CN102399293B - 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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- CN102399293B CN102399293B CN2011103226576A CN201110322657A CN102399293B CN 102399293 B CN102399293 B CN 102399293B CN 2011103226576 A CN2011103226576 A CN 2011103226576A CN 201110322657 A CN201110322657 A CN 201110322657A CN 102399293 B CN102399293 B CN 102399293B
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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 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 for carrying 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 the novel method with site-specific and continuable mode application bFGF just to seem very necessary.Targeted therapy or targeted therapy (targeted therapy), it is a kind of very effective medication, the interaction special by the molecular target of medicine and therapentic part of this method improves result for the treatment of, reduce side effect (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 is exactly a bit to find special molecular target.The great majority damage all is accompanied by angiorrhexis, hemorrhage and form subsequently blood plasma agglutination thing (plasma clot), the main component of this blood plasma agglutination thing is scleroproein (fibrin), also the main component (Wu of thrombus, S.C., Castellino, 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 three-dimensional structure that the microtexture of this agglutinator is a kind of porous, the natural scaffold (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 using following a) or b) c) or d) polypeptide be fused to as fusion partners the recombinant protein that the N-terminal of target protein obtains:
A) polypeptide that sequence 2 forms from the aminoacid sequence shown in the 22nd the-the 107th of N-terminal in sequence table;
B) polypeptide that sequence 4 forms from the aminoacid sequence shown in the 22nd the-the 109th of N-terminal in sequence table;
C) by 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) derivative polypeptide.
D) by 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) derivative polypeptide.
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, can connect label as shown in table 1 at N-terminal or the C-terminal of the protein that the aminoacid sequence shown in sequence 2 or 4 forms in sequence table.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned c) but or the recombinant protein synthetic d), also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned c) or the encoding gene of the recombinant protein d) can by by sequence in sequence table 1 from 5 ' end 1-828 position or sequence 3 lack the codon of one or several amino-acid residue in the DNA sequence dna shown in 5 ' end 1-834 position, 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 table 1.
Wherein, described target protein can be the factor of the regeneration of energy induced tissue and trauma repair.The factor of the regeneration of energy 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 (nerve growth factor) or NT3/4 (NT3/4).
Described in the present invention, target protein is specially Prostatropin.
For the ease of separate and purifying of the present invention can with the recombinant protein of scleroproein specific combination, the N-terminal of described recombinant protein has also added histidine-tagged; In order to guarantee the correct folding of recombinant protein, between Kringle1 albumen or Kringle4 albumen and target protein, also added connection peptides.
Add the recombinant protein after histidine-tagged and connection peptides specifically to can be following 1) to 4) in any albumen:
1) protein that the aminoacid sequence shown in sequence 2 forms in sequence table;
2) protein that the aminoacid sequence shown in sequence 4 forms in sequence table;
3) by 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) derivative protein;
4) by 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) derivative protein.
Above-mentioned by 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) derivative protein specifically can be the polypeptide before histidine-tagged and histidine-tagged is lacked and can with the scleroproein specific combination by 1) derivative protein, aminoacid sequence is the protein from the N-terminal 11-276 position of sequence 2.
Above-mentioned by 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) derivative protein specifically can be the polypeptide before histidine-tagged and histidine-tagged is lacked and can with the scleroproein specific combination by 2) derivative protein, aminoacid sequence is the protein from the N-terminal 11-278 position of sequence 4.
Wherein, in sequence table, sequence 2 is comprised of 276 amino acid, from the 10th of N-terminal 5-, is histidine-tagged, from the 107th of N-terminal 22-, is Kringle1 albumen, from the 276th of N-terminal 123-, being bFGF albumen, is connection peptides from the 122nd of N-terminal 108-; In sequence table, sequence 4 is comprised of 278 amino acid, from the 10th of N-terminal 5-, be histidine-tagged, from the 109th of N-terminal 22-, being Kringle4 albumen, is bFGF albumen from the 278th of N-terminal 125-, from the 124th of N-terminal 110-, is connection peptides.
