CN103342744B - Angiogenesis protein and application thereof - Google Patents

Abstract

The invention discloses an angiogenesis protein and an application thereof, and belongs to the biological technology and medical field. The mRNA coded by the angiogenesis protein is found to be expressed in a vascular endothelial cell, and the expression of the protein in the vascular endothelial cell is determined by preparing polyclonal antiserum of the protein, so that a plurality of methods for expressing and preparing the protein are established. Moreover, the recombinant and expressed protein is firstly found to be capable of prompting the migration and tubular formation of the vascular endothelial cell, so that the generation of vascular endothelial cell NO can be stimulated. The angiogenesis protein disclosed by the invention can be applied in preparing therapeutic drugs of heart disease and atherosclerosis caused by tissue-organ ischemia reperfusion injury repair, myocardial infarction, cerebral infarction and diabetes mellitus as well as drug development during a wound healing process and an organ transplanting process.

Description

A kind of angiogenesis albumen and application thereof

Technical field

The invention belongs to biotechnology and field of medicaments, particularly a kind of angiogenesis albumen and application thereof.

Background technology

The growth of modulating vascular has important clinical meaning, promotes that angiogenesis can improve the ischemia condition of ischemic tissue and organ, alleviates the swelling in lymphedema region, and angiogenesis inhibiting then can the growth of Tumor suppression.In addition, during blood vessel injury, angiogenesis can promote that tunica intima grows, and pachyhymenia in suppression slows down the development of atherosclerotic plaque, can ensure " vascular health " in a broad sense.And new vessel plays a role for promotion skin histology wound healing aspect, on promoting that the survival of graft also produces important impact.

Adiponectin (adiponectin) is a kind of endogenous bioactive polypeptide or the protein of adipocyte secretion, effectively can improve insulin resistance and the arteriosclerosis of mouse; Find the research of human body, adiponectin can indicate the development of type ii diabetes and coronary heart disease, and shows the potentiality of anti-diabetic, anti-atherogenic and inflammation in clinical trial.Ouchi etc. study discovery, adiponectin can promote that huve cell HUVEC forms tubular structure in vitro, and to its migration, there are promoter action (Oucho etc., Adiponectinstimulates angiogenesis by promoting cross-talk between AMP-activated proteinkinase and Akt signaling in endothelial cells, 2004, The Journal of BiologicalChemistry, p1304-1309).Clinical studyes a large amount of is at present verified, and adiponectin can as a kind of mark of cardiovascular disorder, metabolic dysfunction, and strengthens the new direction that the secretion of adiponectin and effect can be used as Cardiovarscular.

Recently, it is found that an adiponectin homology isomer family with high conservative, i.e. C1q tumour necrosis factor associated protein (CTRPs) family.CTRPs comprises 15 members, existing research finds that CTRP3 is as a newfound Adipocyte Factor family important member, directly can suppress apoptosis of cardiac muscle, increase myocyte survival/regeneration, alleviate the rear myocardial fibrosis of heart stalk, raising myocardial cell expresses the cytokine promoting vascularization, thus pathological remodeling of the heart after alleviating heart stalk, improve the rear myocardium shrinkage function of heart stalk.CTRP3 significantly can promote that surface of a wound edge new vessel is formed, thus accelerating wound healing.CTRP9, another adiponectin homology isomer family, activates myocardium AMPK signal path by adiponectin receptors AdipoR I, improves Myocardial Ischemia Reperfusion Injury, improves CTRP9 level and contributes to control myocardial ischemia infarct.

The potentiality of adiponectin in anti-diabetic, anti-atherogenic, inflammation and treating cardiovascular disease, also show that adiponectin homologs also has further drug development and is worth.And the development research of existing adiponectin focuses mostly at great vessels as venous endothelial blood vessel, and the angiogenesis of great vessels is promoted.Other adiponectin homology isomer is if CTRP3 is mainly by promoting that the correlation factor of myocardial cell is secreted thus promotes the angiogenesis of infarcted myocardium marginarium.And CTRP9 mainly acts on the reparation of infarcted myocardium by directly suppressing apoptosis of cardiac muscle and promote vasorelaxation.Direct promotion blood vessel particularly the albumen of Angiogenesis discovery by improve the ischemia condition of ischemic tissue and organ and the heart trouble that cerebral infarction, heart stalk, diabetes are caused, atherosclerotic treatment and wound healing, organ graft survival have great importance and application prospect.The present invention has invented a kind of albumen of promotion angiogenesis, particularly Angiogenesis from adiponectin homology isomer family.The promotion angiogenesis potential of this albumen just contributes to the ischemia condition improving ischemic tissue and organ, and the reparation for the ischemical reperfusion injury such as heart, cerebral tissue has important effect.This albumen can be used for preparing medicine simultaneously, all has certain treatment potential in the survival for the heart trouble caused due to diabetes, heart stalk, cerebral infarction, atherosclerotic treatment and wound healing, organ graft.

