8:523-533.).Therefore, adopt novel method just to seem very necessary with site-specific and continuable mode application of bFGF.Targeted therapy or targeted therapy (targeted therapy); It is a kind of very effective medication; This method improves result of treatment through the special interaction of the molecular target of medicine and therapentic part; Reduce spinoff (Sawyers, C (2004) Targeted cancer therapy.Nature 432:294-297.Garrett, C (2005) Targeted therapy:the fast pace of progress.Cancer Control 12:71-72.).What targeted therapy was very crucial a bit is exactly to seek special molecular target.Great majority damages all is accompanied by angiorrhexis, hemorrhage and form blood plasma agglutination thing (plasma clot) subsequently; The staple of this blood plasma agglutination thing is scleroproein (fibrin); Also be staple (Wu, S.C., the Castellino of thrombus; F.J.; And Wong, S.L. (2003) .A fast-acting, modular-structured staphylokinase fusion with Kringle-1from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy.The Journal of biological chemistry 278:18199-18206.).The microtexture of this agglutinator is a kind of porous three-dimensional structure; Be the natural support (Wu of injury repairing; S.C., et al (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis:effects of molecular chaperones and a wall-bound protease on antibody fragment production.Applied and environmental microbiology 68:3261-3269.).
Summary of the invention
The purpose of this invention is to provide a kind of and recombinant protein and application thereof the scleroproein specific combination.
The recombinant protein of provided by the present invention and scleroproein specific combination, be with following a) or b) c) or d) polypeptide be fused to the recombinant protein that the N-terminal of target protein obtains as fusion partners:
A) polypeptide of forming from the aminoacid sequence shown in the 22nd the-the 107th of the N-terminal by sequence in the sequence table 2;
B) polypeptide of forming from the aminoacid sequence shown in the 22nd the-the 109th of the N-terminal by sequence in the sequence table 4;
C) with the aminoacid sequence that a) limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by a) polypeptides derived.
D) with b) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by b) polypeptides derived.
A) and b) polypeptide be respectively Kringle1 and Kringle4 structural domain.
In order to make recombinant protein of the present invention be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 2 or 4 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | |
| FLAG | ||
| 8 | DYKDDDDK | |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned c) but or the recombinant protein synthetic d), also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Above-mentioned c) or the encoding sox of the recombinant protein d) can through with sequence in the sequence table 1 from 5 ' terminal 1-828 position or sequence 3 in the dna sequence dna shown in the 5 ' terminal 1-834 position, lack the codon of one or several amino-acid residue; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
Wherein, said target protein can be the factor of regeneration of ability induced tissue and trauma repair.The factor of regeneration of ability induced tissue and trauma repair comprises FGF (fibroblast growth factor), BMP3 (bone morphogenetic protein 3), PDGF (Thr6 PDGF BB), EGF (Urogastron), TGF (transforming growth factor), VEGF (vascular endothelial growth factor), NGF (NGFF) or NT3/4 (neurotrophic factor 3/4).
Target protein is specially Prostatropin described in the present invention.
For the ease of separate and purifying of the present invention can with the recombinant protein of scleroproein specific combination, the N-terminal of said recombinant protein has also added histidine-tagged; In order to guarantee the correct folding of recombinant protein, also added connection peptides between Kringle1 albumen or Kringle4 albumen and target protein.
The recombinant protein that adds after histidine-tagged and the connection peptides specifically can be following 1) to 4) in any albumen:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;
3) with 1) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by 1) deutero-protein;
4) with 2) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by 2) deutero-protein.
Above-mentioned with 1) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by 1) deutero-protein specifically can be with the polypeptide before histidine-tagged and histidine-tagged lack and can with the scleroproein specific combination by 1) deutero-protein, promptly aminoacid sequence is the protein from the N-terminal 11-276 position of sequence 2.
Above-mentioned with 2) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by 2) deutero-protein specifically can be with the polypeptide before histidine-tagged and histidine-tagged lack and can with the scleroproein specific combination by 2) deutero-protein, promptly aminoacid sequence is the protein from the N-terminal 11-278 position of sequence 4.
Wherein, Sequence 2 is made up of 276 amino acid in the sequence table, is histidine-tagged from the 10th of N-terminal 5-, is Kringle1 albumen from the 107th of N-terminal 22-; From the 276th of N-terminal 123-is bFGF albumen, is connection peptides from the 122nd of N-terminal 108-; Sequence 4 is made up of 278 amino acid in the sequence table; From the 10th of N-terminal 5-is histidine-tagged; From the 109th of N-terminal 22-is Kringle4 albumen, is bFGF albumen from the 278th of N-terminal 125-, is connection peptides from the 124th of N-terminal 110-.
