CN1225557C - Method for producing blood vessel diseases gene medicine-blood vessel endothelium growth gene-2 naked DNA by microorganism cloning vehicle - Google Patents

Abstract

The method is used for cloning a mouse-derived VEGF-2 (mVEGF-2) gene from an impregnate mouse embryo and then cloning a human-derived VEGF promotor (hVEGF promoter) from a newborn placenta, and the mVEGF-2 is driven by the hVEGF promoter and inserted into an expression vector pEGSH of a mammal so that a vector pEGVPV which generates a large amount of DNA in a prokaryotic cell at a high copy number and both are expressed in a prokaryon and a mammalian cell can be established. After the vector is converted into E. coli DH5 alpha, the obtained DNA copying number is more than 2000/cell, and the DNA output separated from a cell culture can achieve 1.5 mg/l. The present invention also provides a method for producing vascular endothelial growth factor-2 (VEGF-2) DNA medicine by microorganisms. The vascular endothelial growth factor can specially act on a vascular endothelial cell to intensely promote cell proliferation and vascularization, and the vascular endothelial growth factor has the function of enhancing vascular permeability. An ischemic animal model experiment indicates that the naked DNA can promote neovascularization and enhance the establishment of collateral circulation when microinjected to a tissue with ischemia so as to effectively improve the blood supply of the ischemic tissue. The method can provide a new scheme for treating vascular diseases.

Description

A kind of method with microbial cloning carrier production for treating vascular disease genomic medicine vascular endothelial growth factor-2 naked DNA

1. technical field

The present invention relates to a kind of recombinant DNA and structure thereof, and with this recombinant DNA of microorganisms producing, with its method as the DNA medicine.Specifically, the present invention relates to a kind of humanized of including VEGF promotor (hVEGF promoter), can in mammalian cell, express the Construction of eukaryotic of mouse source property VEGF-2cDNA (mVEGF-2), and with the method for microorganisms producing this vascular endothelial growth factor-2 (VEGF-2) DNA medicine.

2. background technology

Vascular disease are common disease, frequently-occurring diseases of serious threat human health, wherein cerebral thrombosis, patients with coronary heart disease are maximum, characteristics with " sickness rate height, recurrence rate height, lethality rate height, disability rate height, complication are many ", be " first killer " who threatens human health, account for total death toll 40.72% (Wuzhong is auspicious. select heath food, should be with reference to expert opinion. Http:// www.cnki.net).According to World Health Organization's conservative estimation, global cardiovascular and cerebrovascular diseases patient has 4,500 ten thousand people at least, singly is that the U.S. just has more than 1,000 ten thousand people to suffer from coronary artery disease, has every year 5000000 people that stenocardia takes place, and has 1,500,000 people that heart trouble takes place; In France, die from every year just the having more than 100,000 people of myocardial infarction (gene therapy treatment coronary heart disease. Http:// www.cnki.net).In recent years, " modern civilization disease " sickness rate such as China's cardiovascular and cerebrovascular diseases rise just year by year, show that according to health ministry medical statistics result cardiovascular and cerebrovascular diseases also accounts for the first place in China's patient death reason, reach 2,000,000 people every year.Critical limb ischemia disease is another kind of artery disease, and also very general in smoker, diabetic subject and essential hypertension people (Cai Xiaohui. the first vascular disease gene therapy in Taiwan begins to enter human experimentation. Http:// www.cnki.netelial), there is 15% people to suffer from the limb artery disease according to estimates among the old man of the U.S. more than 50 years old.Therefore, the specific medicament of exploitation treatment vascular disease is significant to safeguarding human health.

Arteries is narrow to cause thrombosis with infraction, cause local organization ischemic, anoxic and pain occurs, produce the gangrenosum acne focus when serious, being easy to cause dead and disabling is common trait (the Harada K of vascular disease, et al, Am J Physiol, 1996,270 (5 Pt 2): H1791-802).Narrow and the infraction of treatment arteries generally adopts medicine and surgical operation, as adopting blood vessel expansion medicine and fat-reducing medicament, surgery bypass operation of coronary artery and subcutaneous implantation coronary artery urethroptasty etc.In the U.S., the patient who accepts the surgery bypass operation of coronary artery every year reaches more than 450,000, the patient of subcutaneous implantation coronary artery urethroptasty have more than 350,000 (Vascular endothelial growth factor-2 (VEGF-2). Http:// www.hgsi.com/products/vegf.html).Although vascular operation development is very fast, exist the blood vessel repair place narrow and tear open the problem that " Dong Qiang " benefit " Xi Qiang " and blood circulation can not perfect reconstruction (Xiao Jun, Yang Tingshu, Zhang Zhaoshan etc., 2001,12 (3): S41-S42).To suffering from the patient of chronic acute lower limb ischemia, often invade long because of the degree and the anatomical position of angiemphraxis, can't accept the treatment of surgical operation and artery balloon expanding revascularization, thereby these conditions of patients deteriorations are very fast, press for more effective methods of treatment.At present, some treatment vascular disease medicine general effects that use clinically are all undesirable, and toxicity, side effect is bigger.In recent years, people begin to utilize some somatomedin, and it is imported ischemic tissue, promote neovascularity to generate, and to improve the tissue ischemia anoxic condition, treatment has positive meaning to the chronic occlusion vascular disease for this.Therefore, seek the medicine that promotes blood vessel initiatively to rebuild and become one of focus of present medical research.

A series of dependent events are taking place in vasculogenesis, comprise that the differentiation of endotheliocyte, tubulose form and mature blood vessel, regulation and control (Risau W, Flamme I, Annu RevCell Dev Biol, 1995, the 11:73-91 of the inside and outside a series of factors of its generating process acceptor; Risau W, Nature, 1997,386 (6626): 671-674).At present the known factor with angiogenic growth effect has acid and Prostatropin, vascular epidermis somatomedin, α and β transforming growth factor, platelet-derived growth factor, rhIGF-1 and vascular endothelial growth factor (the vascular endothelial growth factor of discovery recently, (Neufeld G such as VEGF), Cohen T, Gengrinovitch S, et al, FASEB J, 1999,13 (1): 9-12).To the research of these several short angiogenesis factors, other all lacks cell-specific except that VEGF according at present.VEGF is the vasoactive endotheliocyte specifically, promotes its propagation strongly, promotes vascularization, and the effect of tool rising vascular permeability (Leung DW, Cachianes G, Sanzo WJ, etal, Science, 1989,246 (4935): 1306-1309).People have produced very big interest to this important biology exceptional function, attempt VEGF is imported the local asphyxia position, to improve vascular permeability, induce new vessel to take place, and improve focus zone blood circulation and oxygen supply situation.This is a kind of brand-new scheme for the treatment of vascular disease, for treatment cerebral arteries infraction, cardiac muscle and limb ischemia disease have been opened up a new way.But the produced in vitro of VEGF be its as a kind of medicinal application in clinical prerequisite, must solve in advance.