In the past few decades, the research of the angiogenesis of every field all achieves great progress, but until in recent years, just has part achievement in research to start to be applied to clinical.Although FDA have approved some anti-angiogenic drugs that can be used for treating tumour, but promote that the clinical application of angiogenesis medicament is had got long long way to go, and promote that the exploitation of angiogenesis medicament is to promoting that the clinical application of angiogenesis medicament has important strategic reserves meaning.

Summary of the invention

For overcoming the shortcoming and defect of above-mentioned prior art, primary and foremost purpose of the present invention is that providing a kind of promotes angiogenesis albumen, particularly promotes the albumen of Angiogenesis.

Another object of the present invention is to the preparation method that above-mentioned angiogenesis albumen is provided.

Another object of the present invention is the application providing above-mentioned angiogenesis albumen, the application of medicine used in the heart trouble preparing the reparation of histoorgan ischemical reperfusion injury, heart stalk, cerebral infarction, diabetes cause, atherosclerosis and wound healing, organ transplantation process.

Object of the present invention is realized by following technical scheme: a kind of promotion angiogenesis albumen, called after C1ql1-gl1(complement component 1q subconstiuent sample albumen 1 globular domain albumen, complement component1q subcomponent-like 1 globular domain) albumen, its aminoacid sequence is as follows:

TYTTVPRVAFYAGLKNPHEGYEVLKFDDVVTNLGNNYDAASGKFTCNIPGTYFFTYHVLMRGGDGTSMWADLCKNGQVRASAIAQDADQNYDYASNSVILHLDAGDEVFIKLDGGKAHGGNSNKYSTFSGFIIYSD；

The nucleotide sequence of the promotion angiogenesis albumen (C1ql1-gl1 albumen) described in coding is as follows:

ACCTATACCACGGTGCCACGCGTGGCCTTCTACGCCGGCCTCAAGAACCCTCATGAGGGTTACGAGGTGCTCAAGTTTGACGACGTGGTCACCAACCTAGGCAACAACTACGATGCGGCCAGCGGCAAGTTTACATGCAACATTCCTGGCACCTACTTTTTCACCTACCACGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGCAAGAACGGCCAGGTGCGGGCCAGTGCAATTGCCCAGGATGCAGACCAGAACTACGACTATGCCAGCAACAGCGTGATCCTGCATCTGGACGCAGGGGATGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACGGGGGCAACAGCAACAAATACAGCACTTTCTCTGGCTTCATCATCTACTCGGATTGA；

The molecular weight of described promotion angiogenesis albumen is 14kDa.

The preparation method of described promotion angiogenesis albumen, is realized by following steps:

(1) amplimer of C1ql1-gl1 albumen is designed, upstream primer P5:5 '-CAGAATTCACCTATACCACGGTGCCA-3 '; Downstream primer P4:5 '-CCGCTCGAGATCCGAGTAGATGATGAAGCCA-3 '; Upstream primer P5 contains EcoRI cleavage site, and downstream primer P4 contains Xho I cleavage site, introduces the globular domain nucleotide sequence of the C1ql1 gene of EcoRI and Xho I restriction enzyme site for increasing;

(2) prepare mouse cDNA template, adopt the total serum IgE of Trizol reagent (invitrogen company of the U.S.) extracting mouse brain tissue, employing First Strand cDNA Synthesis Kit ReverTra Ace-α- ^tM(Toyobo, Japan) synthesizes cDNA mono-chain.CDNA mono-chain synthesized according to this is template, carries out pcr amplification with P5/P4 primer; The Taq enzyme of pcr amplification is purchased from Beijing Ding Guo biotechnology company, and response procedures is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations; 72 DEG C extend 5min.Obtain the pcr amplification product of the introducing EcoRI of specific amplified and the C1ql1-gl1 nucleotide sequence of Xho I restriction enzyme site;

(3) increase P5/P4 the pcr amplification product of C1ql1-gl1 nucleotide sequence of the introducing EcoRI obtained and Xho I restriction enzyme site, by EcoRI and Xho I two enzyme sites, cut, reclaim and be connected rear clone to fusion expression vector pET32a(novagen by enzyme respectively) on, obtain recombinant expression vector pET32a-C1ql1-gl1, by order-checking, its building process as shown in Figure 3, confirms that calling sequence is correct.