The encoding sox of said recombinant protein also belongs to protection scope of the present invention.
Wherein, 1. the encoding sox of said recombinant protein specifically can be arbitrary described gene in 4.;
1. its nucleotide sequence is a sequence 1 in the sequence table;
2. its nucleotide sequence is a sequence 3 in the sequence table;
3. under stringent condition with the dna fragmentation hybridization that 1. or 2. limits and coding can with the dna molecular of the Prostatropin of scleroproein specific combination;
4. with 1. or gene 2. have the homology more than 90%, and coding can with the dna molecular of the Prostatropin of scleroproein specific combination.
The gene of said step in 4., with 1. or gene 2. the homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, sequence 1 is made up of 828 deoxyribonucleotides in the sequence table, is coding (His) from 5 ' terminal the 14th the-the 30th
6The nucleotide sequence of small peptide from 5 ' terminal the 64th the-the 321st Kringle1 structural domain, from 5 ' terminal the 367th the-the 828th nucleotide sequence for the coding Prostatropin, is a catenation sequence from 5 ' the 366th of terminal 322-; Sequence 3 is made up of 834 deoxyribonucleotides in the sequence table, is coding (His) from 5 ' terminal the 14th the-the 30th
6The nucleotide sequence of small peptide from 5 ' terminal the 64th the-the 327th Kringle4 structural domain, from the nucleotide sequence of 5 ' terminal the 373rd the-the 834th Prostatropin, is a catenation sequence from 5 ' the 372nd of terminal 328-.
The recombinant expression vector, transgenic cell line or the reorganization bacterium that contain said encoding sox also belong to protection scope of the present invention.
Recombinant protein of the present invention can be used to preparation and promotes angiogenesis medicament, promotes angiogenesis as can described recombinant protein being combined the formation mixture with the scleroproein support, this mixture being applied in the case that does not have the native plasma agglutinator.Recombinant protein of the present invention also can be used the promotion angiogenesis separately.
The present invention is the target of scleroproein as the factor of regeneration of ability induced tissue and trauma repair, and scleroproein not only plays an important role in cellularstructure and signal transmission, and the while is specifically expressing in some disease also.For the factor specific combination that enables induced tissue regeneration and trauma repair arrives scleroproein; They and scleroproein land (Kringle1 and Kringle4 structural domain) are merged in the present invention, the recombinant protein that obtains (K1bFGF and K4bFG) can with the scleroproein specific combination.Can rest on therapentic part more enduringly with the recombinant protein of scleroproein specific combination in the cell matrix, and work with the mode that continues to discharge.This method has avoided the independent use can induced tissue regeneration and the low target specificity of the factor of trauma repair, and repeatedly-shortcomings such as administering mode of high dosage, thereby significantly strengthens the effect of therapeutic vascularization.Recombinant protein K1bFGF and K4bFG and the case that also is suitable for not having the natural fiber albumen substrate after the scleroproein of external source combines.The scleroproein of external source also can specific combination K1bFGF and K4bFG form multi-functional reparation system; Can the regenerated signal molecule be provided with fixed point, lasting mode on the one hand, the timbering material of external source also provides three-dimensional space for cell proliferation and tissue regeneration simultaneously.Scleroproein/reorganization the K1bFGF and the K4bFG that make up can significantly strengthen angiogenesis in animal model, promote cell proliferation and tissue regeneration, and improve repairing quality.Therefore recombinant protein K1bFGF and K4bFG combine corresponding cell matrix to be expected to improve the treatment of multiple ischemia diseases, like big area ulcer, limb ischemia and myocardial ischemia.Of the present invention and the recombinant protein scleroproein specific combination can be used for trauma repair and utilize the active mass regeneration induction of scleroproein as support; And for therapeutic angiogenesis practice clinically in the future provides new method.
Description of drawings
Fig. 1 is the structural representation of recombinant protein.(His)
6It is the affinity tag that is used for purifying; Kringle represents K1 and K4, is the scleroproein land; BFGF is the functional zone of mitogen and short blood vessel generating effect etc.
Fig. 2 is expression of recombinant proteins, purifying and evaluation
Among A and the C, 1: molecular weight marker, 2: natural bFGF total protein; 3: natural bFG nutrient solution supernatant; 4: the albumen behind the natural bFG purifying; The 5:K1bFGF total protein; 6:K1bFGF nutrient solution supernatant; Albumen behind the 7:K1bFGF purifying; 8, change the contrast of bare.