VEGF is a multigene family, comprises VEGF-A (Neafeld G, et al, Ccancer Metastasis Rev, 1996,15 (2) 153-158), VEGF-B, VEGF-C, VEGF-D (Joukov V, Kaipainen A, JeltschM, et al, J Cell Physiol, 1997,173:206-210), VEGF-E (Ogawa S, Oku A, Sawano A, et al, J Biol Chem, 1998,273:31273-31282) and placenta growth factor (PLGF) (MaglioneD, Guerriero V, Viglietto G, et al, Proc Natl Sci USA, 1991,88:9267-9271) etc., they all are a kind of glycosylation secreted polypeptide factors, connect into homodimer with disulfide linkage, the about 34～46kD of molecular mass.The VEGF-A that is separated to from human body has 5 kinds of varients (isoform), (annotate: the amino-acid residue number of indication does not comprise the hydrophobic signal sequence that 26 amino-acid residues of N-end constitute to contain 121,145,165,189 and 206 amino-acid residues respectively, because these 26 amino-acid residues are all identical at each varient, back VEGF-1/2/3 is also like this), form (NeafeldG, et al, Ccancer Metastasis Rev by the different montages of mRNA, 1996,15 (2) 153-158).The cell of most of VEGF expression can produce multiple VEGF varient simultaneously, but with VEGF ₁₂₁, VEGF ₁₆₅The most common, secondly be VEGF ₁₈₉, and VEGF ₁₄₅Only in the cell of reproductive organ, express.Each varient is different with heparin and heparin sulfate bonding force, and wherein four kinds with heparin and heparin sulfate bonding force size order are: VEGF _189/206＞VEGF ₁₆₅＞VEGF ₁₄₅(Hyder S, Cancer Res, 1998,58 (2): 392-395), and VEGF ₁₂₁Can not connect heparin or heparin sulfate.So, VEGF _121/145/165More easily arrive target cell, VEGF _189/206Can only remain in extracellular matrix.

From mouse embryo tissue, also be separated to VEGF-1, VEGF-2 and VEGF-3 varient, contain 164,120 and 188 amino-acid residues respectively.Compare with VEGF-3, the amino acid of short two kinds of varients (VEGF-2 and VEGF-1) disappearance is positioned at peptide chain carboxy-terminal (Breier G, et al, Development, 1992,114 (2)).

Because people VEGF ₁₄₅Only at reproductive organ cell specific expression, VEGF _189/206Then nearly all be combined on plasma membrane or the basilar membrane and be retained in the extracellular and be difficult to arrive target cell, activity in vivo is not as VEGF ₁₂₁, VEGF ₁₆₅(Neafeld G, et al, Ccancer Metastasis Rev, 1996,15 (2) 153-158) have only VEGF by force _121/165For the most common and more easily arrive target cell, so that adopt mostly in the research of treatment locality ischemic is VEGF ₁₂₁And VEGF ₁₆₅(Yamagishi Si, et al, J Biol Chem, 1997,272 (13): 8723-8730).Among three kinds of VEGF from the mouse embryo, be used for the treatment of the VEGF-2 that has only of locality ischemic research, all the other two kinds are not appeared in the newspapers.Studies show that all succeed with VEGF treatment periphery infraction property vascular disease, cerebral arteries infraction disease and myocardial ischemia research, but the better efficacy of the sort of VEGF does not still have final conclusion actually.Present people VEGF ₁₂₁, VEGF ₁₆₅Still be in preclinical test, have only mouse VEGF-2 to proceed to clinical trial II phase (Yeung PK, Curr Opin Investig Drugs, 2001,2 (6): 796-800).Infer that in view of the above the effect of VEGF-2 on treatment arteries disease may be better.

Using the VEGF treatment matter of utmost importance that vascular disease faced is its validity, and this depends on local VEGF content, vegf receptor (vascular endothelial growth factor receptor, VEGFR) density and both time length in acceptor.Behind the tissue ischemia, local VEGF and Rd thereof raise, but the decline of VEGF amount is too fast, and ischemic tissue's dependence self VEGF can't effective stimulus form collatoral vessel (Bauters C, et al, J Vasc Surg, 1995,21 (2): 314-325).External source VEGF is imported ischemic tissue, ischemic is had better result of treatment, but vegf protein costs an arm and a leg, and unstable in vivo, very easily be degraded, need repetitively administered, thereby do not possess the feasibility of clinical application.With the VEGF gene integration on eukaryon expression plasmid or replication-defective adenoviral DNA; extract purification of Recombinant plasmid DNA or replication-defective adenoviral DNA; naked DNA is injected directly into ischemic tissue; the vegf protein of expressing can be secreted into the outer performance biological effect that combines with its specific receptors of born of the same parents; also can obtain the ideal biological effect even express a small amount of vegf protein, this can overcome the proteic limitation of direct injection of VEGF.Studies have shown that the direct gene transfection can begin to express, and effectively promotes vascularization behind 24h.Its way is to inject artificial simulation ischemic limb, brain or heart part of creating with plasmid as VEGF cDNA carrier or duplicate deficit type recombinant adenovirus gene, result's endotheliocyte in ischemic limb, cardiac muscle and brain reappears and the homonymy vasculogenesis, improved the supply of blood flow of ischemic tissue, the local importing has good therapeutic action (Ono T, Fujino Y, Tsuchiya T, et al, Neurosci Lett, 1990,117:259-263).Be injected directly into after the somebody mixes VEGF DNA and liposome also obtained in the mouse brain cortex genetic expression (Mustonen T, Alitalo K, J Cell Biol, 1995,129:895-898).VEGF naked DNA lead-in mode with regard to three kinds of forms, the naked DNA of duplicate deficit type recombinant adenovirus expression vector and eukaryon expression plasmid adds liposome and mixes the method that imports, have preparation process complexity, shortcomings such as virus diffusion possibility, immunogen and potential toxic side effect inevitably, therefore more people advocates the method with the naked DNA direct injection of eukaryon expression plasmid.

