CN100372572C - Recombinant plasmid with human thymosin Beta-4gene - Google Patents
Recombinant plasmid with human thymosin Beta-4gene Download PDFInfo
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- CN100372572C CN100372572C CNB200510105792XA CN200510105792A CN100372572C CN 100372572 C CN100372572 C CN 100372572C CN B200510105792X A CNB200510105792X A CN B200510105792XA CN 200510105792 A CN200510105792 A CN 200510105792A CN 100372572 C CN100372572 C CN 100372572C
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Abstract
A recombinant plasmid with human extrasin beta4-gene carrier is prepared by inserting human extrasin beta4-gene into eukaryote expression starter to construct recombinant plasmid, entering it into striped muscle cell, synthesizing T beta-4 by cell protein synthetic system and secreting to extracellular region. It can improve wound healed, prevent cell from death and be used to repair skin and heart damages.
Description
One. technical field
The invention belongs to field of gene, specifically, be inserted under the eukaryotic expression promoter a kind of recombiant plasmid that is built into exactly with the human thymosin Beta-4 gene.This plasmid can enter striated muscle cell, and utilizes the protein-synthesizing system of cell itself in cell, synthetic T β 4, and be secreted into outside the born of the same parents, thereby the healing of accelerated in wounds, and the death that stops cell.
Two. background technology
Immune system is the system of defense of human body.Participate in immunoreactive main cell T lymphocyte, bone-marrow-derived lymphocyte and macrophage are arranged.And thymus is the immune maincenter organ that the T lymphocyte is grown, broken up.The excretory thymic factor of thymus (or hormone) is that the T lymphocyte is grown the required a series of essential material of differentiation, and wherein extrasin beta 4 (T β 4) is the existing comparatively clear and definite a kind of main thymic factor of 26S Proteasome Structure and Function.As an institute of making of the activity of T β 4 (a kind of albumen that relates to cell migration and survival among the embryo who is growing) show, the survival ability of embryo and birth back myocardial cell in " thymosin-beta 4 " enhancing tissue culture, this albumen of injection in rat coronary artery ligation posterior peritoneum can successful cardiac stimulus reparation and activation A k t " survival kinase ".These presentation of results, " thymosin-beta 4 " is a possible therapeutic goal for acute coronary occlusion.The research worker of Texas university finds that a kind of protein of making (extrasin beta-4) can help the self-regeneration of heart organ after heart attack in the heart development process.These discoveries that obtain in rat experiment finally may cause the appearance of new cardiotherapy and may change medical personnel to cardiopathic processing mode.
Extrasin beta-the 4th, a kind of embryo is expressed proteins in the heart development process, and it can promote the migration of heart cell and influence the final and decisive juncture of these cells.Recent studies on shows that this albumen can prevent the dead of cell and limit the degree that scar tissue forms after the inductive heart attack of experiment.Extrasin beta-4 has been used to clinical trial, to promote the recovery from illness of skin trauma.Research worker believes that this in the near future albumen will enter the clinical experimental stage of disease treatment.
At present, the T β 4 that uses clinically derives from the albumen form that chemosynthesis or gene recombinaton obtain.Because the 4 albumen form half-life in vivo of T β are very short, this just makes clinical patient must repeatedly use T β 4 in the certain hour section.So only need one to twice use, just gene therapy class T β 4 medicines that 4 pairs of damaged tissues of expression T β work in a period of time in vivo will reduce every cost greatly.But be used to carry T β 4 at present and mainly contain adenovirus vector (AV), gland relevant viral vector (AAV) as Vectors in Gene Therapy, in these two kinds of carriers, AAV uses more, mainly be because their virus characteristic, make transfection efficiency very high, the gene that is easy to carry about with one enters human body cell, yet just because of their virus characteristic, makes the safety of gene therapy become the problem that receives much attention.Compare with AAV with AV, recombiant plasmid is because its no life form, unless enter cell, very fast disappearance makes the safety of gene therapy improve greatly.