The encoding gene of described recombinant protein also belongs to protection scope of the present invention.
Wherein, 1. the encoding gene of described recombinant protein specifically can be to arbitrary described gene in 4.;
1. its nucleotide sequence is sequence 1 in sequence table;
2. its nucleotide sequence is sequence 3 in sequence table;
3. under stringent condition with the DNA fragmentation hybridization 1. or 2. limited and coding can with the DNA molecular of the Prostatropin of scleroproein specific combination;
4. with 1. or gene 2. there is the homology more than 90%, and coding can with the DNA molecular of the Prostatropin of scleroproein specific combination.
The gene of described step in 4., with 1. or gene 2. the homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be at 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridizes under 65 ℃ and washes film.
Wherein, in sequence table, sequence 1 is comprised of 828 deoxyribonucleotides, from the 14th the-the 30th of 5 ' end, is coding (His)
6The nucleotide sequence of small peptide, from the 64th the-the 321st Kringle1 structural domain of 5 ' end, from the 367th the-the 828th nucleotide sequence for the coding Prostatropin of 5 ' end, is catenation sequence from the 366th of 5 ' end 322-; In sequence table, sequence 3 is comprised of 834 deoxyribonucleotides, from the 14th the-the 30th of 5 ' end, is coding (His)
6The nucleotide sequence of small peptide, from the 64th the-the 327th Kringle4 structural domain of 5 ' end, from the nucleotide sequence of the 373rd the-the 834th Prostatropin of 5 ' end, is catenation sequence from the 372nd of 5 ' end 328-.
The recombinant expression vector that contains described encoding gene, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
Recombinant protein of the present invention can be used to preparation and promotes angiogenesis medicament, as described recombinant protein is combined with the scleroproein support, forms mixture, and this mixture is applied in the case that there is no the native plasma agglutinator and promotes angiogenesis.Recombinant protein of the present invention also can be applied separately the promotion angiogenesis.
The target of the present invention using scleroproein as the factor of the regeneration of energy induced tissue and trauma repair, 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 obtained (K1bFGF and K4bFG) can with the scleroproein specific combination.Can rest on more enduringly therapentic part with the recombinant protein of scleroproein specific combination in cell matrix, and work in the mode of sustained release.This method has been avoided the low target specificity of the factor of independent use energy induced tissue regeneration and trauma repair, and the shortcomings such as administering mode of repeatedly-high dosage, thereby significantly strengthens the effect of therapeutic vascularization.Recombinant protein K1bFGF and K4bFG are combined the rear case that also is suitable for not having the natural fiber albumen substrate with the scleroproein of external source.The scleroproein of external source also can specific combination K1bFGF and K4bFG form multi-functional reparation system, on the one hand can provide the regenerated signal molecule with fixed point, lasting mode, the timbering material of external source also provides three-dimensional space for cell proliferation and tissue regeneration simultaneously.The scleroproein built/restructuring K1bFGF and K4bFG can significantly strengthen angiogenesis in animal model, promote cell proliferation and tissue regeneration, and improve repairing quality.Therefore recombinant protein K1bFGF and K4bFG are expected to improve the treatment of multiple ischemia diseases in conjunction with corresponding cell matrix, as ulcer, limb ischemia and myocardial ischemia.Recombinant protein with the scleroproein specific combination of the present invention can and utilize the active mass regeneration induction of scleroproein as support for trauma repair; And for Therapeutic Angiogenesis practice clinically in the future provides new method.
The accompanying drawing explanation
The structural representation that Fig. 1 is recombinant protein.(His)
6It is the affinity tag for purifying; Kringle, represent 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, Purification and Characterization
In A and C, 1: molecular weight marker, 2: natural bFGF total protein; 3: natural bFG nutrient solution supernatant; 4: the albumen after natural bFG purifying; The 5:K1bFGF total protein; 6:K1bFGF nutrient solution supernatant; Albumen after the 7:K1bFGF purifying; 8, turn the contrast of blank carrier.