Among B and the D, 1: molecular weight marker, 2: natural bFGF total protein; 3: natural bFG nutrient solution supernatant; 4: the albumen behind the natural bFG purifying; The 5:K4bFGF total protein; 6:K4bFGF nutrient solution supernatant; Albumen behind the 7:K4bFGF purifying; 8, change the contrast of bare.
Fig. 3 is that K1bFGF, K4bFGF and bFGF are respectively to human fibroblasts's short growth curve
Fig. 4 is ELISA method tested K 1bFGF, K4bFGF and bFGF and blood plasma agglutination thing and fibrinous binding ability
Fig. 5 promotes veins beneath the skin new life for the 5th day K1bFGF in operation back with K4bFG
(A)PBS；(B)bFGF；(C)K1bFGF；(D)K4bFGF。
Fig. 6 is H&E staining evaluating skin wound healing speed and quality
Fig. 7 is that recombinant protein binding fiber albumen promotes angiogenesis
Embodiment
1) acquisition of Kringle1 and Kringle4
Through the complementary amplification Kringle1 fragment that combines of the complementary sequence of primer K1-1, K1-2, K1-3, K1-4 and K1-5.
The addition sequence of primer is: K1-1, K1-2 are used in amplification for the first time, use K1-3, K1-4 for the second time, use K1-1, K1-5 for the third time.
The sequence of primer K1-1, K1-2, K1-3, K1-4 and K1-5 is following:
K1-1：5’-AGA?GGG?ACG?ATG?TCC?AAA?ACA?AAA?AAT?GGC?ATC?ACC?TGT?CAA?AAATGG?AGT?TCC?ACT?TCT?CCC?CAC?AGA?CCT?AGA?TTC?TCA?CCT-3’；
K1-2：5’-CCT?GCG?GAT?CGT?TGT?CTG?GAT?TCC?TGC?AGT?AGT?TCT?CCT?CCA?GTCCCT?CTG?AGG?GGT?GTG?TAG?CAG?GTG?AGA?ATC?TAG?GTC?TGT-3’；
K1-3：5’-TAT?CTC?TCA?GAG?TGC?AAG?ACT?GGG?AAT?GGA?AAG?AAC?TAC?AGAGGGACG?ATG?TCC?AAA?AC-3’：
K1-4：5’-CAT?ATC?TCT?TTT?CTG?GAT?CAG?TAG?TAT?AGC?ACC?AGG?GCC?CCT?GCG?GAT?CGT?TGT?CTG?GA-3’；
K1-5：5’-TTC?CTC?TTC?ACA?CTC?AAG?AAT?GTC?GCA?GTA?GTC?ATA?TCT?CTT?TTC?TGG?ATC?A-3’。
Through the complementary amplification Kringle4 fragment that combines of the complementary sequence of primer K4-1, K4-2, K4-3, K4-4 and K4-5.
The addition sequence of primer is: K4-1, K4-2 are used in amplification for the first time, use K4-3, K4-4 for the second time, use K4-1, K4-5 for the third time.
The sequence of primer K4-1, K4-2, K4-3, K4-4 and K4-5 is following:
K4-1：5’-GGC?ACA?TCC?TCC?ACC?ACC?ACC?ACA?GGA?AAG?AAG?TGT?CAG?TCT?TGG?TCA?TCT?ATG?ACA?CCA?CAC?CGG?CAC?CAG?AAG?ACC?CCA?GAA-3’；
K4-2：5’-AGG?GGC?CTT?TAT?CGG?CAT?CTG?GAT?TCC?TGC?AGT?AGT?TCA?TTG?TCA?GGC?CAG?CAT?TTG?GGT?AGT?TTT?CTG?GGG?TCT?TCT?GGT?GCC-3’；
K4-3：5’-GTC?CAG?GAC?TGC?TAC?CAT?GGT?GAT?GGA?CAG?AGC?TAC?CGA?GGC?ACA?TCC?TCC?ACC?ACC?AC-3’；
K4-4：5’-AGT?ACT?CCC?ACC?TGA?CGC?TGG?GGT?CTG?TGG?TAA?AAC?ACC?AGG?GGC?CTT?TAT?CGG?CAT?CT-3’；
K4-5：5’-AAC?ACT?CGC?TTC?TGT?TCC?TGA?GCA?TTT?TTT?CAG?GTT?GCA?GTA?CTC?CCA?CCT?GAC?GCT?G-3’。
Kringle1 about recovery and purifying 260bp and the dna fragmentation of Kringle4.