Three. summary of the invention
The present invention at first provides a kind of gymnoplasm grain that utilizes as carrier; transportation T Beta-4 gene removes to synthesize, secretes T β 4 albumen in cell; and damaged cell in the surrounding tissue is worked by this albumen; the reorganization gymnoplasm grain gene treatment that stops cell death is with T β 4, especially at diseases such as coronary heart disease, intractable cutaneous ulcer.Present research has proved that T β 4 is a kind of albumen that plays an important role when tissue is impaired, it can stimulate the migration of superficial cell and horn cell, the expression of regulating course Fibronectin, and vasostimulant generation, stop the death of cell even, so, people have begun to be used for T β 4 clinical, the patient who has a superficial wound just treats with T β 4 in RegenRx company such as the U.S., now find that more the effect of T β 4 on heart disease is also fine, but the albumen form of the T β 4 that all is to use during above-described clinical treatment, this mode is the usual way of present disease treatment, half-life in vivo is short but its shortcoming is T β 4 albumen, patient will repeatedly medication in a period of time, this has just increased patient's treatment cost greatly, address this problem, the present invention has expected gene therapy, simple effective method in the gene therapy, select viral vector to carry T β 4 exactly and enter cell and expressing protein, but characteristic just because of viral vector, make the safety of this kind method become very big problem, be afraid of that particularly carrier enters sexual cell, important cells such as stem cell.Everybody thinks safety than higher at present to select plasmid vector for use, and is very difficult but it enters cell, and people usually must select for use external conditions such as virus coat, particle gun, liposome to assist plasmid to enter cell.This just makes complicated operation, and the expression effect of destination protein is undesirable, the present invention constructs a kind of plasmid vector of the T of having Beta-4 gene, this recombinant plasmid vector is a gymnoplasm grain carrier, do not contain virus coat and liposome, also do not use particle gun in use, only the intramuscular that plasmid is expelled to lesions position can produce T β 4 albumen and damaged tissues is worked.
Secondly the present invention provides a kind of recombinant plasmid vector pNL-T β 4, and first feature of this plasmid is the nucleotide sequence that contains T β 4 full length amino acid sequence correspondences; Second feature is that this plasmid contains the self replication district ColEI sequence in the escherichia coli, human cytomegalic inclusion disease virus promoter p (CMV) sequence, kalamycin resistance gene sequence, the polyA signal transcription terminator sequence of bovine growth hormone gene; The 3rd feature is after this plasmid possesses above-mentioned two features, size does not surpass 4kb, make pNL-T β 4 can utilize the ditch of striocellular cell surface to return structure and enter the myocyte, utilize protein expression system to synthesize T β 4 again, be secreted into the outer damaged cell of born of the same parents and work surrounding tissue.
In experimentation, find to have injected in the myocardium meat tissue of pNL-T β 4 continuous release T β 4 in month, and T β 4 is with other two kinds of albumen, by activating migration and the survival that the Akt/ protein kinase B promotes myocardial cell.After having studied the cultured cells activity, the present invention has a heart attack the coronary artery ligation simulation of 20 adult rats, thereby creates rat model.Only then give 10 rat local injection thymosin T β 4250ug/, give 10 rat local injection saline 200uL as blank.The rat of injection pNL-T β 4 after January the cell in the damaged heart seldom dead, and send out the back in heart disease and improved cardiac function several weeks.And the cell mortality of control rats damaged heart.Result of the test shows that plasmid pNL-T β 4 enters the myocyte, and makes the myocyte secrete T β 4, and T β 4 can change the metabolism of cell and create more powerful myocardial cell, and these cells can be resisted the hypoxia situation after heart disease is sent out.If this albumen is effective equally to the mankind, so this albumen will become a kind of strong disease treatment medicine.
Four. description of drawings
Fig. 1 is the structure collection of illustrative plates of pNL-T β 4.
Fig. 2 is the Agarose purity electrophoresis pattern behind the purification of pNL-T β 4.
Fig. 3 is to behind the rat model injection pNL-T β 4, to the result that influences of rat heart wall thickness.
Fig. 4 is to behind the rat model injection pNL-T β 4, to the result that influences of rat heart wall fibrosis.
Fig. 5 is to behind the mouse model injection pNL-T β 4, T β 4 albumen expression in vivo.
Five. the specific embodiment
Following example only is for to explanation of the present invention, and can not cause any restriction to the present invention.