Amount and time length that second aspect that influences VEGF validity is acceptor are because the biologic activity of VEGF plays a role by receptor-mediated.VEGF is by tyrosine kinase receptor mediation (the Petrova TV of the high-affinity of two structurally associateds, et al, Exp Cell Res, 1999,253 (1): 117-130), they are respectively VEGFR-1 (the fms-like tyrosine kinase of 180kD, Flt-1) and the VEGFR-2 of 200kD (kinase insert domain containing receptor/fetal liverkinase, KDR/Flk-1) (Petrova TV, et al, Exp Cell Res, 1999,253 (1): 117-130).The Flt-1 wide expression is in embryo and adult blood endothelial cell, utilization clpp gene division (gene knock out) is rejected the mouse of Flt-1 gene because of there not being vasculogenesis, and cause dead mouse, but what is interesting is that expressing a mouse that lacks the brachymemma Flt-1 gene of Tyrosylprotein kinase can generate normal blood vessels, this explanation Flt-1 gene perhaps be one " bait acceptor " (decoyreceptor), and be not to be a signal transducers (Kim KJ, et al, Nature, 1993,362 (6423): 841-888).KDR mainly is expressed in the vascular endothelial cell of embryonic development period, then expresses decline at the adult blood endothelial cell, though it also wide expression in the vascular endothelial cell of embryonic development period, in adult tissue only for being expressed in lymphatic endothelial cells.Found a new vegf receptor neuropilin-1 recently again, it is the glycoprotein of a cell surface, is the specific acceptor of VEGF165.It not only is expressed in endotheliocyte, and is expressed in the other types cell.Although VEGFR is essential to vascularization, but add the effect (Ma Li that external source VEGFR has reduced VEGF stimulating endothelial cell propagation, king Xiao Ning etc., the Chinese biological goods are learned magazine, 2000,13 (4): 196-200), its reason is considered to external source VEGFR and combines VEGF with the receptor competition on vascular endothelial cell surface, thereby blocked the biologic activity of VEGF, this is to suppressing growth of tumor and transfer, retina and synovium of joint neovascularization, the treatment tumour, diabetic retinopathy and rheumatoid arthritis disease have the clinical medicinal exploitation of potential to be worth, but to seemingly there not being benefit on the cardiovascular disease therapies.

The vegf expression level also is subjected to the influence of other factors, and as Hypoxia, some pathological state such as skin scald, psoriasis, delayed type allergy and nitrogen protoxide (NO) etc. all can promote VEGF synthetic.Hypoxia is the general character that embryonic tissue, some pathological tissues and tumour medium vessels take place, and can't artificially control in the treatment operation.NO is signaling molecule (Michell BJ important in the vascular system, et al, Curr Biol 1999,9 (15): 845-848), when it when the arteries endotheliocyte produces, pass cytolemma rapidly and diffuse to lower floor's muscle cell, VEGF and receptor mrna level thereof are raise, thereby remove the contraction of muscle cell, artery is unfolded, the distribution of controlling blood pressure and blood flow prevents thrombosis.Under anaerobic conditions, when being carried out isolated perfusion, induced lung finds, in perfusate, add SNP (a kind of NO donor), VEGF and receptor mrna level thereof raise, after adding L-NAME (a kind of NO synthetase inhibitors), and VEGF and receptor mrna level decline (Tsurumi Y thereof, et al, Nat Med, 1997,3 (8): 879-886).But NO is how to promote VEGF and the increase of receptor mrna level thereof unknown, and we infer that they may be subjected under the control of NO inductive promotor.NO has the so strong effect of inducing, and but it is the regulatory factor of artificial supply, so separate the inducible promoter that the NO signal drives, make up induction type VEGF expression vector, when injecting VEGF DNA, add the chemicals that produces NO, may play an important role improving VEGF treatment vascular disease effect.

To the progress of VEGF treatment arteries disease, the U.S. maintains the leading position from present countries in the world, Taiwan following closely, other areas still are in the preclinical test stage.U.S. Human Genome Sciences (HGS) utilize studies have shown that of VEGF-2 treatment coronary artery disease (Vascularendothelial growth factor-2 (VEGF-2). Http:// www.hgsi.com/products/vegf.html), when injection of VEGF-2 gene is in ischemic tissue, can make adjacent cells produce VEGF-2 protein, promote the growth of new vessel and lymphatic vessel.The said firm has transferred blood vessel genetics stock company in October, 1997 with the VEGF-2 gene.The blood vessel genetics corporation just carries out the clinical I/II phase on one's body the coronary artery patient to be tested, twice test of suffering from various critical limb part ischemia patients finished, finish for 3 times having in four times of the coronary artery disease tests, the 4th clinical trial is undertaken by food pharmaceuticals office (FDA).It is said, give critical patients with coronary heart disease injection of VEGF gene, patient's cardiac blood flow is increased, thereby patient's daily life quality is improved.Before this, they once accepted various therapies, but all of no avail.In 16 patients that accept this therapy, 9 impaired coronary disease district coronary artery severity of patient have partly or entirely been recovered vigor.After six months, anyone heart trouble of all not reaccessing did not occur than severe complications yet, and anginal weekly average time is reduced to present 2 times by 48 times before treating.In view of the above, they assert that it is effective and safe utilizing the gene therapy of angiogenesis factor treatment cardiovascular disorder.The VEGF preclinical test has also been finished in Taiwan at present, is carrying out the clinical I phase and is testing.From VEGF-2 genomic medicine range of application, it can be used for having severe cardiac angina, the existing irremediable patient of therapeutics, and the lighter patient of coronary artery disease severity.Can replace various cardiac operations with the VEGF-2 gene therapy, or improve the morbidity of having accepted operating patient's curative effect and having alleviated the patient of congested heart obstacle.When being used for the treatment of the limb artery illness, can make patient exempt some resection operation, perhaps reduce the scope of excision.It also can alleviate patient's misery in order to improve the curative ratio of the skin ulcer that is caused by local asphyxia.In addition, also can be used for treating calf pain and the swelling that causes by physical activity.Therefore, the application prospect of VEGF-2 genomic medicine is boundless, has actual exploitation and is worth.