(4) the exogenous sequences C1ql1-gl1 in recombinant expression vector pET32a-C1ql1-gl1 and the TRX tag fusion expressed fusion protein TRX-C1ql1-gl1 on pET32a carrier.By pET32a-C1ql1-gl1 transformation of E. coli BL21(DE3).Picking list colony inoculation is in the 2 × YT substratum containing 100ng/ml ammonia Bian, 37 DEG C of jolting overnight incubation activated spawn, 500ml2 × YT liquid nutrient medium is inoculated into according to the volume ratio of 1:100, when 37 DEG C of amplification culture are about 0.9 to OD600, adding IPTG to final concentration is 1mM, collected by centrifugation thalline after 18 DEG C of inducing culture 24h.By total thalline Tris damping fluid (50mM TrisCl, 150mM NaCl, pH7.5) wash, again according to Tris damping fluid (the 50mM TrisCl of 1g thalline 10ml, 150mM NaCl, the thalline collected suspends by ratio pH7.5), after supersound process 20min, by the supernatant liquor after ultrasonic through Ni ²⁺chelating Sepharose affinity chromatography purifying, first washes Ni with the imidazoles of 75mM ²⁺post, abandons elution peak, then directly washes Ni with the imidazoles of 150mM ²⁺post, collects the elution peak of recombinant protein, obtains containing the higher elutriant of fusion rotein TRX-C1ql1-gl1 purity.

(5) fusion rotein TRX-C1ql1-gl1 enzyme is cut purifying C1ql1-gl1 albumen.The elutriant that step (4) obtains is crossed the desalination of G25 gel column and changes enzyme cutting buffering liquid, then, in normal temperature water-bath, with recombinant enterokinase (Guangdong Zhongda South China Sea Ocean Biotechnology Engineering Center Co.Ltd), cut through to fusion rotein TRX-C1ql1-gl1 enzyme night, enzyme cut after C1ql1-gl1 protein solution carry out Ni ²⁺the column purification of again going up of Chelating Sepharose post removes the TRX companion body.Enzyme cut after sample when upper prop, TRX major part is adsorbed on Ni ²⁺on Chelating Sepharose post, C1ql1-gl1 albumen, in percolation peak, collects percolation peak.Albumen in the percolation peak solution of collection is crossed to obtain after Pierce High-Capacity Endoxin Removal Resin pillar (Thermo company of the U.S.) removes intracellular toxin purifying without the C1ql1-gl1 albumen of endotoxic purity more than 95%, namely prepared promotion angiogenesis albumen.

In step (2), mouse used is KM mouse, purchased from Guangdong Province's Experimental Animal Center;

Tris damping fluid described in step (4) is for containing 50mM TrisCl, 150mM NaCl, pH7.5;

The condition of the supersound process described in step (4) is that 300W, 10s are ultrasonic, 10s interval;

Enzyme cutting buffering liquid described in step (5) is 20mM TrisCl, 100mM NaCl, pH8.0.

Application in the drug development in the heart trouble preparing the reparation of histoorgan ischemical reperfusion injury, heart stalk, cerebral infarction, diabetes cause, atherosclerotic medicine and wound healing, organ transplantation process of described promotion angiogenesis albumen.

The present invention has following advantage and effect relative to prior art:

Promotion angiogenesis albumen of the present invention can promote the migration of rat heart microvascular endothelial cell, significantly promote that vascular endothelial cell formation tubular structure and promotion heart microvascular endothelial cell are to the release of NO, therefore have important application prospect in preparation with the medicine of vascular repair relative disease.

Accompanying drawing explanation

Fig. 1 is that RT-PCR analyzes the expression identification figure of C1ql1 in rat heart microvascular endothelial cell.1,2 is the heart microvascular endothelial cell being separated two the different rats obtained; M is DNA molecular amount standard.

Fig. 2 is that C1ql1-gl1 protein expression vector pET21b-C1ql1-gl1 builds schematic diagram.A, pcr amplification C1ql1 gene globular domain electrophorogram; B, pET21b-C1ql1-gl1 build schema; C, carrier universal primer screening pET21b-C1ql1-gl1 positive colony, swimming lane 2 and 4 is positive colony.

Fig. 3 is that C1ql1-gl1 protein expression vector pET32a-C1ql1-gl1 builds schematic diagram.A, pcr amplification C1ql1 gene globular domain electrophorogram; B, pET32a-C1ql1-gl1 build schema; C, C1ql1 gene specific primer screening pET32a-C1ql1-gl1 positive colony; Swimming lane 1,2,4 is positive colony; 3 and 5 is negative clone.

Fig. 4 be e. coli bl21 containing pET21b-C1ql1-gl1 plasmid through IPTG abduction delivering detection figure, swimming lane 1 is bacterium total before induction; Swimming lane 2 is the total bacterium after induction; Swimming lane 3 be ultrasonic after precipitation; Swimming lane 4 be ultrasonic after supernatant.

Fig. 5 is C1ql1 polyclonal antiserum qualification figure.Swimming lane 1 is preimmune serum 1:2,000 dilution; Swimming lane 2 is anti-C1ql1 negative serum 1:2,000 dilution; Swimming lane 3 is preimmune serum 1:5,000 dilution; Swimming lane 4 is anti-C1ql1 negative serum 1:5,000 dilution.

