CN101597335B - Recombined protein specially combined with fiber protein and application thereof - Google Patents
Recombined protein specially combined with fiber protein and application thereof Download PDFInfo
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Abstract
The invention discloses recombined protein specially combined with fiber protein and the application thereof. The recombined protein is fusion protein which is obtained by fusing polypeptide a, b, c or d to the amino terminal of target protein, wherein the polypeptide a is formed by an amino acid sequence which is shown by the 22nd-107th site from the amino terminal in the second amino acid sequence of an amino acid sequence table; the polypeptide b is formed by an amino acid sequence which is shown by the 22nd-109th site from amino terminal in the fourth amino acid sequence of the amino acid sequence table; the polypeptide c is deviated from the polypeptide a, is specially combined with the fiber protein and is formed by substituting and/or deleting one or a plurality of amino acid from the limited amino acid sequence of the polypeptide a and/or adding one or a plurality of amino acid to the limited amino acid sequence of the polypeptide a; and the polypeptide d is deviated from the polypeptide b, is specially combined with the fiber protein and is formed by substituting and/or deleting one or a plurality of amino acid from the limited amino acid sequence of the polypeptide b and/or adding one or a plurality of amino acid to the limited amino acid sequenceof the polypeptide b. The recombined protein specially combined with fiber protein can be used for wound healing and active tissue inducing regeneration.
Description
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 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 in vivo rapid diffusion of recombinant bfgf 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 improvessurvival and vascularization in a model of acute skin flap ischemia.Genetherapy 8:523-533.).Therefore, adopt the novel method of using bFGF with site-specific and continuable mode just to seem very necessary.Targeted therapy or targeted therapy (targeted therapy), it is a kind of very effective medication, this method improves result for the treatment of by the special interaction of the molecular target of medicine and therapentic part, 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 a bit is exactly to seek 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 be the main component (Wu of thrombus, S.C., Castellino, F.J., and Wong, S.L. (2003) .A fast-acting, modular-structuredstaphylokinase fusion with Kringle-1 from human plasminogen as thefibrin-targeting domain offers improved clot lysis efficacy.The Journalof biological chemistry 278:18199-18206.).The microtexture of this agglutinator is a kind of three-dimensional structure of porous, the natural scaffold (Wu of injury repairing, S.C., et al (2002) Functionalproduction and characterization of a fibrin-specific single-chain antibodyfragment from Bacillus subtilis:effects of molecular chaperones and awall-bound protease on antibody fragment production.Applied andenvironmental 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 that is formed from the aminoacid sequence shown in the 22nd the-the 107th of the N-terminal by sequence in the sequence table 2;
B) polypeptide that is formed 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 the polypeptide of a) deriving.
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) polypeptide of deriving.
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, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in the sequence 2 or 4 forms 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) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep- |
8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned c) but or the recombinant protein synthetic d), also can synthesize first its encoding gene, carry out again biological expression and obtain.Above-mentioned c) or the encoding gene of the recombinant protein d) can by 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, 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).
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 described 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 the histidine-tagged and connection peptides specifically can be following 1) to 4) in any albumen:
1) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
2) protein that is formed 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) protein of deriving;
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) protein of deriving.
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) protein of deriving specifically can be the polypeptide before histidine-tagged and histidine-tagged lacked and can with the scleroproein specific combination by 1) protein of deriving, namely 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) protein of deriving specifically can be the polypeptide before histidine-tagged and histidine-tagged lacked and can with the scleroproein specific combination by 2) protein of deriving, namely aminoacid sequence is the protein from the N-terminal 11-278 position of sequence 4.
Wherein, sequence 2 is comprised 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-, being bFGF albumen from the 276th of N-terminal 123-, is connection peptides from the 108 122nd of N-terminal; Sequence 4 is comprised of 278 amino acid in the sequence table, be histidine-tagged from the 10th of N-terminal 5-, being Kringle4 albumen from the 109th of N-terminal 22-, is bFGF albumen from the 278th of N-terminal 125-, is connection peptides from the 124th of N-terminal 110-.
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 arbitrary described gene in 4.;
1. its nucleotide sequence is sequence 1 in the sequence table;
2. its nucleotide sequence is 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 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. homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be at 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1%SDS, hybridizes under 65 ℃ and washes film.