In B and D, 1: molecular weight marker, 2: natural bFGF total protein; 3: natural bFG nutrient solution supernatant; 4: the albumen after natural bFG purifying; The 5:K4bFGF total protein; 6:K4bFGF nutrient solution supernatant; Albumen after the 7:K4bFGF purifying; 8, turn the contrast of blank carrier.
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 for operation after the 5th day K1bFGF and K4bFG promote veins beneath the skin new life
(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
Complementary in conjunction with amplification Kringle1 fragment by 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 for the second time K1-3, K1-4, use for the third time K1-1, K1-5.
The sequence of primer K1-1, K1-2, K1-3, K1-4 and K1-5 is as follows:
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’。
Complementary in conjunction with amplification Kringle4 fragment by 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 for the second time K4-3, K4-4, use for the third time K4-1, K4-5.
The sequence of primer K4-1, K4-2, K4-3, K4-4 and K4-5 is as follows:
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’。
Reclaim and the Kringle1 of purifying 260bp left and right and the DNA fragmentation of Kringle4.
2) build pET28a-6His-K1bFGF and pET28a-6His-K4bFGF expression vector
By NdeI and KpnI difference double digestion Kringle1 and Kringle4 fragment, after reclaiming purifying, be inserted into respectively pET28a-(His)
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, after recovery, be connected into respectively in the pMD18-T carrier, obtain pMD18-T-6His-K1bFGF and pMD18-T-6His-K4bFGF carrier, checked order respectively, sequencing result shows that the DNA fragmentation of 6His-K1bFGF has the nucleotide sequence shown in sequence 1 in sequence table, 828 deoxyribonucleotides, consisting of, is coding (His) from the 14th the-the 30th of 5 ' end
6The nucleotide sequence of small peptide, from the 64th the-the 321st Kringle 1 structural domain of 5 ' end, from the 367th the-the 828th nucleotide sequence for the coding Prostatropin of 5 ' end, is catenation sequence from the 366th of 5 ' end 322-.In sequence table, sequence 1 is encoding sequence from 5 ' end 1-828 position, the albumen shown in sequence 2 in the code sequence list.In sequence table, sequence 2 is comprised of 276 amino acid, from the 10th of N-terminal 5-, be histidine-tagged, from the 107th of N-terminal 22-, being Kringle1 albumen, is bFGF albumen from the 276th of N-terminal 123-, from the 122nd of N-terminal 108-, is connection peptides.
According to the method described above the DNA fragmentation of 6His-K4bFGF is checked order, sequencing result shows, the DNA fragmentation of His-K4bFGF has the nucleotide sequence shown in sequence 3 in sequence table, by 834 deoxyribonucleotides, formed, and from the 14th the-the 30th of 5 ' end, be coding (His)
6The nucleotide sequence of small peptide, from the 64th the-the 327th Kringle4 structural domain of 5 ' end, from the nucleotide sequence of the 373rd the-the 834th Prostatropin of 5 ' end, is catenation sequence from the 372nd of 5 ' end 328-.In sequence table, sequence 3 is encoding sequence from 5 ' end 1-834 position, the albumen shown in sequence 4 in the code sequence list.In sequence table, sequence 4 is comprised of 278 amino acid, from the 10th of N-terminal 5-, be histidine-tagged, from the 109th of N-terminal 22-, being Kringle4 albumen, is bFGF albumen from the 278th of N-terminal 125-, from the 124th of N-terminal 110-, is connection peptides.
Recombinant expression vector pET28a-6His-K1bFGF and pET28a-6His-K4bFGF are proceeded to respectively in e. coli bl21 (D3), obtain recombinant bacterium BL21 (D3)-K1bFGF and BL21 (D3)-K4bFGF.