One), the structure of gymnoplasm grain pNL-T β 4
At first by the synthetic complete genome sequence that amplifies extrasin beta 4 of full gene:
GCT?AGC?ATG?TCT?GGT?GAC?AAA?CCC?GAT?ATG?GCT?GAG?ATC?GAG?AAA?TTC?GAT
AAG?TCG?AAA?CTG?AAG?AAG?ACA?GAG?ACG?CAA?GAG?AAA?AAT?CCA?CTG?CCT?TCC
AAA?GAA?ACG?ATT?GAA?CAG?GAG?AAG?CAA?GCA?GGC?GAA?TCG?TAA?CTC?GAG
The purpose fragment reclaims small fragment respectively behind NdeI, XhoI double digestion, plasmid pNL reclaims big fragment behind XhoI and EcoR I double digestion, 18 ℃ in T4 ligase system three sections genes of interest segments connect recombiant plasmid called after pNL-T β 4 (concrete structure is seen Fig. 1) that obtain through screening.Use CaCl2 method transformed into escherichia coli DH5a then, be coated on the LB flat board that contains the 50ug/ml kanamycin, select positive bacteria and drop on that to be cultured to OD600 among the LK be 1.5 o'clock centrifugal collection thalline, use the alkaline lysis method of extracting plasmid, identify that through the 1%Agarose electrophoresis expression is greater than 1mg plasmid/gram thalline.Through NdeI, the calibrating of XhoI double digestion, cut out the 150bp segment.Obtained engineering bacteria called after pNL-T β 4/DH5a.
Two), the production of gymnoplasm grain pNL-T β 4
1. fermentation
1). shake bottle and express: the pNL-T β 4/DH5a that contains the extrasin beta 4 gene, in the LB culture medium that contains 50 μ g/ml kanamycin, shake bottle and spend the night (37 ℃, 200rpm), contain in the LB culture medium of 50 μ g/ml kanamycin by inoculation in 1: 30 again, cultivated 10 hours for 37 ℃, collect thalline through the 1%Agarose electrophoretic analysis, expression is about 1mg pNL-T β 4/ gram bacterium.
2) fermentation technology:
A. culture medium:
A) seed liquor culture medium (LK):
Tryptone: 10 grams per liters, yeast powder: 5 grams per liters, sodium chloride: 10 grams per liters, kanamycin: 50 mcg/ml.
B) breeding base (15 liters):
Sodium hydrogen phosphate: 315 grams, potassium dihydrogen phosphate: 100 grams, sodium chloride: 10.05 grams, ammonium chloride: 50 grams, tryptone: 90 grams.
Above composition is sterilized in jar together; After sterilizing separately, following composition adds fermentation tank.
Magnesium sulfate: 15 grams, glucose: 450 grams, feed supplement (500 grams per liter): glucose
B. sweat:
1. seed culture:
Draw from the plate identified and to get strain, be inoculated among 50 milliliters of (250 milliliters triangular flasks) LC, behind 36 degree, 200 rev/mins, 8 hours these 50 milliliters of seed liquor are transferred among 700 milliliters of LC, 36 degree, 200 rev/mins spend the night.
2. sweat:
Cultured seed liquid (OD600=3-4) is added in the jar, mix up each parameter, 36 degree, 150 change, dissolved oxygen 100%,
Begin fermentation; Beginning adds 3 hours with 2.5 fast streams after 7 hours, adds 1500 milliliters of feed supplements approximately, collects thalline.
2. pre-treatment and chromatography:
(1) pre-treatment
Adopt the broken bacterium of alkaline lysis, behind centrifugal and the membrane filtration with 0.45um, chromatography in the preparation.
(2) gel permeation chromatography
Sepharose 6 Fast Flow chromatographic columns on the broken bacterium liquid of the clarification of previous step, balance liquid is 100mM Tris-HCl, 10mM EDTA, 2M (NH
4)
2SO
4, pH7.0, leading peak is collected in the 260nm monitoring.
(3) affinity chromatograph
Adopt the Plasmidselect chromatography media, balance liquid is 100mM Tris-HCl, 10mM EDTA, 2M (NH
4)
2SO
4, pH7.0, the sample of previous step is directly gone up sample, and eluent is 100mM Tris-HCl, 10mM EDTA, 2M (NH
4)
2SO
4, 1.4M NaCl, pH7.0 collects eluting peak.