Wherein, sequence 1 is comprised 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 catenation sequence from 5 ' the 366th of terminal 322-; Sequence 3 is comprised of 834 deoxyribonucleotides in the sequence table, is coding (His) from 5 ' terminal the 14th the 30th
6The nucleotide sequence of small peptide from 5 ' terminal the 64th the 327th Kringle4 structural domain, from the nucleotide sequence of 5 ' terminal the 373rd the-the 834th Prostatropin, is catenation sequence from 5 ' the 372nd of terminal 328-.
The recombinant expression vector, transgenic cell line or the recombinant bacterium that contain described encoding gene also belong to protection scope of the present invention.
Recombinant protein of the present invention can be used to preparation and promotes angiogenesis medicament, forms mixture as described recombinant protein being combined with the scleroproein support, this mixture is applied in the case that does not have the native plasma agglutinator promote angiogenesis.Recombinant protein of the present invention also can be used separately the promotion angiogenesis.
The present invention is the target of scleroproein as the factor of the regeneration of energy 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 more enduringly therapentic part with the recombinant protein of scleroproein specific combination in the cell matrix, and work in the mode of sustained release.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 also are suitable for not having the case of natural fiber albumen substrate after the scleroproein of external source is combined.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, the mode that continues, the timbering material of external source also provides three-dimensional space for cell proliferation and tissue regeneration simultaneously.The scleroproein that makes up/restructuring K1bFGF and K4bFG can significantly strengthen angiogenesis in animal model, promote cell proliferation and tissue regeneration, and improve repairing quality.Therefore the corresponding cell matrix of recombinant protein K1bFGF and K4bFG combination is expected to improve the treatment of multiple ischemia diseases, such as ulcer, limb ischemia and myocardial ischemia.Recombinant protein with the scleroproein specific combination of the present invention can be used for trauma repair and utilize scleroproein as active mass's regeneration induction of 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 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, Purification and Characterization
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, turn the contrast of blank carrier.
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, 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 newborn
(A)PBS；(B)bFGF；(C)K1bFGF；(D)K4bFGF。
Fig. 6 is H﹠amp; 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 for the first time amplification, use K1-3, K1-4 for the second time, 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 GCGGAT CGT TGT CTG GA-3’；
K1-5：5’-TTC CTC TTC ACA CTC AAG AAT GTC GCA GTA GTC ATA TCT CTT TTCTGG 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 for the first time amplification, use K4-3, K4-4 for the second time, 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 TGGTCA 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 TCAGGC 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 ACATCC TCC ACC ACC AC-3’；
K4-4：5’-AGT ACT CCC ACC TGA CGC TGG GGT CTG TGG TAA AAC ACC AGG GGCCTT TAT CGG CAT CT-3’；
K4-5：5’-AAC ACT CGC TTC TGT TCC TGA GCA TTT TTT CAG GTT GCA GTA CTCCCA 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 respectively pET28a-(His)
6The NdeI of-bFGF expression vector (Novagen) and KpnI restriction enzyme site obtain recombinant expression vector pET28a-6His-K1bFGF and pET28a-6His-K4bFGF.
With NdeI and KpnI difference double digestion pET28a-6His-K1bFGF and pET28a-6His-K4bFGF, obtain 6His-K1bFGF and 6His-K4bFGF fragment, be connected into respectively in the pMD18-T carrier after the recovery, 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, formed 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 Kringle1 structural domain, from 5 ' terminal the 367th the-the 828th nucleotide sequence for the coding Prostatropin, is catenation sequence from 5 ' the 366th of terminal 322-.Sequence 1 is 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 comprised of 276 amino acid in the sequence table, be histidine-tagged from the 10th of N-terminal 5-, being Kringle1 albumen from the 107th of N-terminal 22-, is bFGF albumen from the 276th of N-terminal 123-, is connection peptides from the 122nd of N-terminal 108-.
Dna fragmentation with 6Hi s-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 comprised 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 catenation sequence from 5 ' the 372nd of terminal 328-.Sequence 3 is 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 comprised of 278 amino acid in the sequence table, be histidine-tagged from the 10th of N-terminal 5-, being Kringle4 albumen from the 109th of N-terminal 22-, 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), 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 fresh LB substratum of 10ml, at 37 ℃, 200rpm cultivated about 12 hours, then in the ratio of 2% (volume percent) the recombinant bacterium BL21 (D3) that cultivates-K1bFGF and BL21 (D3)-K4bFGF are inoculated into respectively the fresh LB substratum of 150ml, 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 precipitation 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 the company in Amersham Biosciences) purifying.Then carry out SDS-PAGE and Western blot, detect purity of protein.