Fig. 6 adopts the C1ql1 polyclonal antibody of preparation to detect C1ql1 at rat heart microvascular endothelial cell expression figure.1, the cell strain NIH3T3 of the C1ql1 positive is expressed in contrast; 2,3 is the rat heart microvascular endothelial cell of separation and Culture.

Fig. 7 is the SDS-PAGE detection figure of the Expression and purification of TRX-C1ql1-gl1 fusion rotein.Swimming lane 1 is the total bacterium before induction; Swimming lane 2 is the total bacterium after induction; Swimming lane 3 is the ultrasonic supernatant after induction; Swimming lane 4 is the ultrasound precipitation after induction; Swimming lane 5 is through Ni ²⁺the albumen of post affinity purification.

Fig. 8 is the SDS-PAGE detection figure that enteropeptidase enzyme cuts front and back C1ql1-gl1 albumen.Swimming lane 1, enzyme cut before TRX-C1ql1-gl1 albumen; Swimming lane 2, enzyme cut after albumen.

Fig. 9 be C1ql1-gl1 albumen rat heart microvascular endothelial cell is moved affect result figure.A, takes cell migration figure under opticmicroscope.B, Image-Pro Plus5.0 analyzes relative migration Area comparison histogram.BSA, negative control; C1ql1-gl1,2.5 μ g/ml.After Dual culture 12h and 24h, C1ql1-gl1 albumen is compared with BSA, and the area of cell migration has the significance of difference.

Figure 10 is that C1ql1-gl1 albumen affects result figure to rat heart microvascular endothelial cell angiogenesis.A, takes vascularization figure under opticmicroscope.BSA, negative control; C1ql1-gl1,2.5 μ g/ml; Vascular endothelial growth factor (VEGF), 100ng/ml, positive control.B, Image-Pro Plus5.0 analyzes the relative length schematic diagram of tubule, and C1ql1 and VEGF, compared with contrast BSA, has the significance of difference to the impact of length of vessel.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Embodiment 1: the separation of rat heart microvascular endothelial cell and cultivation

The female sd inbred rats (150 ~ 200g) of 2 months, after etherization, dislocation is put to death, heart is taken out under aseptic condition, put into 37 DEG C of PBS, remove left and right atrium and right ventricle, cut off left room along left ventricle antetheca, PBS rinses until washing fluid is limpid, volume fraction is 70% alcohol immersion 30s, deactivation visceral pericardium and endocardial cell; A large amount of PBS removes great vessels after rinsing, and residual myocardium shreds, and adds collagenase II, 37 DEG C of shaking bath 30-60min that mass fraction is 0.1% preheating, piping and druming 10min; After 100 μm of strainer filterings, the centrifugal 15min of 100g, remove supernatant, with percoll gradient centrifugation 5min, get middle layer, Gibco company of the DMEM(U.S. with serum-free) make the centrifugal 15min of cell suspension 100g, the ECGF(U.S. sigma with containing the 20%FBS of volume percent and 0.5% of mass percent) DMEM substratum resuspended.Be inoculated in culture dish.Impurity is removed with the PBS washed cell of preheating after 4h.Change liquid every other day once, 5-7d cell is the intensive paving stone sample growth of individual layer later.

Embodiment 2:RT-PCR analyzes the expression of C1ql1 in rat heart microvascular endothelial cell

The total serum IgE of 2 the SD rat heart microvascular endothelial cells adopting Trizol reagent (invitrogen company of the U.S.) extracting to be separated, employing First Strand cDNA Synthesis Kit ReverTra Ace-α- ^tM(Toyobo, Japan) synthesizes cDNA mono-chain.With P1:5 '-ACCTATACCACGGTGCCACGC-3 ' and P2:5 '-ATCCGAGTAGATGATGAAGCCA-3 ' primer amplification C1ql1 gene globular domain, the Taq enzyme of pcr amplification is purchased from Beijing Ding Guo biotechnology company, and response procedures is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations; 72 DEG C extend 5min.The sepharose of PCR primer mass percent 1% carries out electrophoresis and takes pictures.Result as shown in Figure 1, can detect the expression of C1ql1mRNA in rat heart microvascular endothelial cell.

The structure of embodiment 3:C1ql1 prokaryotic expression carrier

According to globular domain (NP_035925.2,122-258aa) two ends sequent synthesis two pairs of primers of mouse C1ql1 gene.Wherein the upstream primer (P3:5 '-GGAATTCCATATGACCTATACCACGGTGCCACGC-3 ') of pair of primers is containing Nde I cleavage site, downstream primer (P4:5 '-CCGCTCGAGATCCGAGTAGATGATGAAGCCA-3 ') containing XhoI cleavage site, for building the C1ql1-gl1 albumen of non-fusion expression, for the preparation of polyclonal antiserum.Containing EcoRI cleavage site, downstream primer is P4 to the upstream primer (P5:5 '-CAGAATTCACCTATACCACGGTGCCA-3 ') of pair of primers in addition, for the preparation of solvable C1ql1 albumen.