2) make up pET28a-6His-K1bFGF and pET28a-6His-K4bFGF expression vector
With NdeI and KpnI difference double digestion Kringle1 and Kringle4 fragment, behind the recovery purifying, be inserted into pET28a-(His) respectively
6The NdeI of-bFGF expression vector (Novagen) and KpnI restriction enzyme site obtain recombinant expression vector pET28a-6Hi s-K1bFGF and pET28a-6Hi s-K4bFGF.
With NdeI and KpnI difference double digestion pET28a-6His-K1bFGF and pET28a-6His-K4bFGF; Obtain 6His-K1bFGF and 6His-K4bFGF fragment; Be connected in the pMD18-T carrier after the recovery respectively; Obtain pMD18-T-6His-K1bFGF and pMD18-T-6His-K4bFGF carrier, check order respectively, sequencing result shows that the dna fragmentation of 6His-K1bFGF has the nucleotide sequence shown in the sequence 1 in the sequence table; Form by 828 deoxyribonucleotides, be coding (His) from 5 ' terminal the 14th the-the 30th
6The nucleotide sequence of small peptide from 5 ' terminal the 64th the-the 321st Kringle 1 structural domain, from 5 ' terminal the 367th the-the 828th nucleotide sequence for the coding Prostatropin, is a catenation sequence from 5 ' the 366th of terminal 322-.Sequence 1 is an encoding sequence from 5 ' terminal 1-828 position in the sequence table, the albumen shown in the sequence 2 in the code sequence tabulation.Sequence 2 is made up of 276 amino acid in the sequence table; From the 10th of N-terminal 5-is histidine-tagged; From the 107th of N-terminal 22-is Kringle1 albumen, is bFGF albumen from the 276th of N-terminal 123-, is connection peptides from the 122nd of N-terminal 108-.
Dna fragmentation with 6His-K4bFGF checks order according to the method described above; Sequencing result shows; The dna fragmentation of His-K4bFGF has the nucleotide sequence shown in the sequence 3 in the sequence table, is made up of 834 deoxyribonucleotides, is coding (His) from 5 ' terminal the 14th the-the 30th
6The nucleotide sequence of small peptide from 5 ' terminal the 64th the-the 327th Kringle4 structural domain, from the nucleotide sequence of 5 ' terminal the 373rd the-the 834th Prostatropin, is a catenation sequence from 5 ' the 372nd of terminal 328-.Sequence 3 is an encoding sequence from 5 ' terminal 1-834 position in the sequence table, the albumen shown in the sequence 4 in the code sequence tabulation.Sequence 4 is made up of 278 amino acid in the sequence table; From the 10th of N-terminal 5-is histidine-tagged; From the 109th of N-terminal 22-is Kringle4 albumen, is bFGF albumen from the 278th of N-terminal 125-, is connection peptides from the 124th of N-terminal 110-.
Recombinant expression vector pET28a-6His-K1bFGF and pET28a-6His-K4bFGF are changed over to respectively in the e. coli bl21 (D3), and bacterium BL21 (D3)-K1bFGF and BL21 (D3)-K4bFGF obtain recombinating.
3) expression and purification recombinant protein K1bFGF and K4bFGF
Reorganization bacterium BL21 (D3)-K1bFGF and BL21 (D3)-K4bFGF are inoculated in the fresh LB substratum of 10ml respectively; At 37 ℃; 200rpm cultivated about 12 hours, and reorganization bacterium BL21 (the D3)-K1bFGF and BL21 (the D3)-K4bFGF that will cultivate in the ratio of 2% (volume percent) then are inoculated into the fresh LB substratum of 150ml respectively, at 37 ℃; 200rpm cultivated about 3 hours, worked as OD
600Reach 0.6-0.8, add inductor IPTG (IPTG final concentration 1mM) and induce.The centrifugal 10min collecting precipitation of 8000g.With the deposition of the above-mentioned collection of 15mlPBS (containing 0.5M NaCl) dissolving, ultrasonication, centrifugal 30 minutes of 13000g separates supernatant, 0.45um membrane filtration supernatant; With agarose-Ni
2+Post (buying company) purifying in Amersham Biosciences.Carry out SDS-PAGE and Western blot then, detect purity of protein.
Western blot uses anti-his (Sigma) antibody.