(4) ion-exchange chromatography
Adopt the Source30Q chromatography media, balance liquid is 100mM Tris-HCl, 10mM EDTA, 0.4M NaCl, pH7.0, the samples with water dilution of previous step is gone up sample for 5 times, and eluent is 100mM Tris-HCl, 10mM EDTA, 1MNaCl, pH7.0 collects eluting peak, is the pure product of pNL-T β 4 plasmids.
The pNL-T β 4 that obtains detects by Agarose purity detecting and reversed-phase HPLC, and purity is greater than 95% (see figure 2).
Three), the treatment effectiveness evaluation of 4 pairs of rat heart ischemia models of gymnoplasm grain pNL-T β
1, ill Animal Model Making and test method
In order to estimate the drug effectiveness of gymnoplasm grain pNL-T β 4, set up that the ill animal model of acute myocardial infarction---the rat coronary artery left anterior descending branch is the perfusion model again.
Intramuscular injection xylazine 5mg/kg; Ketamine 50mg/kg anesthesia is with thoracic wall before iodine tincture, the alcohol disinfecting rat; Shop aseptic operation towel exposes operative site behind the bone dry, undergos surgery under the integral asepsis state.Cut the preceding thoracic wall skin of rat, cut off the 3rd to the 6th rib, expose the thoracic cavity, push lung to a side, open pericardium, expose heart.,, pour into again after 1 hour to 3mm from the left anterior descending coronary artery starting point with 6-0 polypropylene (polypropylene) ligation silk and NELATON ligation blood vessel.Confirm no hemorrhage after, with rib and breastbone stitching, the skin suture again that cuts off.Operation back intramuscular injection gentamycin 3mg/kg/ days 3 days, is protected from infection totally.
2, utilize relatively left ventricle antetheca thickness of ultrasoundcardiogram
After setting up the rat heart ischemia model, injection negative control thing (normal saline), substances (gymnoplasm grain PNL-T β 4).Utilized cardiac ultrasonic kinetocardiogram (Live3D Echo7500) and probe (probe15-16L) on the 14th, 28,56 day after the injection, measure and respectively organize left ventricle antetheca thickness.
Basic value (the 0th day, before the administration), normal saline group 0.2352 ± 0.0332,4 group 0.2413 ± 0.025 of gymnoplasm grain pNL-T β, there was no significant difference between each group.
The 14th day physiology saline group 0.1265 ± 0.0021 of administration, 4 group 0.1278 ± 0.014 of gymnoplasm grain pNL-T β, there was no significant difference between each group.
Administration the 28th day, normal saline group 0.1221 ± 0.0125,4 group 0.1295 ± 0.009 of gymnoplasm grain pNL-T β, there was no significant difference between each group.
Administration 56 days, normal saline group 0.1189 ± 0.0135,4 group 0.1574 ± 0.008 of gymnoplasm grain pNL-T β, 4 groups of heart left ventricle of gymnoplasm grain pNL-T β antetheca thickness increases (p＜0.05).
These results show, gymnoplasm grain pNL-T β 4 can have the minimizing (see figure 3) of the left ventricular wall thickness that the efficient recovery myocardial infarction causes.
3, utilize histology's picture relatively at the ventricle fibrosis
Above-mentioned experimental animal the 56th day, it is dirty to core, and is as the criterion with mamillary muscle, and the crosscut heart is made section.With 1% dipped into formalin 24 hours, make paraffin wax tissue slice and slide, carry out Masson ' trichrom dyeing.Relatively adopted Image-pro PLUS ver.4.1 (Media Cybernetics) method between each group.
The ratio that myocardial fibrosis partly accounts for full myocardium of left ventricle is normal saline group 28.13 ± 2.1%, 4 group 22.91 ± 2.3% of pN L-T β (P＜0.05); Between 4 groups of the normal saline groups, gymnoplasm grain pNL-T β significant difference is arranged.These results show that myocardial injection gymnoplasm grain pNL-T β 4 can effectively reduce myocardial damage and the myocardial fibrosis (see figure 4) that myocardial infarction causes.
4, the mensuration of T β 4 albumen expression in vivo
4.1 the formulation of zoological specimens
4.1.1 get 4 age in week male mice, raise a week, eliminate the bad mice of general situation.