Adopt the total serum IgE of Trizol reagent (invitrogen company of the U.S.) extracting KM mouse (purchased from Guangdong Province's Experimental Animal Center) brain tissue, employing First Strand cDNA Synthesis Kit ReverTraAce-α- ^tM(Toyobo, Japan) synthesizes cDNA mono-chain.CDNA mono-chain synthesized with this is template, carries out pcr amplification respectively with P3/P4 and P5/P4 two pairs of primers.The Taq enzyme of pcr amplification is purchased from Beijing Ding Guo biotechnology company, and response procedures is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations; 72 DEG C extend 5min.Obtain the single band of specific amplified, product size is at about 400bp.P3/P4 is increased the pcr amplification product that obtains by Nde I and Xho I two enzyme sites, cut by enzyme and connect rear clone to prokaryotic expression carrier pET21b(novagen) on, carry out PCR screening by universal primer T7 and T7T on pET21b carrier and obtain recombinant expression vector pET21b-C1ql1-gl1, its building process as shown in Figure 2.P5/P4 is increased the pcr amplification product that obtains by EcoRI and Xho I two enzyme sites, cut by enzyme and connect rear clone to fusion expression vector pET32a(novagen) on, obtain recombinant expression vector pET32a-C1ql1-gl1, its building process as shown in Figure 3.Foreign gene in expression vector pET21b-C1ql1-gl1 and pET32a-C1ql1-gl1 is correct through order-checking qualification.

The preparation of embodiment 4:C1ql1 polyclonal antiserum and qualification

PET21b-C1ql1-gl1 transformation of E. coli BL21(DE3 prepared by embodiment 3) after select mono-clonal and express 8h through 1mM IPTG at 37 DEG C of inducible proteins, collect total bacterium, Western blot detection is carried out by His monoclonal antibody (novagen), find that bacterial strain has obvious specifically expressing product band after induction, molecular weight conforms to the theoretical value of prediction.Resuspended with sonication buffer (50mM TrisCl, 150mM NaCl, pH7.4) after a large amount of abduction delivering, showing through SDS-PAGE electrophoretic analysis after supersound process (300W, 10s are ultrasonic, 10s interval) 20min, mainly there is (Fig. 4) with inclusion bodies in albumen.

Inclusion body protein purifying, owing to mostly being inclusion body, is directly cut glue as antigen by the protokaryon albumen that restructuring obtains.Concrete operations are as follows: by the total bacterium ultrasonication after induction, and the precipitation obtained is first with the TBS(pH7.4 containing 2M urea) washing, then carry out Tricine SDS-PAGE electrophoretic separation, stand-by after cutting target protein band PBS balance.Quantitative roughly with the trypsinogen (trypsinogen, 24kDa) of different concns.Target protein antigen (1mg, gel state) is added the PBS damping fluid of 1ml, in ice bath, be about 1h with mortar grinder until the gel particle in suspension is tiny and even.Sample for fundamental immunity slowly need add isopyknic Freund's complete adjuvant (sigma company of the U.S.), and the sample for booster immunization then adds isopyknic Freund's incomplete adjuvant (sigma company of the U.S.).Continue grinding 1h or repeatedly lash with syringe to make complete emulsification, then subcutaneous injection immunize New Zealand White Rabbit.Getting serum before fundamental immunity is negative control.The antigen protein dosage of fundamental immunity is 1mg, and within after fundamental immunity one month, carry out booster immunization, each booster immunization dosage is 0.5mg antigen protein.Carry out booster immunization week about, get serum after continuous booster immunization four times and carry out tiring and specific detection.As shown in Figure 5, the polyclonal antiserum of preparation can specific recognition C1ql1 albumen, and have higher titre for detected result.And there is high homology (>90%) in rat, mouse and people due to the antigen adopted, therefore this antiserum(antisera) can specific recognition rat, mouse and people C1ql1 albumen, and through experimental verification.

Embodiment 5: Western blot detects the expression of C1ql1 albumen in rat heart microvascular endothelial cell

Get the rat heart microvascular endothelial cell cultivated on 6 orifice plates, adding final concentration is 1mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (1/100 dilution) (Calbiochem, Merck Millipore company of the U.S.) Western and the IP cell pyrolysis liquid (Mei Jin biotech firm) of 100 μ l, ice bath cracking.Get supernatant after 4 DEG C of 12,000rpm centrifugal 15min, carry out carrying out Western blot detection quantitatively to the total protein content of each tissue with BAC protein quantification test kit.The primary antibodie adopted is that polyclonal antiserum volume ratio 1:2000 prepared in embodiment 4 carries out diluting rear detection, and two resist the goat anti-rabbit igg (volume ratio 1:2000, sigma) for horseradish peroxidase-labeled.Detect with ECL chemiluminescence detection kit (Thermo company of the U.S.) and find, the expression (Fig. 6) of C1ql1 albumen can be detected at rat heart microvascular endothelial cell.Detect using NIH3T3 cell as positive cell strain Western blot.