3) expression and purification recombinant protein K1bFGF and K4bFGF
Recombinant bacterium BL21 (D3)-K1bFGF and BL21 (D3)-K4bFGF are inoculated in respectively the LB substratum that 10ml is fresh, at 37 ℃, 200rpm cultivates about 12 hours, then the ratio in 2% (volume percent) is inoculated into respectively the fresh LB substratum of 150ml by the recombinant bacterium BL21 (D3) of cultivation-K1bFGF and BL21 (D3)-K4bFGF, at 37 ℃, 200rpm cultivates about 3 hours, works as OD
600Reach 0.6-0.8, add inductor IPTG (IPTG final concentration 1mM) to induce.The centrifugal 10min collecting precipitation of 8000g.Dissolve the precipitation of above-mentioned collection with 15mlPBS (containing 0.5M NaCl), ultrasonication, centrifugal 30 minutes of 13000g, separate supernatant, 0.45um membrane filtration supernatant; With agarose-Ni
2+Post (buying the company in Amersham Biosciences) purifying.Then carry out SDS-PAGE and Western blot, detect purity of protein.
Western blot is used anti-his (Sigma) antibody.
Experimental result as shown in Figure 2, the SDS-PAGE detected result that Fig. 2 A is K1bFGF, the Western blot detected result that Fig. 2 C is K1bFGF, show that recombinant protein K1bFGF is at expression in escherichia coli, and through separating and purifying acquisition recombinant protein K1bFGF; The SDS-PAGE detected result that Fig. 2 B is K4bFGF, the Western blot detected result that Fig. 2 D is K4bFGF, show that recombinant protein K4bFGF is at expression in escherichia coli, and through separating and purifying acquisition recombinant protein K4bFGF.
The biologic activity of embodiment 2, Validation in vitro K1bFGF and K4bFGF
1) recombinant protein mitogen activity identification
Test the mitogen activity of recombinant protein K1bFGF and K4bFGF with the human fibroblasts.
The human fibroblasts is inoculated into 48 orifice plates by 3000/hole, with containing 10% (volume ratio) foetal calf serum (FBS), 1% (mass ratio) glutaminase, 1% (mass ratio) non-essential amino acid, 100U penicillin/ml and 100 μ g Streptomycin sulphates/ml DMEM in high glucose substratum (H-DMEM), at 37 ℃, 5%CO
2And cultivate 24 hours under saturated humidity.After 24 hours, the FBS concentration of substratum is replaced with to 2% (volume ratio), other condition is constant.Then to adding successively final concentration in each culture hole, be 0,30,120,600,1500 and K1bFGF or the K4bFGF of 3000pmol/L, each concentration is established 4 Duplicate Samples, continue to cultivate 3 days, to adding final concentration in each culture hole, be 1mg/ml MTT (methylthiazoletetrazolium, Sigma company), 37 ℃ are continued to cultivate, discard supernatant after 4 hours, add wherein 400 μ l DMSOs (dimethl sulfoxid), after fully dissolving, read the OD value under the 492nm microplate reader.In Fig. 3, the absolute increased value that ordinate zou is the OD value, represent the increasing amount of cell density; X-coordinate is the recombinant protein K1bFGF that adds in substratum or the final concentration (μ M) of K4bFGF, and numerical value means with mean+SD.
Experimental result shows, the activity of recombinant protein K1bFGF and K4bFGF and the bFGF of natural 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 by the ELISA method.