Western blot uses anti-his (Sigma) antibody.
Experimental result as shown in Figure 2, Fig. 2 A is the SDS-PAGE detected result of K1bFGF, 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 biologic activity of embodiment 2, Validation in vitro 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 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 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.Then add successively final concentration in each culture hole and 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, adding final concentration in each culture hole is 1mg/ml MTT (methylthiazoletetrazolium, Sigma company), 37 ℃ are continued to cultivate, discard supernatant after 4 hours, to wherein adding 400 μ l DMSOs (dimethl sulfoxid), after fully dissolving, under the 492nm microplate reader, 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 represents with mean+SD.
Experimental result shows that 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 with the ELISA method.
A) the combination experiment of somatomedin and blood plasma agglutination thing (plasma clot)
(1) separates 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); Other gets the centrifugal 15min of 10ml anti-freezing rabbit blood 1500g, collects supernatant liquor as not containing thrombocyte blood plasma (PPP), and-80 ℃ frozen;
(2) preparation blood plasma agglutination thing: 4 ℃ of thaw PPP and PRP; Add first the concentrated CaCl of 3 μ l to the every hole of 96 orifice plates (NUNC company)
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 front 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 precipitation 4 times in (3) with PBS, add confining liquid (containing mass percent is that the 2.5%BSA+ percent by volume is the PBS solution of 0.1%Tween20), the every hole of 100 μ l/, 37 ℃, the centrifugal 1.5hr of 60rpm;
(5) wash precipitation 4 times in (4) with PBS, add primary antibodie (anti-his is dilution in 1: 2000 with PBS by volume, Sigma company), the every hole of 100 μ l/, 37 ℃, centrifugal 100 minutes of 60rpm;
(6) wash precipitation 4 times in (5) with PBS, add two anti-(anti-mouse IgG is dilution in 1: 10000 with PBS by volume, Sigma company), the every hole of 100 μ l/, 37 ℃, the centrifugal 1hr of 60rpm;
(7) wash (6) middle precipitation 4 times with PBS, add the AP chromogenic substrate p-oil of mirbane phosphoric acid of solution (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), with the OD value under the microplate reader mensuration 405nm.
B) the combination experiment of somatomedin and scleroproein (fibrin)
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 the 0.125IU factor XIII.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 coated enzyme plate is not outwelled, and washes 3 times with PBS.Add 3% (mass percent) BSA (not containing Tween20 with the PBS preparation) sealing, the every hole of 100 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 contain 30mM CaCl
2), the every hole of 100 μ l/, 37 ℃, centrifugal 2 hours of 60rpm;
4) thoroughly wash 3 with PBS) in precipitation add afterwards 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 for 4 times;
5) wash 4 with PBS) in precipitation 4 times, add primary antibodie (anti-his is dilution in 1: 2000 with PBS by volume).The every hole of 100 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 every hole of 100 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 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), with the OD value under the microplate reader mensuration 405nm.
Experimental result shows that the scleroproein binding ability of K1bFGF and K4bFGF will be significantly higher than the bFGF (Sigma, 106096-93-9) of natural form as shown in Figure 4.
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 represents the concentration of recombinant protein, and ordinate zou represents 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 at random ischemia model (the model of random skin flap) 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 then observing angiogenesis in this model to recombinant protein.
Recombinant protein is applied to rat skin, and the method for ischemia model is as follows at random:
1) animal model: rat " at random skin lid " (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
2The skin lid that take both sides as the basis;
2) K1bFGF and the bFGF that respectively 580pmol (being equivalent to approximately the natural bFGF of 10 μ g) are measured 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) the postoperative rat is fed at cleaning level Animal House;
5) 5 days after operation is injected excessive narcotic to rat and is put to death, and cuts whole skin skin of back the apparent situation of observed and recorded angiogenesis open from ridge alignment both sides solution.
Experiment divides four groups: control group, K1bFGF group, K4bFG group and bFGF group.
Control group was processed rat 5 days with PBS; The K1bFGF group was processed rat 5 days with K1bFGF; The K4bFGF group was processed rat 5 days with K4bFGF; The bFGF group was processed rat 5 days with bFGF.Process 5 rats for every group 5, experiment repeats 3 times.