4.1.2 use the etherization mice, with the slow intramuscular injection of insulin syringe (5-10/ second) medicine 0.1ml, the syringe needle degree of depth is 3-5mm, has injected etc. and to have dialled pin 5 seconds again, presses 5 seconds after dialling pin.
4.1.3, got muscular tissue (cut off at both sides tendon place, get a complete muscle) and detect in 2,3,4,5,6,7,8 days respectively at 0,1.(as Fig. 5)
4.2T the mensuration of β 4 protein contents
4.2.1 get muscular tissue and internal organs, weigh, and write down body weight
4.2.2 be contained in the 1.5ml test tube (conical tube)-80 ℃ of preservations (liquid nitrogen is freezing)
4.2.3 thaw under the room temperature, dress muscle in vitro put into 500 μ l cell buffer, make homogenate (manually) with homogenizer
4.2.44 ℃ placement 16 hours
4.2.54 centrifugal 30 minutes of ℃ 5000rpm gets supernatant.
4.2.6 survey the total protein of supernatant
4.2.7 measure box explanation mensuration supernatant T β 4 protein contents (ELISA method) are arranged by HGF
4.2.8 be converted into intramuscular T β 4 protein contents of every 100mg with measuring T β 4 protein contents that come out, conversion method is as follows:
The protein content ÷ that the T β 4 albumen=ELISA method of 100mg muscle records (100/ muscle body weight mg * total protein concentration)
The albumen unit that the ELISA method records is pg/ml, should be converted into ng/ml, 1000pg=1ng.
Claims (8)
1. a class is by plasmid vector mediated gene therapy recombinant vector, and be characterized as: this plasmid can enter striated muscle cell, and synthetic in the myocyte, secretion human thymosin beta 4; Synthetic extrasin beta 4 aminoacid sequence is: Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser.
2. the gene order of described human thymosin beta 4 aminoacid sequences of the claim 1 of encoding out.
3. the recombiant plasmid that comprises the described gene of claim 2.
4. the described recombiant plasmid of claim 3 is PNL-T β 4.
5. use the carrier microorganism transformed of claim 4.
6. the microorganism of claim 5 is PNL-T β 4/DH5 α that the plasmid in claim 4 transforms.
7. with the described recombiant plasmid of claim 4, be used to the medicine for preparing treatment or prevent limbs ulcer, erosion.
8. with the described recombiant plasmid of claim 4, be used to prepare the medicine of treatment or prevents heart disease.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002074193A2 (en) * | 2001-03-15 | 2002-09-26 | Regenerx, Biopharmaceuticals, Inc. | METHODS OF TREATING DISORDERS OF THE EYE AND SURROUNDING TISSUE WITH THYMOSIN ss4 (Tss4), ANALOGUES, ISOFORMS AND OTHER DERIVATIVES |
| CN1547480A (en) * | 2001-08-29 | 2004-11-17 | 雷根内克斯生物制药有限公司 | Methods of healing or preventing inflammation, damage and other changes that occur prior to, during or immediately after a myocardial event with thymosin beta 4, analogues, isoforms and other derivati |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002074193A2 (en) * | 2001-03-15 | 2002-09-26 | Regenerx, Biopharmaceuticals, Inc. | METHODS OF TREATING DISORDERS OF THE EYE AND SURROUNDING TISSUE WITH THYMOSIN ss4 (Tss4), ANALOGUES, ISOFORMS AND OTHER DERIVATIVES |
| CN1547480A (en) * | 2001-08-29 | 2004-11-17 | 雷根内克斯生物制药有限公司 | Methods of healing or preventing inflammation, damage and other changes that occur prior to, during or immediately after a myocardial event with thymosin beta 4, analogues, isoforms and other derivati |
Non-Patent Citations (2)
| Title |
|---|
| 胸腺素β_4与非小细胞肺癌转移和预后的关系. 侯萍，于敏.中国肺癌杂志，第7卷第6期. 2004 * |
| 胸腺素β_4及胸腺素β_(10)与非小细胞肺癌转移和预后关系. 侯萍，宋志国.中国组织化学与细胞化学杂志，第14卷第3期. 2005 * |
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