A) the combination experiment of somatomedin and blood plasma agglutination thing (plasma clot)
(1) separate rabbit plasma: from healthy Rabbit central ear artery blood sampling 20ml, suck 3.8% (quality percentage composition) Trisodium Citrate of 1/10 volume as antithrombotics at syringe in advance.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); Separately get the centrifugal 15min of 10ml anti-freezing rabbit blood 1500g, collect supernatant liquor as not containing thrombocyte blood plasma (PPP) ,-80 ℃ frozen;
(2) prepare the blood plasma agglutination thing: 4 ℃ of thaw PPP and PRP; First to the every hole of 96 orifice plates (NUNC company), add the concentrated CaCl of 3 μ l
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 is solidified 2-3 hour, with front PBS, washes 2 times;
(3) add K1bFGF, bFGF (0,0.1,1,2,3,4 and 5uM) the 100 every holes of μ l/ of different gradients, 37 ℃, the centrifugal 2hr of 60rpm to the blood plasma agglutination thing of above-mentioned preparation;
(4) wash precipitation in (3) 4 times with PBS, add confining liquid (containing mass percent is the PBS solution that the 2.5%BSA+ percent by volume is 0.1%Tween20), the 100 every holes of μ l/, 37 ℃, the centrifugal 1.5hr of 60rpm;
(5) wash precipitation in (4) 4 times with PBS, add primary antibodie (anti-hi s, be dilution in 1: 2000 with PBS by volume, Sigma company), the 100 every holes of μ l/, 37 ℃, centrifugal 100 minutes of 60rpm;
(6) wash precipitation in (5) 4 times with PBS, add two anti-(anti-mouse IgG, are dilution in 1: 10000 with PBS by volume, Sigma company), the 100 every holes of μ l/, 37 ℃, the centrifugal 1hr of 60rpm;
(7) wash in (6) and precipitate 4 times with PBS, add the AP chromogenic substrate p-oil of mirbane phosphoric acid of solution (P-NPP, Amersham) (2mg/ml), the 80 every holes of μ l/, lucifuge reaction 8.5 minutes, add equal-volume 0.2M NaOH termination reaction.
8) get the solution after termination reaction in 100 μ l (7), by microplate reader, measure the OD value under 405nm.
B) the combination experiment of somatomedin and scleroproein (fibrin)
1) test dry powder human fibrinogen used and buy (trade(brand)name Kang Puxin) from Hualan Bio-Engineering Co Ltd., every milligram of this sample is containing the 0.125IU factor XIII.Be made into the solution of 100 μ g/ml with PBS, by the 100 every holes of μ l/, add 96 hole enzyme plates, 4 ℃ of absorption of spending the night;
2) the not protein solution of absorption that will spend the night next day in coated enzyme plate is outwelled, and with PBS, washes 3 times.Add 3% (mass percent) BSA (with the PBS preparation, not containing Tween20) sealing, the 100 every holes of μ l/, 37 ℃, centrifugal 1.5 hours of 60rpm;
3) thoroughly wash 2 with PBS) in precipitation 4 times after add thrombin solution 1NIH unit/ml (the aseptic double-distilled water preparation, containing 30mM CaCl
2), the 100 every holes of μ l/, 37 ℃, centrifugal 2 hours of 60rpm;
4) thoroughly wash 3 with PBS) in precipitation 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 100 every holes of μ l/, 37 ℃, centrifugal 2.5 hours of 60rpm;
5) wash 4 with PBS) in precipitation 4 times, add primary antibodie (anti-hi s is dilution in 1: 2000 with PBS by volume).The 100 every holes of μ l/, 37 ℃, centrifugal 1.5 hours of 60rpm;
6) wash 5 with PBS) in precipitation 4 times, add two anti-(anti-mouse IgG is dilution in 1: 10000 with PBS by volume), the 100 every holes of μ l/, 37 ℃, the centrifugal 1hr of 60rpm;
7) wash 6 with PBS) in precipitation 4 times, add AP nitrite ion P-NPP (2mg/ml), the 80 every holes of μ l/, lucifuge reaction 8.5 minutes, add equal-volume 0.2M NaOH termination reaction.
8) get the solution 100 μ l after termination reaction in 100 μ l (7), by microplate reader, measure the OD value under 405nm.
Experimental result as shown in Figure 4, shows that the scleroproein binding ability of K1bFGF and K4bFGF will be significantly higher than the bFGF (Sigma, 106096-93-9) of natural form.
In 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 represents the concentration of recombinant protein, and ordinate zou means to be combined in the relative quantity of the recombinant protein on scleroproein or 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 the K4bFG Effect Evaluation in the random ischemia model of rat skin
The random ischemia model of rat skin (the model of random skin flap) can be used to detect the new life of blood vessel.Owing to there being blood plasma agglutination thing (plasma clot) to form in this model, can be applied directly to the situation of then observing angiogenesis in this model to recombinant protein.