Experimental result is as shown in Figure 2, and Fig. 2 A is the SDS-PAGE detected result of K1bFGF, and Fig. 2 C is the Western blot detected result of K1bFGF, shows recombinant protein K1bFGF at expression in escherichia coli, and through separating and purifying acquisition recombinant protein K1bFGF; Fig. 2 B is the SDS-PAGE detected result of K4bFGF, and Fig. 2 D is the Western blot detected result of K4bFGF, shows recombinant protein K4bFGF at expression in escherichia coli, and through separating and purifying acquisition recombinant protein K4bFGF.
The BA of embodiment 2, external checking K1bFGF and K4bFGF
1) recombinant protein mitogen activity identification
The mitogen of testing recombinant protein K1bFGF and K4bFGF with the human fibroblasts is active.
The human fibroblasts is inoculated into 48 orifice plates by 3000/hole; With containing the high sugared DMEM substratum (H-DMEM) of 10% (volume ratio) foetal calf serum (FBS), 1% (mass ratio) glutaminase, 1% (mass ratio) non-essential amino acid, 100U penicillium mould/ml and 100 μ g Streptomycin sulphates/ml; At 37 ℃, 5%CO
2And cultivated 24 hours under the saturated humidity.FBS concentration with substratum after 24 hours is replaced with 2% (volume ratio), and other condition is constant.In each culture hole, add final concentration then successively and be 0,30,120,600,1500 and K1bFGF or the K4bFGF of 3000pmol/L, each concentration is established 4 parallel appearance, continues to cultivate 3 days; In each culture hole, adding final concentration is 1mg/ml MTT (methylthiazoletetrazolium, Sigma company), and 37 ℃ are continued to cultivate; Discard supernatant after 4 hours; After wherein adding the inferior maple (dimethl sulfoxid) of 400 μ l diformazans, treating fully dissolving, under the 492nm ELIASA, read the OD value.Among Fig. 3, ordinate zou is the absolute increased value of OD value, represents the increasing amount of cell density; X-coordinate is the recombinant protein K1bFGF that adds in the substratum or the final concentration (μ M) of K4bFGF, and numerical value is represented with mean+SD.
Experimental result shows that the activity of recombinant protein K1bFGF and K4bFGF and the bFGF of crude form do not have marked difference.
2) test of the binding ability of recombinant protein
Detect recombinant protein and the human fibrin of purifying and the binding ability of rabbit plasma with the ELISA method.
A) growth factor and blood plasma agglutination thing (plasma clot) combines experiment
(1) separate rabbit plasma: from healthy rabbit ear central artery blood sampling 20ml, 3.8% (quality percentage composition) Trisodium Citrate that sucks 1/10 volume at syringe in advance is as antithrombotics.Get the centrifugal 15min of 10ml anti-freezing rabbit blood 100g, collect the upper strata turbid solution as being rich in thrombocyte blood plasma (PRP); Other gets the centrifugal 15min of 10ml anti-freezing rabbit blood 1500g, collects supernatant as not containing thrombocyte blood plasma (PPP), and-80 ℃ frozen;
(2) preparation blood plasma agglutination thing: 4 ℃ of thaw PPP and PRP; Add 3 μ l to the every hole of 96 orifice plates (NUNC company) earlier and concentrate CaCl
2And thrombin (zymoplasm, Roche company) mixture (CaCl
2Final concentration is 30mM, and the thrombin final concentration is 1.0NIH unit/ml), every hole adds 50 μ l PPP or PRP, and room temperature was solidified 2-3 hour, washed 2 times with preceding PBS;
(3) add K1bFGF, bFGF (0,0.1,1,2,3,4 and 5uM) the every hole of 100 μ l/ of different gradients to the blood plasma agglutination thing of above-mentioned preparation, 37 ℃, the centrifugal 2hr of 60rpm;
(4) wash the deposition 4 times in (3) with PBS, add confining liquid (containing mass percent is the PBS solution of 0.1%Tween20 for the 2.5%BSA+ percent by volume), the every hole of 100 μ l/, 37 ℃, the centrifugal 1.5hr of 60rpm;
(5) wash the deposition 4 times in (4) with PBS, add one anti-(anti-hi s, using PBS is dilution in 1: 2000 by volume, Sigma company), the every hole of 100 μ l/, 37 ℃, centrifugal 100 minutes of 60rpm;
(6) wash the deposition 4 times in (5) with PBS, add two anti-(anti-mouse IgG, using PBS is dilution in 1: 10000 by volume, Sigma company), the every hole of 100 μ l/, 37 ℃, the centrifugal 1hr of 60rpm;
(7) wash in (6) deposition 4 times with PBS, add AP chromogenic substrate solution right-oil of mirbane phosphoric acid (P-NPP, Amersham) (2mg/ml), the every hole of 80 μ l/, lucifuge reaction 8.5 minutes adds equal-volume 0.2M NaOH termination reaction.