3. summary of the invention

An object of the present invention is: structure contains humanized VEGF promotor (each varient common promotor of human body VEGF-A, it can drive each gene transcription of VEGF-A) expression mouse source property VEGF-2 expression carrier, but its promotor has NO and anoxic induction regulating controlling element, it both can duplicate production VEGF gene with multiple copied in microorganism, can in mammalian cell, efficiently express again, when giving the VEGF of local asphyxia sex organization gene naked DNA, be aided with the NO donor chemicals, just can improve VEGF and promote the new vessel generative capacity, more effectively improve the blood supply situation of ischemic tissue, reach the purpose of more effectively treating vascular disease.

Another object of the present invention provides the microbial cloning of a kind of NO of comprising and hypoxia inducible specific expression promoter and VEGF-2 gene.

Another object of the present invention provides a kind of naked DNA with microorganisms producing and is expelled in the local asphyxia Mammals model, and it is promoted the method that vasculogenesis, the ability of improving the blood supply situation and toxic side effect are identified.

The present invention has at first cloned mouse source property VEGF-2 (mVEGF-2) gene (SEQID NO 1) from the mice embryonic of becoming pregnant, then from newborn infant's placenta clone humanized's VEGF promotor (hVEGF promoter) (SEQ ID NO 3), and mVEGF-2 is driven by hVEGF promoter, insert then among the mammalian expression vector pEGSH, construct the carrier pEGVPV that can in prokaryotic cell prokaryocyte, produce a large amount of DNA and in protokaryon and mammalian cell, all can express with high copy number.Behind this carrier Transformed E .coli DH5 α, obtaining the DNA copy number has more than the 2000/cell, and separated DNA output can reach 1.5mg/L from cell culture.Ischemic type animal model experiment shows, can promote new vessel to form to this naked DNA of ischemic tissue microinjection, strengthens the foundation of collateral circulation, has improved the blood supply of ischemic tissue effectively.Present method may provide a kind of new departure for the treatment of vascular disease, makes vascular endothelial growth factor vasoactive endotheliocyte specifically, promotes its propagation and vascularization strongly, and the effect of rising vascular permeability is arranged.

To influencing the factor of VEGF effect, particularly NO and ischemic all have certain understanding to the hormesis people of VEGF effect performance, but at present in the naked DNA treatment ischemic angiopathy research with the VEGF gene both at home and abroad, all do not have to consider how to utilize this regulatory factor of NO to strengthen the effect of VEGF gene therapy, and what adopt mostly in the carrier that makes up is some viral promotors of can promotor gene in mammalian cell expressing, what started is constitutive expression, and in fact the new vessel generation is a condition of growth of tumour cell, a kind of like this expression vector that does not have control imports the potentially dangerous whether human body has induced tumor, still can't expect.Utilization of the present invention has NO regulatable humanized VEGF promotor and mouse source property VEGF gene, is used the pharmaceutical chemicals that discharges NO when importing ischemic tissue, has improved the result of treatment of VEGF effectively; What adopt simultaneously is the promotor with humanized, and it is dangerous potentially to have got rid of viral promotors, knows from experience more safe and reliable to the people.Such vegf expression carrier at home and abroad there is no report.

4. description of drawings

Fig. 1 mice embryonic RNA RT-PCR product agarose electrophoresis result

Lane1：DGL2000 DNA Markers

Lanes2～4: the pcr amplification product of not isotype pulling (cDNA first chain) consumption, wherein the template amount of Jia Ruing is: 2#1ul; 3#1.5ul; 4#2ul

Fig. 2 BamHI and EcoRI cut the result to two germplasm granzymes

Lane1：DL2000 DNA Markers

Lane2：pUC-V450

Lane3：pET-28c(+)

Lane 4：λDNA/EcoRI+Hind III Markers

Fig. 3 people's placenta dna is made the template pcr amplification product

The Lane1:PCR amplified production

Lane2：λDNA/EcoRI+Hind III Markers

The uciferase activity fluorescence microscopy is observed among Fig. 4 transfection Hela cells

The A.phrGFPVP transfection, the NO stimulation is induced; The B.phrGFPVP transfection, anoxic stimulates; The C.phrGFPVP transfection, the NO+ anoxic stimulates;

The D.phrGFPVP transfection, normal oxygen level; E. unloaded plasmid transfection, anoxic stimulates; F. unloaded plasmid transfection, normal oxygen supply

The formation of the ischemic brain organization center blood vessel of four kinds of processing of Fig. 5 relatively

The mVEGF-2 mammalian expression vector that Fig. 6 has the hVEGF promotor makes up synoptic diagram

5. embodiment

Embodiment 1. mouse source property VEGF-2 gene and the Identification of Fusion Protein in the microbial expression system thereof

(GenBank:S38100 gi:249860) designs a pair of special primer, has introduced BamH I and EcoR I site respectively at 5 ' end of this a pair of primer according to known mVEGF-2 nucleotide sequence.

Bam HI

VP-1：5’- GCC TCC GGA TCCATG AAC TTT CTG-3’

VP-2：5’- GAA TTCACC GCC TCG GCT TGT C-3’

Eco RI

In becoming pregnant the mouse body in 2 weeks, execution takes out the embryo, with the total RNA extraction reagent box (product-TRIZOL of GIBCO company ^Reagent Cat.No.15596) extracts total RNA.With the total RNA of embryo is template, Oligo (dT) ₁₅Make primer, synthetic cDNA first chain under AMV Reverase (AMV-RT) effect.Then, the reverse transcription product dilution is made template for 50 times, add synthetic primer (VP-1 and VP-2), dNTP and Taq DNA polymerase, the laggard performing PCR amplification of mixing.The pcr amplification reaction parameter is:

Amplification obtains three fragments (Fig. 1) of the about 450bp of molecular weight, 580bp and 650bp.