The Expression and purification of embodiment 6:TRX-C1ql1-gl1 fusion rotein

Exogenous sequences gC1ql1 in recombinant expression vector pET32a-gC1ql1 and the TRX tag fusion expressed fusion protein TRX-C1ql1-gl1 on carrier.

By pET32a-gC1ql1 transformation of E. coli BL21(DE3).After cultivating breeding, after genetic engineering bacterium ultrasonication (300W, 10s are ultrasonic, 10s interval) 20min, supernatant liquor shows through SDS-PAGE electrophoretic analysis with precipitation, bacterial strain has obvious specifically expressing product band (Fig. 7) after induction, and molecular weight conforms to the theoretical value of prediction.

To incubation time, induced concentration, the culture condition of genetic engineering bacterium of groping to draw of the conditions such as temperature is: picking list colony inoculation is in the 2 × YT substratum containing 100ng/ml ammonia Bian, 37 DEG C of jolting overnight incubation activated spawn, 500ml2 × YT liquid nutrient medium is inoculated into according to the volume ratio of 1:100, when 37 DEG C of amplification culture are about 0.9 to OD600, adding IPTG to final concentration is 1mM, collected by centrifugation thalline after 18 DEG C of inducing culture 24h.By total thalline Tris damping fluid (50mM Tris, 150mM NaCl, pH7.5) wash, appropriate Tris damping fluid (50mM Tris, 150mM NaCl, pH7.5) is used to suspend again, after supersound process, cracking supernatant liquor and ultrasonic after precipitation show through SDS-PAGE electrophoretic analysis, the expression amount of TRX-C1ql1-gl1 fusion rotein accounts for more than 80% of bacterial protein with this understanding, and major part is in solvable state.

By the supernatant liquor after ultrasonic through Ni ²⁺chelating Sepharose affinity chromatography purifying, collects the elution peak of recombinant protein.Experiment finds that TRX-C1ql1-gl1 can by Ni ²⁺post adsorbed, and washes Ni with the imidazoles of 75mM concentration ²⁺during post, the foreign protein that major part is adsorbed on post can be washed down, and target protein TRX-C1ql1-gl1 can be washed down with the imidazoles of 100mM, 150mM concentration.In order to improve yield and the concentration of recombinant protein, simplifying experimental procedure, shortening the treatment time to recombinant protein, with Ni ²⁺during post affinity chromatography purification of recombinant proteins, we adopt single stage method to carry out purifying.It is foreign protein expressed by vector plasmid pET32a and Ni that described single stage method carries out purifying ²⁺the feature that the binding ability of affinity column is stronger, we have selected the elution requirement comparatively simplified: first wash Ni with the imidazoles of 75mM ²⁺post, abandons elution peak, then directly washes Ni with the imidazoles of 150mM ²⁺post, collects the elution peak of recombinant protein, obtains containing the higher elutriant of fusion rotein TRX-C1ql1-gl1 purity.In elutriant, the SDS-PAGE of TRX-C1ql1-gl1 albumen detects figure as shown in Figure 7.

Above-mentioned acquisition elutriant is crossed the desalination of G25 gel column and changes enzyme cutting buffering liquid (20mM TrisCl, 100mMNaCl, pH8.0), then in normal temperature water-bath, night is cut through with recombinant enterokinase (Guangdong Zhongda South China Sea Ocean Biotechnology Engineering Center Co.Ltd) enzyme to TRX-C1ql1-gl1, SDS-PAGE electrophoresis detection enzyme cuts effect, and result as shown in Figure 8.Major part albumen can be good at reorganized enteropeptidase enzyme and cuts.

Enzyme cut after C1ql1-gl1 protein solution carry out Ni ²⁺the column purification of again going up of Chelating Sepharose post removes the TRX companion body.Enzyme cut after sample when upper prop, TRX major part is adsorbed on Ni ²⁺on ChelatingSepharose post, target protein, in percolation peak, is collected percolation peak and is carried out subsequent experimental further.

Crossing Pierce High-Capacity Endoxin RemovalResin pillar (Thermo company of the U.S.) to the albumen in the percolation peak solution of above-mentioned collection goes intracellular toxin to do follow-up functional experiment.