The method that recombinant protein is applied to the random ischemia model of rat skin is as follows:
1) animal model: rat " random skin lid " (random pattern skin flap), male Wistar rat, the 180g left and right, 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 back
2Take both sides as basic skin lid;
2) respectively K1bFGF and the bFGF of 580pmol (approximately being equivalent to the natural bFGF of 10 μ g) amount are applied to the surface of a wound.The position of each sample is different in different rats, to eliminate the impact of position;
3) sew up a wound, each open side is stitched a pin;
4) postoperative rat is fed at clean level Animal House;
5) 5 days after operation, inject excessive narcotic to rat and put to death, and whole skin skin of back cut open to the apparent situation of observed and recorded angiogenesis from ridge alignment both sides solution.
Experiment divides four groups: control group, K1bFGF group, K4bFG group and bFGF group.
Control group is processed rat 5 days with PBS; The K1bFGF group is processed rat 5 days with K1bFGF; The K4bFGF group is processed rat 5 days with K4bFGF; The bFGF group is processed rat 5 days with bFGF.Process 5 rats for every group 5, experiment repeats 3 times.
Experimental result as shown in Figure 5, show operation latter the 5th day, recombinant protein K1bFGF and K4bFG can significantly strengthen the new life of blood vessel, illustrate that recombinant protein energy 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 for estimating the classical model of wound healing, recombinant protein K1bFGF and K4bFGF are applied directly in this model, can estimate recombinant protein K1bFGF and the K4bFGF impact on wound healing.
The method that recombinant protein is applied in full skin incision model is as follows:
1) animal model: full skin incision (full skin incision), male Wistar rat, the 200g left and right, 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 respectively K1bFGF, K4bFGF and bFGF and the PBS processing otch of 300pmol (approximately suitable 5 μ g natural form bFGF), do not sew up;
3) postoperative rat is fed at clean level Animal House;
4) after operation 1,3,6 week, to inject excessive narcotic to animal and put to death, the otch of take is cut both sides 1.5cm as center line with interior full skin, then from the direction vertical with otch, sample is divided into two.A copy of it is fixed for histologic analysis with 4% formaldehyde, and another part also carries out the tear strength test in time with the PBS preservation of precooling.
The tear strength testing method is as follows:
1) instrument: digital mechanical meaurement instrument (Instron 5848microtester, Japan), useful range and precision are 1000 ± 0.01N;
2) the sample two ends are fixed with the transfer arm of survey meter and the fixture on 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 represents tear strength, for statistical analysis.
Experiment divides four groups: control group, K1bFGF group, K4bFGF group and bFGF group.
Control group is processed rat one week with PBS; The K1bFGF group is processed rat one week with K1bFGF; The K4bFGF group is processed rat one week with K4bFGF; The bFGF group is processed rat one week with bFGF.Process 5 rats for every group 5, experiment repeats 3 times.
Experimental result as shown in Figure 6, is processed the otch that uses recombinant protein K1bFGF or K4bFGF after 1 week, and the speed that its promoting epidermization and granulation tissue form will and be used the bFGF group higher than control group, and there were significant differences for statistical result showed.After processing 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.
In Fig. 6, A-D is that Histological method evaluataion epidermis newborn (arrow) and granulation (GT) form, and A:PBS processes 1 week epidermis newborn (arrow) and granulation (GT) formation; B:bFGF processes 1 week epidermis newborn (arrow) and granulation (GT) formation; C:K1bFGF processes 1 week epidermis newborn (arrow) and granulation (GT) formation; D:K4bFGF processes 1 week epidermis newborn (arrow) and granulation (GT) formation.Dotted line means the otch original boundaries, scale, 100 μ m.E-H is H& E staining analysis collagen deposition and cicatrization, E:PBS processes 6 weeks; F:bFGF processes 6 weeks; G:K1bFGF processes 6 weeks; H:K4bFGF processes 6 weeks.Dotted line means the scar sideline, scale, 100 μ m.I: 1 week granulation tissue of quantitative analysis forms.J: 6 weeks scar areas of quantitative analysis, numerical value means (n=5) with mean value ± standard error.K: over time, numerical value means (n=5) with mean value ± standard error to tear strength, and " * " represents p<0.05.