Because the VEGF of mice embryonic has three kinds of varients (VEGF-1, VEGF-2 and VEGF-3), contain 164,120 and 188 amino-acid residues (not comprising the signal peptide that 26 amino acid of N end are formed) respectively.Compare with VEGF-3, the amino acid of short two kinds of varients (VEGF-2 and VEGF-1) disappearance is positioned at the peptide chain carboxy-terminal, difference on the cDNA base sequence is that the difference disappearance of nearly 3 ' end causes, their threes are identical with 3 ' terminal sequence 5 ', therefore, 3 special bands have been gone out with identical a pair of primer amplification.

The specific band (because the cDNA length of coding VEGF-2 varient is 450bp) that is about 450bp with molecular weight in the PGR Fragment Recovery Kit recovery PCR product.Under the effect of T4 dna ligase, will reclaim fragment and be connected, transformed competence colibacillus E.coli DH5 α with pUCm-T Vector.(available from ancient cooking vessel state biotechnology limited liability company product, Cat.No.Y009) select white single bacterial plaque and shake bacterium, adopt the alkaline lysis trace to extract plasmid.By hysteresis plasmid screening, enzyme is cut and PCR identifies, tentatively is defined as recon, submits to Shanghai United Gene Science Co., Ltd to check order.Because pUCm-T Vector is used for carrier (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product that the PCR product inserts and clones, Cat.No.D0211), can check order to the PCR product with M13 universal primer and T7 promoter primer, we adopt the M13 universal primer when requiring Shanghai connection polygene company to check order.Sequencing result is SEQ ID NO 1, and its amino acid sequence coded is SEQ ID NO 2, with the VEGF-2 sequence (S38100 among the GenBank, gi:249860) carry out homology relatively, identical rate reaches 98.5%, and conclusive evidence obtains the VEGF-2 gene, with this recon called after pUC-V450.

Cultivate pUC-V450/E.coli DH5 α and pET-28c (+)/E.coli BL21 (available from U.S. Novagen company, Cat.No.69258 and 69387-3) respectively, extract plasmid.According to the restriction enzyme site of pUC-V450 and pET-28c (+), select for use Bam HI and Eco RI respectively this two germplasms granzyme to be cut (Fig. 2); From pUC-V450 plasmid enzyme restriction liquid, reclaim about 470bp (comprising restriction enzyme site) fragment, reclaim the 5363bp fragment in pET-28c (+) the plasmid enzyme restriction liquid; Under the effect of T4 dna ligase, reclaim fragment with above-mentioned two and connect, be built into prokaryotic expression carrier, with this recombinant plasmid called after pET-V450.With pET-V450 transformed competence colibacillus e. coli bl21, be applied to overnight incubation on the LB solid medium that adds kantlex (50mg/L) then.Next day, picking from the transformed bacteria coating plate was isolated 8 of good single bacterium colonies, extracted plasmid after shaking bacterium on a small quantity, and PCR identifies all positive.

According to pET-28c (+) Kit test kit (U.S. Novagen company-pET System Manual, Www.novagen.com)The method of specification sheets is carried out abduction delivering with IPTG to the clone bacterium, and the 20kD protein molecular of outcome expectancy is able to high level expression.

The clone of embodiment 2. humanized VEGF promotors and green fluorescent protein (GFP) gene transient expression vector construction and Function Identification thereof

(GenBank:AF095785 gi:4154290), is 3401bp to the existing report of humanized VEGF gene (hVEGF) upstream sequence, and big like this fragment cloning difficulty is bigger.According to the general characteristic of eukaryotic promoter, the main element of promotor is usually about the 1kb of transcription initiation site upstream.According to Hideo etc. (Hideo K, et al.Blood, 2000, research 95:189-197), we think that further the fragment of-1041～-1 (from the transcription initiation site orderings) has comprised the primary element of promotor.But at (Hideo K such as Hideo, et al.Blood, 2000, do not consider in research 95:189-197) 5 ' end non-translational region (5 '-UTR) (and refer to transcription initiation site after beginning+1～+ 1038 fragment), people recognize that more and more 5 '-UTR plays important effect to gene expression regulation now.Therefore, we also clone 5 '-UTR fragment in the lump, and its expression regulation ability is identified.

1.hVEGF the clone of promotor

According to (Brogan IJ such as Brogan, et al, Hum.Immunol.1999,60 (12): 1245-1249) Bao Dao humanized VEGF gene and promoter sequence (GenBank:AF095785, gi:4154290), design and synthesize following two primers after the analysis-by-synthesis:

Not I

Primer VPP-1:5 '-GC G CGG CCG CCT GTC TGC

Bam HI

Primer VPP-2:5 '-CGC GGA TCCGGT TTC GGA GGC CCG ACC GGG-3 '

5 ' end at this a pair of primer has been introduced Not I site and Bam HI site respectively.

With the total DNA of people's placenta is template, by pcr amplification (94 ℃, 3min; 94 ℃ of 40sec, 65 ℃ of 40sec, 72 ℃ of 2min circulate 30 times; 72 ℃ of 10min mend flat) obtain the product (Fig. 3) of about 2.06kb, be connected with pUCm-T Vector after reclaiming purifying, with connecting product Transformed E .coli DH5 α competent cell, screening positive clone also extracts recombinant plasmid, carry out enzyme and cut evaluation and pcr amplification evaluation, preliminary definite hVEGF promoter fragment has been inserted into T-Vector, submits to genome company to check order, and its sequence measurement and use primer are as aforementioned.Sequencing result is shown in SEQ ID NO 3, with humanized VEGF gene (hVEGF) upstream sequence (AF095785 among the GenBank, gi:4154290) carried out homology relatively, the rate of coming to the same thing reaches 98.2%, prove conclusively this plasmid thus and contain the hVEGF promoter sequence, with this recombinant plasmid called after pUCVP.

2.hVEGF promoter function and CHARACTERISTICS IDENTIFICATION

(U.S. STRATAGENE company product, German MERCK corporate agent is Cat.No.#240063) as mammalian expression vector, in order to identify hVEGF promoter function and characteristic to select phrGFP Vector for use.This carrier has green fluorescent protein (hrGFP) reporter gene of hommization, is low toxicity to transducer cell, does not influence cell activity and be convenient to expression level to detect; Have multiple clone site at the hrGFP upstream region of gene, be convenient to promotor or enhanser are inserted, its function is identified.With Bam HI and Not I, respectively phrGFP Vector and pUCVP enzyme are cut, reclaim 3670bp (the big fragment of phrGFP Vector after Bam HI and Not I enzyme are cut) and 2078bp fragment (comprising the Not I of hVEGF promotor 2064bp fragment and adding and the 14bp of Bam HI restriction enzyme site), mix, under the effect of T4DNA ligase enzyme, connect, import E.coli DH5 α, be applied on the plate of the LB solid medium that contains Amp and cultivate 14h.Select single bacterium colony and shake bacterium, extract plasmid, carry out PCR and identify that positive recombinant is the mammalian expression vector with hVEG promotor, called after phrGFPVP.