8) get the solution after the termination reaction among the 100 μ l (7), measure the OD value under the 405nm with ELIASA.
B) growth factor and scleroproein (fibrin) combines experiment
1) the used dry powder human fibrinogen of experiment buys (trade(brand)name Kang Puxin) from Hualan Bio-Engineering Co Ltd., and every milligram of this sample contains 0.125IU Hageman factor I.Be made into the solution of 100 μ g/ml with PBS, add 96 hole enzyme plates by the every hole of 100 μ l/, 4 ℃ of absorption of spending the night;
2) protein solution of absorption that will spend the night next day in the enzyme plate that encapsulates is not outwelled, and washes 3 times with PBS.Add 3% (mass percent) BSA (not containing Tween20) sealing, the every hole of 100 μ l/, 37 ℃, centrifugal 1.5 hours of 60rpm with the PBS preparation;
3) thoroughly wash 2 with PBS) in deposition 4 times after add thrombin solution 1NIH unit/ml (the aseptic double-distilled water preparation contain 30mM CaCl
2), the every hole of 100 μ l/, 37 ℃, centrifugal 2 hours of 60rpm;
4) thoroughly wash 3 with PBS) in deposition 4 times after add bFGF, K1bFGF or the K4bFGF (0,0.2,0.5,1,2,3,4,5 μ M) of different concns, the every hole of 100 μ l/, 37 ℃, centrifugal 2.5 hours of 60rpm;
5) wash 4 with PBS) in deposition 4 times, add one anti-(anti-hi s, using PBS is dilution in 1: 2000 by volume).The every hole of 100 μ l/, 37 ℃, centrifugal 1.5 hours of 60rpm;
6) wash 5 with PBS) in deposition 4 times, add two anti-(anti-mouse IgG, using PBS is dilution in 1: 10000 by volume), the every hole of 100 μ l/, 37 ℃, the centrifugal 1hr of 60rpm;
7) wash 6 with PBS) in deposition 4 times, add AP colour developing liquid P-NPP (2mg/ml), the every hole of 80 μ l/, lucifuge reaction 8.5 minutes adds equal-volume 0.2M NaOH termination reaction.
8) get the solution 100 μ l after the termination reaction among the 100 μ l (7), measure the OD value under the 405nm with ELIASA.
Experimental result is as shown in Figure 4, show the scleroproein binding ability of K1bFGF and K4bFGF to be significantly higher than the bFGF of crude form (Sigma, 106096-93-9).
Among Fig. 4, A be K1bFGF, K4bFGF and bFGF respectively with fibrinous binding curve, B be K1bFGF, K4bFGF and bFGF respectively with the binding curve of blood plasma agglutination thing.X-coordinate is represented the concentration of recombinant protein, and ordinate zou representes to be combined in the relative quantity of the recombinant protein on scleroproein or the blood plasma agglutination thing; " * * " represents p＜0.005; " * * * ", p＜0.001; Data are expressed as mean+SD.
The functional evaluation in animal body of embodiment 3, recombinant protein
1) K1bFGF and K4bFG are in the rat skin Effect Evaluation in the ischemia model at random
Rat skin ischemia model (the model of random skin flap) at random can be used to detect the new life of blood vessel.Owing to have blood plasma agglutination thing (plasma clot) to form in this model, can be applied directly to the situation of observing angiogenesis in this model then to recombinant protein.
Recombinant protein is applied to rat skin, and the method for ischemia model is following at random:
1) animal model: rat " skin lid at random " (random pattern skin flap), male Wistar rat about 180g, is anaesthetized from abdominal injection (i.p.) vetanarcol by the dosage of 40mg/kg body weight; Remove dorsal body setae, alcohol disinfecting; Cut 4 15*15mm at the back
2Be the skin lid on basis with both sides;
2) K1bFGF and the bFGF that respectively 580pmol (being equivalent to the natural bFGF of 10 μ g approximately) are measured are applied to the surface of a wound.The position of each sample is different in different rats, to eliminate position effects;
3) sew up a wound, each open side is stitched a pin;
4) the postoperative rat is fed at cleaning level Animal House;
5) postoperative is 5 days, injects excessive narcotic to rat and puts to death, and whole skin skin of back is separated from ridge alignment both sides cut open the apparent situation of observed and recorded angiogenesis.
Experiment divides four groups: control group, K1bFGF group, K4bFG group and bFGF group.