Above-mentioned experimental result shows that recombinant protein K1bFGF and K4bFGF can significantly accelerate wound healing, improve repairing quality.Illustrate that restructuring K1bFGF and K4bFGF can reach with the natural fiber albumen specific combination of site of injury the purpose that promotes angiogenesis.
3) K1bFGF and K4bFGF are combined the effect to revascularization with the scleroproein support
Scleroproein is a kind of good and ripe biomaterial, and recombinant protein and biomaterial are estimated to the impact on revascularization in conjunction with being transplanted to subcutaneous rat again.
Recombinant protein is combined on the testing method of revascularization impact as follows with the scleroproein support:
Human fibrinogen (fibrinogen, FN) buys from Hua Lan biotech firm (Henan, Xinxiang), wherein contains factor XIII, the 0.125U/mg human fibrinogen;
2) dissolve human fibrinogen's (before dissolving, first PBS and FN being preheating to 37 ℃ again by the two mixing in water-bath) 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.During operation first by concentrated zymoplasm and CaCl
2Solution adds in the hole of 48 orifice plates in proportion, then gets 200 μ l FN solution (containing 870pmol somatomedin, the approximately bFGF of 15 μ g natural forms) and adds in hand-hole and mix rapidly, in 37 ℃ of thermostat containers, places 1 hour.
Experiment divides four groups: control group, scleroproein support/K1bFGF group, scleroproein support/K4bFGF group and scleroproein support/bFGF group.
Control group: by subcutaneous 5 days of scleroproein support/PBS transplanting rat; Scleroproein support/K1bFGF group: by subcutaneous 5 days of scleroproein support/K1bFGF transplanting rat; Scleroproein support/K4bFGF group: by subcutaneous 5 days of scleroproein support/K4bFGF transplanting rat; Scleroproein support/bFGF group: by subcutaneous 5 days of scleroproein support/bFGF transplanting rat.Process 5 rats for every group 5, experiment repeats 3 times.
Experimental result as shown in Figure 7, show in recombinant protein K1bFGF and K4bFGF treatment group to observe a large amount of new blood vessels that form, few in control group, the blood vessel of new formation is also arranged in the bFGF treatment group, but fewer than recombinant protein K1bFGF and K4bFGF treatment group, there were significant differences for statistics.
In 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 energy specific combination scleroproein support effectively improves the regeneration of blood vessel.
Claims (8)
1. a recombinant protein, be the protein that the aminoacid sequence shown in sequence 4 forms in sequence table.
2. the encoding gene of the described recombinant protein of claim 1.
3. gene according to claim 2, it is characterized in that: the nucleotide sequence of the encoding gene of described recombinant protein is sequence 3 in sequence table.
4. the recombinant expression vector that contains claim 2 or 3 described encoding genes.
5. the transgenic cell line that contains claim 2 or 3 described encoding genes.
6. the recombinant bacterium that contains claim 2 or 3 described encoding genes.
7. recombinant protein claimed in claim 1 promotes the application in angiogenesis medicament in preparation.
8. recombinant protein claimed in claim 1 is combined the mixture formed with the scleroproein support.
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| CN1990871A (en) * | 2005-12-30 | 2007-07-04 | 上海新生源医药研究有限公司 | Preparation method of recombinant human plasminogen Kringle 5(hk5) |
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| 抗菌肽Cecropin B—肿瘤血管生长抑制因子Kringle5融合蛋白表达载体的构建及其表达;樊钰虎等;《药物生物技术》;20051231;第12卷(第5期);281-284 * |
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