3. the evaluation of transient expression

In Φ 10cm tissue culture ware, add 20 μ l lipofection thing (Life Technologies, Rockville, MD) and the cell of the heLa cells of concentrated 20%-30% (Hela cells), (5 μ g phrGFPVP) carries out transfection with vector plasmid, compares with empty carrier phrGFP Vector simultaneously.Under 37 ℃, transfection is hatched and is cultivated 15h.After the ticket reserving time is cultivated in transfection, replace the substratum that contains DNA, give aerobic (21%O with normal substratum ₂), anoxic (1%O ₂), NO donor chemical reagent S-nitroso-group-N-ethanoyl-D, the processing such as (SNAP) of L-Trolovol continues down to cultivate 12h at 37 ℃.

Scrape cell, be dissolved in 200 μ l 0.25mol/L Tris-Cl (pH7.5), at 4 ℃ of centrifugal 10min collecting cells of 270g.With the resuspended precipitation of damping fluid, place 10min on ice, aspirate 5-8 time repeatedly so that homogeneity with syringe then.With 10 * damping fluid (40mmol/L Tris-Cl[pH7.9], 10mmol/LEDTA[pH8.0], 150mmol/L NaCl) cell collected, be resuspended in whole cell and extract damping fluid (10mmol/L N-2-hydroxyethyl piperazine-N '-2 ethane sulfonic aicd [pH7.9]; 400mmol/L NaCl; 0.1mmol/L EDTA; 5%[vol/vol] glycerine; 1mmol/L DTT; And 1mmol/L phenylmethyl sulfonylfluoride).Then under 4 ℃ of conditions, 16, the centrifugal 30min of 000g.Supernatant liquor be housed in-70 ℃ standby.

100 μ l cell extracts and 225 μ l luciferase reagents are mixed, in order to identify uciferase activity.Behind 37 ℃ of hatching 0.5-1h, add 50 μ l 1mol/L Na ₂CO ₃Termination reaction.Observe and take pictures at the fluorescence microscopy mirror image, measure A simultaneously ₄₂₀Absorbancy.Measure the plain enzymic activity (mean value ± standard error) of relative fluorescence with betagalactosidase activity as the standard of uciferase activity, with the plain enzymic activity of stimulated cells relative fluorescence to the ratio of non-stimulation contrast as the induced activity of measuring conversion.

According to observed result, uciferase activity difference in the transfectional cell of different treatment is very obvious, and several no luciferase expressions in the usefulness empty carrier cells transfected (Fig. 4-E, F), and luciferase expression (Fig. 4-A is arranged all in having hVEGF promotor phrGFPVP cells transfected, B, C, D), anoxic and NO stimulate the expression (Fig. 4-A that can improve luciferase significantly, B C), illustrates that clone's hVEGF promotor has the characteristic of induction regulating controlling.The absorbance measurement of uciferase activity (result slightly) also obtains same result.

Embodiment 3.hVEGF promotor and VEGF-2 mammalian expression vector make up and the experiment of ischemia animal pattern

1.hVEGF the structure of promotor and mVEGF-2 mammalian expression vector

(U.S. STRATAGENE company product, German MERCK corporate agent is Cat.No.#217461) for inserting the expression vector of hVEGF promotor and mVEGF-2 to select pEGSH for use.Why select this carrier, mainly be the consideration for three aspects: 1. this plasmid has ColE1 replication origin and Amp selective marker, be relaxed plasmid, can in microorganism, exist, help utilizing microorganism to come High-efficient Production hVEGF promotor and mVEGF-2 naked DNA with the multiple copied form; The existence of selective marker can prevent that recombinant plasmid from losing in the host bacterium, help keeping engineering bacteria stability; 2. have multiple clone site (MCS), be convenient to the insertion of foreign gene; 3. the virus-free promotor of this plasmid, the poly-adenosine effect section that has simian vacuolating virus 40 (SV40) in the MCS downstream, after being transcribed, the external source functional gene adds poly (A), this is very important to keeping the transcription of foreign genes product stability and improving translation skill, introduces this plasmid according to products instruction and can make the exogenous gene expression level improve 1200～1700 times.

Respectively pUCVP plasmid and pEGSH enzyme are cut digestion with Not I and Bam HI, reclaim the 2078bp fragment and (comprise hVEGF promotor 2064bp fragment, and comprise the Not I of adding and the 14bp of Bam HI restriction enzyme site) and 4812bp (the big fragment after the pEGSH enzyme is cut) fragment, under the effect of T4DNA ligase enzyme, connect, import competence E.coli DH5 α, being coated on the solid medium that contains Amp screens, picking is isolated good single bacterium colony and is shaken bacterium, extract plasmid, the plasmid that lags behind screening and PCR identify, positive recombinant promptly is the plasmid that contains the hVEGF promotor, called after pEGVP.

Then with Bam HI and Eco RI respectively enzyme cut pEGVP and pUC-V450, reclaim 6890bp (the big fragment of pEGVP after Bam HI and Eco RI enzyme are cut) and 474bp (VEGF-2cDNA and Bam HI and Eco RI site) fragment, connect as stated above, transform and identify, positive colony called after pEGVPV (Fig. 6).

2. ischemia animal pattern result of experiment

(1) animal model is made and grouping: selecting rat for use is laboratory animal.For examination rat brain ischemia model make the line bolt method of pressing Longa etc. (Longa EZ et al.Stroke, 1989,20:84-91) carry out; The ischemic animal model is divided into four groups: inject naked pEGVPV plasmid DNA (containing hVEGF promotor and 5 ' UTR and mVEGF-2cDNA) for one group; another organizes the naked pEGVPV plasmid DNA of injection injection (containing hVEGF promotor and 5 ' UTR and mVEGF-2cDNA)+NO donor chemical reagent S-nitroso-group-N-ethanoyl-D; L-Trolovol (SNAP); inject naked pEGSH (zero load) plasmid DNA+S-nitroso-group-N-ethanoyl-D for the 3rd group; L-Trolovol (SNAP); the 4th group of injecting normal saline (contrasts, CK).Each treated animal all will reach 4～6.