Experimental result as shown in Figure 5, showed operation rear 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 the classical model of estimating wound healing, recombinant protein K1bFGF and K4bFGF are applied directly in this model, can estimate recombinant protein K1bFGF and K4bFGF to the impact of wound healing.
The method that recombinant protein is applied in the full skin incision model is as follows:
1) animal model: full skin incision (full skin inci sion), 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 respectively K1bFGF, the K4bFGF of 300pmol (suitable approximately 5 μ g natural form bFGF) and bFGF and PBS to process otch, 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, take otch as center line both sides 1.5cm is cut with interior full skin, from the direction vertical with otch sample is divided into two again.A copy of it is fixed for histologic analysis with 4% formaldehyde, and another part also in time carries out the tear strength test with the PBS preservation of precooling.
The tear strength testing method is as follows:
1) instrument: digital mechanical meaurement instrument (Instron 5848 microtester, 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 anchor 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 namely represents tear strength, is used for statistical analysis.
Experiment divides four groups: control group, K1bFGF group, K4bFGF group and bFGF group.
Control group is processed one week of rat with PBS; The K1bFGF group is processed one week of rat with K1bFGF; The K4bFGF group is processed one week of rat with K4bFGF; The bFGF group is processed one week of rat with bFGF.Process 5 rats for every group 5, experiment repeats 3 times.
Experimental result is processed the otch of the rear recombinant protein K1bFGF of use of 1 week or K4bFGF as shown in Figure 6, and the speed that its promoting epidermization and granulation tissue form will be higher than control group and use bFGF to organize, and there were significant differences for statistical result showed.After processing 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 processes 1 all epidermises newborn (arrow) and granulation (GT) forms; B:bFGF processes 1 all epidermises newborn (arrow) and granulation (GT) forms; C:K1bFGF processes 1 all epidermises newborn (arrow) and granulation (GT) forms; D:K4bFGF processes 1 all epidermises newborn (arrow) and granulation (GT) forms.Dotted line represents the otch original boundaries, scale, 100 μ m.E-H is H﹠amp; E staining analysis collagen deposition and cicatrization, E:PBS processed for 6 weeks; F:bFGF processed for 6 weeks; G:K1bFGF processed for 6 weeks; H:K4bFGF processed for 6 weeks.Dotted line represents the scar sideline, scale, 100 μ m.I: quantitative analysis 1 all granulation tissues form.J: quantitative analysis 6 all scar areas, numerical value represents (n=5) with mean value ± standard error.K: tear strength over time, numerical value represents (n=5) with mean value ± standard error, " * " 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 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 (first 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.During operation first with concentrated zymoplasm and CaCl
2Solution adds in the hole of 48 orifice plates in proportion, then gets 200 μ l FN solution (containing the 870pmol somatomedin, the bFGF of about 15 μ g natural forms) 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.Process 5 rats for every group 5, experiment repeats 3 times.
Experimental result 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 energy specific combination scleroproein support effectively improves the regeneration of blood vessel.
Sequence table
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉a kind of recombinant protein of and scleroproein specific combination
<130>CGGNARW81369
<160>4
<210>1
<211>828
<212>DNA
＜213〉Genus Homo people (homo sapiens)
<400>1
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgtatctct cagagtgcaa gactgggaat ggaaagaact acagagggac gatgtccaaa 120
acaaaaaatg gcatcacctg tcaaaaatgg agttccactt ctccccacag acctagattc 180
tcacctgcta cacacccctc agagggactg gaggagaact actgcaggaa tccagacaac 240
gatccgcagg ggccctggtg ctatactact gatccagaaa agagatatga ctactgcgac 300
attcttgagt gtgaagagga aggtaccggt agcgcgggca gtgctgcggg ttctggcggt 360
gtcgacgcag ccgggagcat caccacgctg cccgccttgc ccgaggatgg cggcagcggc 420
gccttcccgc ccggccactt caaggacccc aagcggctgt actgcaaaaa cgggggcttc 480
ttcctgcgca tccaccccga cggccgagtt gacggggtcc gggagaagag cgaccctcac 540
atcaagctac aacttcaagc agaagagaga ggagttgtgt ctatcaaagg agtgtgtgct 600
aaccgttacc tggctatgaa ggaagatgga agattactgg cttctaaatg tgttacggat 660
gagtgtttct tttttgaacg attggaatct aataactaca atacttaccg gtcaaggaaa 720
tacaccagtt ggtatgtggc actgaaacga actgggcagt ataaacttgg atccaaaaca 780