The impact that embodiment 7:C1ql1-gl1 albumen is bred rat heart microvascular endothelial cell

Collect rat heart microvascular endothelial cell logarithmic phase cell, adjustment concentration of cell suspension, bed board 96 well culture plate, every hole adds 100 μ l, makes cell to be measured adjust density 1,000-10,000/ hole.Volume fraction is 5%CO ₂, hatch for 37 DEG C, in the DMEM substratum containing volume fraction 20%FBS, be cultured to cell monolayer be paved with at the bottom of hole, be now changed to the hungry 12h of plasma-free DMEM medium and make Growth of Cells synchronization.Add after 12h concentration gradient albumen (TRX-C1ql1-gl1 albumen and enzyme cut after the concentration of C1ql1-gl1 albumen be 0,0.15,1,5 μ g/ml, contrast vascular endothelial growth factor (VEGF) concentration is 50ng/ml) each concentration establishes 6 multiple holes.Volume fraction is 5%CO ₂, hatch 48h for 37 DEG C.Adopt promega company of the cell proliferation detecting kit ITS(U.S.) detect.Every hole adds 20 μ l nitrite ions, softly mixes with rifle head.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD490nm place after leaving standstill 3h.Experimental result display C1ql1-gl1 albumen is on the not impact of rat heart microvascular endothelial cell propagation.

The impact that embodiment 8:C1ql1-gl1 albumen moves rat heart microvascular endothelial cell

First use marker pen at 6 orifice plates behind, compare with ruler, draw horizontal line equably, approximately every 0.5-1cm together, cross via hole.Every hole is at least through 5 lines.About 5 × 10 are added in hole ⁵individual cell.Within second day, compare ruler with rifle head, perpendicular to horizontal line cut behind.Wash cell 3 times with PBS, remove the cell under drawing, control group adds serum free medium, and experimental group adds containing final concentration the serum free medium of the C1ql1-gl1 albumen being 2.5 μ g/ml.Put into 37 DEG C, volume fraction is 5%CO ₂incubator is cultivated.By 0,12,24h observes, take pictures, and with software I mage-Pro Plus5.0, the area do not healed analyzed.Under Fig. 9 is presented at the treatment condition of BSA negative control and 2.5 μ g/ml C1ql1-gl1, after Dual culture 12h and 24h, C1ql1-gl1 albumen is compared with BSA, and the area of cell migration has the significance of difference.Experimental result shows that experimental group C1ql1-gl1 can promote the migration of rat heart microvascular endothelial cell.

The impact that embodiment 9:C1ql1-gl1 albumen is formed rat heart microvascular endothelial cell tubulose

Tubule forms the Matrigel of the low somatomedin of experiment reagent purchased from BD Bioscience company ,-20 DEG C of preservations.Before using, test kit is put in 4 DEG C to spend the night, makes solid dissolve into liquid; In laminar flow sterile incubator, 48 hole sterile culture plates of precooling before opening, mark each group with marking pen; In 48 orifice plates, every hole adds the liquid glue 100 μ l dissolved on ice, and put into 37 DEG C, volume fraction is 5%CO ₂1h in sterile culture case, makes liquid glue be frozen into solid-state glue; Plantation endotheliocyte selects rat heart microvascular endothelial cell in 10 generations to carry out little tube formation assay, and before plantation, desirable cell density is 70-80%; Trysinization, prepares endotheliocyte suspension, centrifugal, hangs vascular endothelial cells with serum-free Endothelial cell culture basic weight.Grouping, adds corresponding process factor, i.e. experimental group C1ql1-gl1(2.5 μ g/ml respectively in each group), BSA negative control group and positive control VEGF(100ng/ml, sigma company of the U.S.); Endotheliocyte suspension is added, μ l(5 × 10, every hole 500 in 48 orifice plates completing glue ⁴individual cell), put into 37 DEG C, volume fraction is 5%CO ₂24h in sterile culture case, observe and take a picture, result as shown in Figure 10.Measure little length of tube with software I mage-Pro Plus5.0, carry out statistical study.Experimental result shows that external use C1ql1-gl1 significantly can promote that vascular endothelial cell forms tubular structure.

Embodiment 10:C1ql1-gl1 promotes that rat heart microvascular endothelial cell tubulose forms the change of the content of rear NO

Get the C1ql1-gl1 experimental group in above-described embodiment 9 and BSA control group nutrient solution supernatant 100 μ l respectively, add nitrogen protoxide test kit (Science and Technology Ltd. is built up in Nanjing, article No. A013) related reagent, and in 550nm microplate reader colorimetric, measure each hole OD value.By production standard curve, R ²0.9986, curve can use.After C1ql1-gl1 process, the content that rat heart microvascular endothelial cell tubulose forms rear NO is 11.53 ± 1.25 μm of ol/L, and the NO content of control group BSA is respectively 8.64 ± 0.89 μm of ol/L, show that C1ql1-gl1 albumen can promote that heart microvascular endothelial cell is to the release of NO.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (6)

1. promote an angiogenesis albumen, it is characterized in that called after C1ql1-gl1 albumen, its aminoacid sequence is as shown in SEQ ID NO.1.

