CN1896096A - Production of high-activity extrasin beta 4 derivative by gene engineering - Google Patents
Production of high-activity extrasin beta 4 derivative by gene engineering Download PDFInfo
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- CN1896096A CN1896096A CN 200510083894 CN200510083894A CN1896096A CN 1896096 A CN1896096 A CN 1896096A CN 200510083894 CN200510083894 CN 200510083894 CN 200510083894 A CN200510083894 A CN 200510083894A CN 1896096 A CN1896096 A CN 1896096A
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Abstract
A high-activity extrasin beta-4 derivative by gene engineering method is prepared by expressing in bacterium, combing protein with natural extrasin beta-4, dissolved expressing in colibacillus, cutting and processing by fusion protease. It has stable and efficient expression, higher purity and can be used to repair skin and heart damage.
Description
One. technical field
The invention belongs to the generation field of recombinant protein drug production and derivative.Make up by the genetically engineered to the precursor fusion rotein, realized efficiently expressing of target protein, this method has the expression amount height, and is easy and simple to handle, the characteristics of finished product Ala-T β 4 purity height (greater than 98%).
Two. background technology
Immunity system is the system of defense of human body.Participate in immunoreactive main cell T lymphocyte, bone-marrow-derived lymphocyte and scavenger cell are arranged.And thymus gland is the immune maincenter organ that the T lymphocyte is grown, broken up.Thymus gland excretory thymic factor (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 comparatively clear and definite a kind of main thymic factor of present 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, embryo and birth back myocardial cell's survival ability 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 Akt " survival kinase ".These presentation of results, " thymosin-beta 4 " is a possible therapeutic goal for acute coronary occlusion.The researchist 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 heart attack of experiment inductive.Extrasin beta-4 has been used to clinical trial, to promote the recovery from illness of skin wound.The researchist believes that this in the near future albumen will enter the clinical experimental stage of disease treatment.
T β 4 is peptide species of separating in the thymosin fraction 5 (TF5) that extracts from calf thymus the earliest such as Goldstein, contains 43 amino acid, molecular weight 4.963KD, no disulfide linkage and glycosylation.
At present, the production of T β 4 mainly is to derive from chemosynthesis.Bulk drug adopts solid-phase synthesis to make in the chemical synthesis process, uses the high performance liquid chromatography purification.This preparation method requires high to material purity, synthetic instrument and facility investment are big, production technique (synthetic and chemically modified etc.) complexity, and the product activity is lower, the production cost height.And conventional gene engineering method makes up the extrasin beta 4 expression vector, at expression in escherichia coli, to make through separation and purification, the product purity that obtains is with active high, but because little peptide molecule easily is degraded after expressing and is difficult for detectedly, its productive rate and yield all do not reach industrialized requirement.
Three. summary of the invention
Characteristics of the present invention are to utilize the albumen of one section energy a large amount of stably express, existence in bacterial body to do the guide, T β 4 is brought out in the expression process, produce " T β 4 artificial precursor proteins ", cut back to close through vitro enzyme again, obtain highly purified Ala-T β 4, and Ala-T β 4 biological activity of comparing with T β 4 is higher, but on production cost far below chemical synthesis process and conventional gene engineering expression method.At first, the pilot protein that we select for use is a section of Glutathione S transferase (GST), and this section albumen is in the kytoplasm of e. coli bl21, can be a large amount of with the soluble form stable existence, express, exist the high 1-2 of rate doubly than GST complete sequence soluble form, maximum is about the 45%-60% of tropina total amount.Just T β 4 can not be in intestinal bacteria the reason of a large amount of soluble-expressions, probably be the structure and the race relation of itself, can not be in intestinal bacteria high-yield expression, this just greatly reduces output and has increased producting process difficulty.After we receive the pilot protein back to T β 4, find " T β 4 artificial precursor proteins " also can be in intestinal bacteria overexpression and exist with soluble form.Again because this section pilot protein that we select for use, still can combine under certain condition with the gsh affinity column, this has just further improved turnout, has reduced technology difficulty, thereby reduce production costs, use this cover technology to make the 1g wet thallus can finally obtain about 1mg finished product albumin A la-T β 4, purity is greater than 98%.Though the N of finished product has not held many Ala, it is that small molecules amino acid not only can not exert an influence to T β 4 structures, but also the structure of T β 4 is played stabilization, and this raising on the biological activity as can be seen.Secondly, we have changed the penbritin of routine into kantlex when selecting the screening resistance, make this technology difficulty and cost descend greatly.Ala-T β 4 activity that the present invention produces are higher than the T β 4 of chemosynthesis.The present invention is by expressing the method for T β 4 artificial precursor proteins, and High-efficient Production people T β 4 reduces production cost greatly in intestinal bacteria, is more conducive to substituted chemistry synthetic T β 4, is expected to provide for extensive patients the medicine of high-quality low-cost.