ggacctgggc agaaagctat actttttctt ccaatgtctg ctaagagc 828
<210>2
<211>276
<212>PRT
＜213〉Genus Homo people (homo sapiens)
<400>2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys
20 25 30
Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln
35 40 45
Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr
50 55 60
His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn
65 70 75 80
Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr
85 90 95
Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Gly Thr Gly Ser Ala
100 105 110
Gly Ser Ala Ala Gly Ser Gly Gly Val Asp Ala Ala Gly Ser Ile Thr
115 120 125
Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro
130 135 140
Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe
145 150 155 160
Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys
165 170 175
Ser Asp Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val
180 185 190
Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu
195 200 205
Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe
210 215 220
Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys
225 230 235 240
Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu
245 250 255
Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met
260 265 270
Ser Ala Lys Ser
275
<210>3
<211>834
<212>DNA
＜213〉Genus Homo people (homo sapiens)
<400>3
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggtccagg actgctacca tggtgatgga cagagctacc gaggcacatc ctccaccacc 120
accacaggaa agaagtgtca gtcttggtca tctatgacac cacaccggca ccagaagacc 180
ccagaaaact acccaaatgc tggcctgaca atgaactact gcaggaatcc agatgccgat 240
aaaggcccct ggtgttttac cacagacccc agcgtcaggt gggagtactg caacctgaaa 300
aaatgctcag gaacagaagc gagtgttggt accggtagcg cgggcagtgc tgcgggttct 360
ggcggtgtcg acgcagccgg gagcatcacc acgctgcccg ccttgcccga ggatggcggc 420
agcggcgcct tcccgcccgg ccacttcaag gaccccaagc ggctgtactg caaaaacggg 480
ggcttcttcc tgcgcatcca ccccgacggc cgagttgacg gggtccggga gaagagcgac 540
cctcacatca agctacaact tcaagcagaa gagagaggag ttgtgtctat caaaggagtg 600
tgtgctaacc gttacctggc tatgaaggaa gatggaagat tactggcttc taaatgtgtt 660
acggatgagt gtttcttttt tgaacgattg gaatctaata actacaatac ttaccggtca 720
aggaaataca ccagttggta tgtggcactg aaacgaactg ggcagtataa acttggatcc 780
aaaacaggac ctgggcagaa agctatactt tttcttccaa tgtctgctaa gagc 834
<210>4
<211>278
<212>PRT
＜213〉Genus Homo people (homo sapiens)
<400>4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
20 25 30
Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
35 40 45
Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr
50 55 60
Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
65 70 75 80
Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
85 90 95
Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Gly Thr Gly
100 105 110
Ser Ala Gly Ser Ala Ala Gly Ser Gly Gly Val Asp Ala Ala Gly Ser
115 120 125
Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe
130 135 140
Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly
145 150 155 160
Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg
165 170 175
Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg
180 185 190
Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met
195 200 205
Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys
210 215 220
Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser
225 230 235 240
Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr
245 250 255
Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys AlaIle Leu Phe Leu
260 265 270
Pro Met Ser Ala Lys Ser
275
Claims (8)
1. a recombinant protein is the protein that is comprised of the aminoacid sequence shown in the sequence in the sequence table 2.
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 1 in the 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. the application of the described recombinant protein of claim 1 in preparation promotion angiogenesis medicament.
8. the described recombinant protein of claim 1 is combined the mixture that forms with the scleroproein support.
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| CN1824775A (en) * | 2005-02-25 | 2006-08-30 | 东北师范大学遗传与细胞研究所 | Preparation technology of recombination human blood vessel inhibitor K1-3 and its application in medicine for treating tumour |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1824775A (en) * | 2005-02-25 | 2006-08-30 | 东北师范大学遗传与细胞研究所 | Preparation technology of recombination human blood vessel inhibitor K1-3 and its application in medicine for treating tumour |
| 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) |
Non-Patent Citations (2)
| Title |
|---|
| 樊钰虎等人.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.《药物生物技术》.2005,第12卷(第5期),281-284. * |
| 王健琪等人.简化的人纤溶酶原饼环区5基因的克隆及其在大肠杆菌中的融合表达.《四川大学学报(自然科学版)》.2006,第43卷(第2期),435-440. * |
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