2. promotion angiogenesis albumen according to claim 1, is characterized in that: the nucleotide sequence of the C1ql1-gl1 albumen described in coding is as shown in SEQ ID NO.2.

3. the preparation method of promotion angiogenesis albumen according to claim 1, is characterized in that being realized by following steps:

(1) amplimer of C1ql1-gl1 albumen is designed, upstream primer P5:5 '-CAGAATTCACCTATACCACGGTGCCA-3 '; Downstream primer P4:5 '-CCGCTCGAGATCCGAGTAGATGATGAAGCCA-3 '; Upstream primer P5 contains EcoRI cleavage site, and downstream primer P4 contains Xho I cleavage site, introduces the globular domain nucleotide sequence of the C1ql1 gene of EcoRI and Xho I restriction enzyme site for increasing;

(2) prepare mouse cDNA template, adopt the total serum IgE of Trizol reagent extracting mouse brain tissue, employing First Strand cDNA Synthesis Kit ReverTra Ace-α- ^tMsynthesis cDNA mono-chain; CDNA mono-chain synthesized according to this is template, carries out pcr amplification with P5/P4 primer; Pcr amplification reaction program is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations; 72 DEG C extend 5min; Obtain the pcr amplification product of the introducing EcoRI of specific amplified and the C1ql1-gl1 nucleotide sequence of Xho I restriction enzyme site;

(3) increase P5/P4 the pcr amplification product of C1ql1-gl1 nucleotide sequence of the introducing EcoRI obtained and Xho I restriction enzyme site, by EcoRI and Xho I two enzyme sites, cut, reclaim and be connected rear clone on fusion expression vector pET32a by enzyme respectively, obtain recombinant expression vector pET32a-C1ql1-gl1, confirm that calling sequence is correct by order-checking;

(4) the exogenous sequences gC1ql1-gl1 in recombinant expression vector pET32a-gC1ql1-gl1 and the TRX tag fusion expressed fusion protein TRX-C1ql1-gl1 on pET32a carrier; By pET32a-gC1ql1-gl1 transformation of E. coli BL21 (DE3); Picking list colony inoculation is in the 2 × YT substratum containing 100ng/ml ammonia Bian, 37 DEG C of jolting overnight incubation activated spawn, 2 × YT liquid nutrient medium of 500ml is inoculated into according to the volume ratio of 1:100, when 37 DEG C of amplification culture are 0.9 to OD600, adding IPTG to final concentration is 1mM, collected by centrifugation thalline after 18 DEG C of inducing culture 24h; By total thalline Tris buffer solution, then according to the ratio of the Tris damping fluid of 1g thalline 10ml, the thalline collected is suspended, after supersound process 20min, by the supernatant liquor after ultrasonic through Ni ²⁺chelating Sepharose affinity chromatography purifying, first washes Ni with the imidazoles of 75mM ²⁺post, abandons elution peak, then directly washes Ni with the imidazoles of 150mM ²⁺post, collects the elution peak of recombinant protein, obtains containing the higher elutriant of fusion rotein TRX-C1ql1-gl1 purity;

(5) fusion rotein TRX-C1ql1-gl1 enzyme is cut purifying C1ql1-gl1 albumen; The elutriant that step (4) obtains is crossed the desalination of G25 gel column and changes enzyme cutting buffering liquid, then, in normal temperature water-bath, with recombinant enterokinase, cut through to fusion rotein TRX-C1ql1-gl1 enzyme night, enzyme cut after C1ql1-gl1 protein solution carry out Ni ²⁺the column purification of again going up of Chelating Sepharose post removes the TRX companion body; Enzyme cut after sample when upper prop, TRX major part is adsorbed on Ni ²⁺on Chelating Sepharose post, C1ql1-gl1 albumen, in percolation peak, collects percolation peak; Albumen in the percolation peak solution of collection is crossed to obtain after Pierce High-Capacity EndoxinRemoval Resin pillar removes intracellular toxin purifying without the C1ql1-gl1 albumen of endotoxic purity more than 95%, namely prepared promotion angiogenesis albumen.

4. the preparation method of promotion angiogenesis albumen according to claim 3, is characterized in that:

In step (2), mouse used is KM mouse, purchased from Guangdong Province's Experimental Animal Center.

5. the preparation method of promotion angiogenesis albumen according to claim 3, is characterized in that:

Tris damping fluid described in step (4) is for containing 50mM TrisCl, 150mM NaCl, pH 7.5;

The condition of the supersound process described in step (4) is that 300W, 10s are ultrasonic, 10s interval;

Enzyme cutting buffering liquid described in step (5) is 20mM TrisCl, 100mM NaCl, pH8.0.

6. the promotion angiogenesis albumen described in claim 1 or 2 is preparing the application in Angiogenesis or the exploitation of capillary blood vessel repair medicine.