Control group was handled rat 5 days with PBS; The K1bFGF group was handled rat 5 days with K1bFGF; The K4bFGF group was handled rat 5 days with K4bFGF; The bFGF group was handled rat 5 days with bFGF.Handle 5 rats for every group 5, experiment repetition 3 times.
Experimental result is as shown in Figure 5; Show operation back the 5th day, recombinant protein K1bFGF and K4bFG can significantly strengthen the new life of blood vessel, explain that recombinant protein ability specific combination is on the blood plasma agglutination thing of wound site; Increase the concentration of local factors, thereby promoted the new life of blood vessel.
2) K1bFGF and the K4bFGF Effect Evaluation in the full skin incision model of rat
Full skin incision model (full-skin incision model) is the classical model that is used for estimating wound healing, is applied directly to recombinant protein K1bFGF and K4bFGF in this model, can estimate recombinant protein K1bFGF and the K4bFGF influence to wound healing.
The method that recombinant protein is applied in the full skin incision model is following:
1) animal model: full skin incision (full skin incision), male Wistar rat about 200g, is anaesthetized from abdominal injection (i.p.) vetanarcol by the dosage of 40mg/kg body weight; Remove dorsal body setae, alcohol disinfecting; Cut off the long full skin incision of 4 2cm in exposed area with operating scissors;
2) use K1bFGF, K4bFGF and the bFGF of 300pmol (suitable approximately 5 μ g crude form bFGF) and PBS to handle otch respectively, do not sew up;
3) the postoperative rat is fed at cleaning level Animal House;
4) 1,3,6 weeks after operation, inject excessive narcotic to animal and put to death, be that center line is cut both sides 1.5cm with interior full skin with the otch, again from sample being divided into two with the vertical direction of otch.A copy of it is used for histologic analysis with 4% formaldehyde fixed, and another part also in time carries out the tear strength test with the PBS preservation of precooling.
The tear strength testing method is following:
1) instrument: digital mechanical meaurement appearance (Instron 5848microtester, Japan), useful range and precision are 1000 ± 0.01N;
2) the sample two ends are used the transfer arm of survey meter and the clamps on the fixed arm respectively, transfer arm is with the constant speed tractive skin samples of 10mm/s.Sample is broken required maximum, force by computer record from the otch center line;
3) every group has 5 samples, and the maximum, force of each sample is promptly represented tear strength, is used for statistical analysis.
Experiment divides four groups: control group, K1bFGF group, K4bFGF group and bFGF group.
Control group is handled one week of rat with PBS; The K1bFGF group is handled one week of rat with K1bFGF; The K4bFGF group is handled one week of rat with K4bFGF; The bFGF group is handled one week of rat with bFGF.Handle 5 rats for every group 5, experiment repetition 3 times.
Experimental result is as shown in Figure 6, handles the otch of 1 week back use recombinant protein K1bFGF or K4bFGF, and the speed that its promoting epidermization and granulation tissue form will be higher than control group and use the bFGF group, and there were significant differences for statistical result showed.After handling for 6 weeks, the skin tear strength of recombinant protein K1bFGF or K4bFGF treatment group will be significantly higher than control group and bFGF treatment group, shows that recombinant protein K1bFGF or K4bFGF treatment group repairing quality will be higher than control group and bFGF treatment group.
Among Fig. 6, A-D is that Histological method evaluataion's epidermis newborn (arrow) and granulation (GT) form, and A:PBS handles 1 all epidermises newborn (arrow) and granulation (GT) forms; B:bFGF handles 1 all epidermises newborn (arrow) and granulation (GT) forms; C:K1bFGF handles 1 all epidermises newborn (arrow) and granulation (GT) forms; D:K4bFGF handles 1 all epidermises newborn (arrow) and granulation (GT) forms.Dotted line is represented the otch original boundaries, scale, 100 μ m.E-H is H&E staining analysis collagen deposition and cicatrization, and E:PBS handled for 6 weeks; F:bFGF handled for 6 weeks; G:K1bFGF handled for 6 weeks; H:K4bFGF handled for 6 weeks.Dotted line is represented the scar sideline, scale, 100 μ m.I: quantitative analysis 1 all granulation tissues form.J: quantitative analysis 6 all scar areas, numerical value is represented (n=5) with MV ± standard error.K: tear strength over time, numerical value is represented (n=5) with MV ± standard error, " * " represents p＜0.05.
Above-mentioned experimental result shows that recombinant protein K1bFGF and K4bFGF can significantly quicken wound healing, improve repairing quality.Explain that reorganization K1bFGF and K4bFGF can reach the purpose that promotes angiogenesis with the natural fiber albumen specific combination of site of injury.