(2) gene transfection:, in analogue formation, inject corresponding naked plasmid dna (or physiological saline and SNAP) to zone of ischemia immediately with microsyringe according to above four groups of designs.In cerebrum ischemia animal model group, cranium is opened an aperture before should being drilled in the hand sth. made by twisting earlier, injects naked plasmid dna, unloaded plasmid or SNAP then, sews up a wound.

(3) the VEGF-2 gene expression dose detects: gene imported after 5 days, extracted RNA from ischemic tissue, and one group of amplimer with VEGF-2 behind the reverse transcription carries out pcr amplification, carries out electrophoretic analysis then.

(4) the VEGF-2 expression of gene protein detects: gene imported after 7 days, got animal model group ischemic region tissue, after fixing, embedding, the quick-frozen, cut into slices on freezing-microtome, carried out immunochemistry with polyclonal antibody and immuning tissue's detection kit and dyeed microscopy.

(5) vascular counts: 10d after the administration, get an animal for every group and do angiography.Anesthesia down 4F conduit is injected 76% Compound Diatrzoatc Meglumlne, speed 3ml/s, usefulness X-ray sheet Taking Pictures recording behind the 4s through left common carotid artery 3cm place to the aorta abdominalis.

(6) embolism stereometry: tissue carries out freezing microtome section, dyeing after fixing, embedding, quick-frozen, measures the infarct size and the total area on ias, is converted to volume then.

(7) analysis of statistical data: enumeration data is represented with relative number, with two groups of independent sample X ²Statistical procedures is carried out in (card) check; Measurement data is represented with x ± s, carries out statistical procedures with two groups of independent sample t checks.Statistical software adopts the SAS software package.

Experimental result shows, compare with the control group that shifts unloaded plasmid and injecting normal saline, transfer has behind the mVEGF-2 gene of hVEGF promoters driven in 5 days the rat cerebral tissue expression of mRNA is arranged, and the expression amount of injecting mRNA in the experimental group rat cerebral tissue of SNAP simultaneously is only to inject 2～3 times that contain hVEGF promotor and mVEGF-2 gene plasmid.The visible VEGF-2 protein expression level of VEGF-2 immunohistochemical staining increases, cerebrovascular number increases (Fig. 5), infarct volume dwindles (table 1), and it is the most obvious to have the experimental group effect of the mVEGF-2 gene plasmid naked DNA of hVEGF promotor and NO releasing agent (SNAP) with injection simultaneously especially.

Four kinds of treatment combinations of table 1 are to reducing the rat cerebral infarction volume

The ischemic tissue of brain injection treatment	Infarct volume/big brain volume (%)				X±s(％)
						1	2	3	4
	Naked pEGVPV DNA naked pEGVPV DNA+SNAP naked pEGSH DNA+SNAP physiological saline (CK)	11.3 8.2 16.3 26.1	10.7 6.0 18.4 23.3	8.6 5.3 14.2 21.7						9.4 5.7 18.8 20.5	10.0±1.2 * 6.3±1.3 ** 16.8±2.0 22.9±2.4

^*Contrast reaches the conspicuous level of P＜0.05;

^*Contrast reaches the conspicuous level of P＜0.01.

The information of SEQ ID NO 1

Sequence signature:

(A) (length): 457 bases

(B) type: nucleic acid

(C) chain: two strands

(D) (topological framework): linearity

Sequence description: SEQ ID NO 1:

Initiation codon ↓

1 GCCTCCGGAT CC ATGAACTT TCTGCTGTCT TGGGTGCACT GGACCCTGGC

51 TTTACTGCTG TACCTCCACC ATGCCAAGTG GTCCCAGGCT GCACCCACGA

101 CAGAAGGAGA GCAGAAGTCC CATGAAGTGA TCAAGTTCAT GGACGTCTAC

151 CAGCGAAGCT ACTGCCGTCC AATTGAGACC CTGGTGGACA TCTTCCAGGA

201 GTACCCCGAC GAGATAGAGT ACATCTTCAA GCCGTCCTGT GTGCCGCTGA

251 TGCGCTGTGC AGGCTGCTGT AACGATGAAG CCCTGGAGTG CGTGCCCACG

301 TCAGAGAGCA ACATCACCAT GCAGATCATG CGGATCAAAC CTCACCAAAG

351 CCAGCACATA GGAGAGATGA GCTTCCTACA GCACAGCCGA TGTGAATGCA

401 GACCAAAGAA AGACAGGACA AAGCCAGAAA AATGTGACAA GCCGAGGCGG

451 TGAATTC

↑ termination codon

The information of SEQ ID NO 2

Sequence signature:

(A) (length): 146 amino acid

(B) type: polypeptide

(C) chain: strand

(D) (topological framework): linearity

Sequence description: SEQ ID NO 2:

Signal peptide

1 MNFLLSWVHW TLALLLYLHH AKWSQAAPTT FGEQKSHEVI KFMDVYQRSY

51 CRPIETLVDI FQEYPDEIEY IFKPSCVPLM RCAGCCNDEA LECVPTSESN

101 ITMQIMRIKP HQSQHIGEMS FLQHSRCECR PKKDRTKPEK CDKPRR

The information of SEQ ID NO 3

Sequence signature:

(A) (length): 2064 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) (topological framework): linearity

Sequence description: SEQ ID NO 3:

The NF-kB-like sequence

1 CTGTCTGCCC AGCTGCCTCC CCCTTT GGGT TTTGCCAGAC TCCACAGTGC

HIF-1 binding site HIF-1 auxiliary sequencel AP-1 site

51 A TACGTGGGC TCCA ACAGGT CCTCTTCCCT CCCAGTCAC T GACTAACCCC

101 GGAACCACAC AGCTTCCCGT TCTCAGCTCC ACAAACTTGG TGCCAAATTC

151 TTCTCCCCTG GGAAGCATCC CTGGACACTT CCCAAAGGAC CCCAGTCACT

201 CCAGCCTGTT GGCTGCCGCT CACTTTGATG TCTGCAGGCC AGATGAGGGC

251 TCCAGATGGC ACATTGTCAG AGGGACACAC TGTGGCCCCT GTGCCCAGCC

301 CTGGGCTCTC TGTACATGAA GCAACTCCAG TCCCAAATAT GTAGCTGTTT

351 GGGAGGTCAG AAATAGGGGG TCCAGGAGCA AACTCCCCCC ACCCCCTTTC

401 CAAAGCCCAT TCCCTCTTTA GCCAGAGCCG GGGTGTGCAG ACGGCAGTCA

451 CTAGGGGGCG CTCGGCCACC ACAGGGAAGC TGGGTGAATG GAGCGAGCAG

501 CGTCTTCGAG AGTGAGGACG TGTGTGTCTG TGTGGGTGAG TGAGTGTGTG

551 CGTGTGGGGT TGAGGGTGTT GGAGCGGGGA GAAGGCCAGG GGTCACTCCA

601 GGATTCCAAC AGATCTGTGT GTCCCTCTCC CCACCCGTCC CTGTCCGGCT

651 CTCCGCCTTC CCCTGCCCCC TTCAATATTC CTAGCAAAGA GGGAACGGCT

701 CTCAGGCCCT GTCCGCACGT AACCTCACTT TCCTGCTCCC TCCTCGCCAA

751 TGCCCCGCGG GCGCGTGTCT CTGGACAGAG TTTCCGGGGG CGGATGGGTA

801 ATTTTCAGGC TGTGAACCTT GGTGGGGGTC GAGCTTCCCC TTCATTGCGG

851 CGGGCTGCGG GCCAGGCTTC ACTGGGCGTC CGCAGAGCCC GGGCCCGAGC

901 CGCGTGTGGA GGGGCTGAGG CTCGCCTGTC CCCGCCCCCC GGGGCGGGCC

951 GGGGGCGGGG TCCCGGCGGG GCGGAGCCAT GCGCCCCCCC CTTTTTTTTT

1001 TAAAAGTCGG CTGGTAGCGG GGAGG TCGC GGAGGCTTGG GGCAGCCGGG

-1+1 (transcription initiation site)

1051 TAGCTCGGAG GTCGTGGCGC TGGGGGCTAG CACCAGCGCT CTGTCGGGAG

1101 GCGCAGCGGT TAGGTGGACC GGTCAGCGGA CTCACCGGCC AGGGCGCTCG

1151 GTGCTGGAAT TTGATATTCA TTGATCCGGG TTTTATCCCT CTTCTTTTTT

1201 CTTAAACATT TTTTTTTAAA ACTGTATTGT TTCTCGTTTT AATTTATTTT

1251 TGCTTGCCAT TCCCCACTTG AATCGGGCCG ACGGCTTGGG GAGATTGCTC

1301 TACTTCCCCA AATCACTGTG GATTTTGGAA ACCAGCAGAA AGAGGAAAGA

1351 GGTAGCAAGA GCTCCAGAGA GAAGTCGAGG AAGAGAGAGA CGGGGTCAGA

1401 GAGAGCGCGC GGGCGTGCGA GCAGCGAAAG CGACAGGGGC AAAGTGAGTG

1451 ACCTGCTTTT GGGGGTGACC GCCGGAGCGC GGCGTGAGCC CTCCCCCTTG

1501 GGATCCCGCA GCTGACCAGT CGCGCTGACG GACAGACAGA CAGACACCGC

1551 CCCCAGCCCC AGCTACCACC TCCTCCCCGG CCGGCGGCGG ACAGTGGACG

1601 CGGCGGCGAG CCGCGGGCAG GGGCCGGAGC CCGCGCCCGG AGGCGGGGTG

1651 GAGGGGGTCG GGGCTCGCGG CGTCGCACTG AAACTTTTCG TCCAACTTCT

1701 GGGCTGTTCT CGCTTCGGAG GAGCCGTGGT CCGCGCGGGG GAAGCCGAGC

1751 CGAGCGGAGC CGCGAGAAGT GCTAGCTCGG GCCGGGAGGA GCCGCAGCCG

1801 GAGGAGGGGG AGGAGGAAGA AGAGAAGGAA GAGGAGAGGG GGCCGCAGTG

1851 GCGACTCGGC GCTCGGAAGC CGGGCTCATG GACGGGTGAG GCGGCGGTGT

1901 GCGCAGACAG TGCTCCAGCC GCGCGCGCTC CCCAGGCCCT GGCCCGGGCC

1951 TCGGGCCGGG GAGGAAGAGT AGCTCGCCGA GGCGCCGAGG AGAGCGGGCC

2001 GCCCCACAGC CCGAGCCGGA GAGGGAGCGC GAGCCGCGCC GGCCCCGGTC

2051 GGGCCTCCGA AACC

Claims (8)

1, a kind of carrier that includes humanized VEGF promotor and mouse source property VEGF-2 cDNA, it is characterized in that: the hVEGF promotor and the mVEGF-2 orientation of this carrier link together, the hVEGF promotor is positioned at the mVEGF-2 upstream, and the mVEGF-2 gene is driven by the hVEGF promotor.

2, a kind of carrier as claimed in claim 1, wherein mouse source property VEGF-2 gene nucleotide series is SEQ ID NO 1.

3, a kind of carrier as claimed in claim 1, wherein the nucleotides sequence of humanized VEGF promotor is classified SEQ ID NO 3 as.

4, a kind of method that makes up as claim 1-3 carrier as described in each.

5, a kind of microbial cloning that contains just like each described carrier of claim 1-3.

6, a kind of method of each described carrier of production claim 1-3 is characterized in that, each described carrier of claim 1-3 is transformed into recipient bacterium, recipient bacterium is shaken bacterium activation after, therefrom extract plasmid DNA purification in a large number.

7, a kind of purposes of each described carrier of claim 1-3 is characterized in that this carrier is as the application in the genomic medicine of preparation treatment vascular disease.

8, a kind of purposes of each described carrier of claim 1-3, it is characterized in that this carrier in the mode of naked DNA as the application in the genomic medicine of preparation treatment vascular disease.

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