3) K1bFGF and K4bFGF combine the effect to revascularization with the scleroproein support
Scleroproein is a kind of good and sophisticated biomaterial, recombinant protein and biomaterial is combined to be transplanted to subcutaneous rat again estimate the influence to revascularization.
Recombinant protein combines the testing method of revascularization influence following with the scleroproein support:
(fibrinogen FN) buys from Hua Lan biotech firm (Henan, Xinxiang) human fibrinogen, wherein contains Hageman factor I, the 0.125U/mg human fibrinogen;
2) dissolve human fibrinogen's (earlier PBS and FN being preheating to 37 ℃ again with the two mixing in water-bath before the dissolving) with PBS.Fibrin gel has FN solution, zymoplasm and CaCl
2Mix cohesion and form, three's final concentration is followed successively by: 50mg/ml, 20IU/ml, 40mM.Elder generation is with spissated zymoplasm and CaCl during operation
2Solution adds in the hole of 48 orifice plates in proportion, gets 200 μ l FN solution (containing the 870pmol growth factor, the bFGF of about 15 μ g crude forms) then and adds in the hand-hole and rapid mixing, places 1 hour in 37 ℃ of thermostat containers.
Experiment divides four groups: control group, scleroproein support/K1bFGF group, scleroproein support/K4bFGF group and scleroproein support/bFGF group.
Control group: with subcutaneous 5 days of scleroproein support/PBS transplanting rat; Scleroproein support/K1bFGF group: with subcutaneous 5 days of scleroproein support/K1bFGF transplanting rat; Scleroproein support/K4bFGF group: with subcutaneous 5 days of scleroproein support/K4bFGF transplanting rat; Scleroproein support/bFGF group: with subcutaneous 5 days of scleroproein support/bFGF transplanting rat.Handle 5 rats for every group 5, experiment repetition 3 times.
Experimental result is as shown in Figure 7; Show and can observe a large amount of new blood vessels that form in recombinant protein K1bFGF and the K4bFGF treatment group, then few in the control group, the blood vessel of new formation is also arranged in the bFGF treatment group; But lack than recombinant protein K1bFGF and K4bFGF treatment group, there were significant differences for statistics.
Among Fig. 7, A: scleroproein support/PBS; B: scleroproein support/bFGF; C: scleroproein support/K1bFGF; D: scleroproein support/K4bFGF; Scale 3mm.
Thereby The above results explanation recombinant protein ability specific combination scleroproein support effectively improves the regeneration of blood vessel.
Claims (10)
1. a recombinant protein is with following b) or polypeptide d) be fused to the fusion rotein that the N-terminal of target protein obtains as fusion partners:
B) polypeptide of forming from the aminoacid sequence shown in the 22nd the-the 109th of the N-terminal by sequence in the sequence table 4;
D) with b) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by b) polypeptides derived.
2. recombinant protein according to claim 1 is characterized in that: said target protein is the factor of regeneration of ability induced tissue and trauma repair.
3. recombinant protein according to claim 1 and 2 is characterized in that: said target protein is fibroblast growth factor, bone morphogenetic protein 3, Thr6 PDGF BB, Urogastron, transforming growth factor, vascular endothelial growth factor, NGFF or neurotrophic factor 3/4.
4. recombinant protein according to claim 3 is characterized in that: said target protein is a Prostatropin.
5. recombinant protein according to claim 4 is characterized in that: said recombinant protein is following 2) or 4) in any albumen:
2) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;
4) with 2) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and can with the scleroproein specific combination by 2) deutero-protein.
6. the encoding sox of arbitrary said recombinant protein in the claim 1 to 5.
7. gene according to claim 6 is characterized in that: the encoding sox of said recombinant protein is 2. arbitrary described gene in 4.;
2. its nucleotide sequence is a sequence 3 in the sequence table;
3. under stringent condition with the dna fragmentation hybridization that 2. limits and coding can with the dna molecular of the Prostatropin of scleroproein specific combination;
4. have the homology more than 90% with 2. gene, and coding can with the dna molecular of the Prostatropin of scleroproein specific combination.
8. the recombinant expression vector, transgenic cell line or the reorganization bacterium that contain claim 6 or 7 said encoding soxs.
9. arbitrary described recombinant protein promotes the application in the angiogenesis medicament in preparation in the claim 1 to 5.
10. arbitrary described recombinant protein combines the mixture that forms in the claim 1 to 5 with the scleroproein support.
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