The generation of T β 4 artificial precursors and the preparation method of Ala-T β 4 are among the present invention: be that template is selected synthetic T β 4 dna sequence dnas of the full gene of intestinal bacteria preference codon for use and added restriction enzyme site at N, C-terminal with T β 4 aminoacid sequences earlier, with the GST aminoacid sequence is that template is selected the synthetic leading peptide dna sequence dna of the full gene of intestinal bacteria preference codon for use and added restriction enzyme site at N, C-terminal, again leading peptide, T β 4 and screening plasmid β BSK are handled well with restriction enzyme, press certain mol proportion as calculated behind each sample concentration and add the T4 linked system.Be PSK-T β 4 through transforming, filter out a successful recon life.Downcut antigen-4 fusion protein gene with Restriction Enzyme, (this plasmid is the kalamycin resistance selection markers with the plasmid PGM of our company that handles well with Restriction Enzyme with this gene again, energy great expression GST class fusion rotein is that our company makes up) connect, obtain correct recon called after PGMT β 4 through screening.PGMT β 4 is transformed among the expressive host bacterium BL21, go out high expression engineering PGM T β 4/BL21 through expression screening, in 30 liters of fermentor tanks, PGMT β 4/BL21 is carried out high density fermentation then, through fermentation in 12 hours, finally obtain the engineering thalline that the target protein expression amount accounts for bacterial protein amount 60% (see figure 1), the about 100g/L of weight in wet base, again through centrifugal collection thalline, high-pressure homogeneous crusher machine bacterium also centrifugally obtains containing purpose precursor protein supernatant liquor, with sample on the supernatant liquor to the GST affinity column, wash-out purpose precursor protein is further purified sample after also cutting with the zymoplasm enzyme again by anion column, make final purity of protein greater than 98%.
In animal experiment study, find thymosin Ala-T β 4 with other two kinds of albumen, by activating migration and the survival that the Akt/ protein kinase B promotes the myocardial cell.After having studied the cultured cells activity, the researchist has a heart attack the coronary artery ligation simulation of 60 adult rats, thereby creates rat model.Only then give 20 rat local injection thymosin Ala-T β 4 2ug/, give 20 rat local injection thymosin T β 4 2ug/ only, and in addition 20 rat pump pickle 200uL as blank.Continuously the cell in the damaged heart of injection thymosin Ala-T β 4 and the rat in 4 January of T β is seldom dead, and sends out the back in heart trouble and improved heart function several weeks, and Ala-T β 4 results of treatment are more obvious than T β 4.The researchist believes that thymosin Ala-T β 4 can change the metabolism of cell and create more powerful myocardial cell, and these cells can be resisted the hypoxemia situation after heart trouble is sent out.Next, the researchist will determine the Best Times of this proteic most effective dose, medication.This research will be opened up the approach that makes new advances for cardiopathic treatment.If this albumen is effective equally to the mankind, so this albumen will become a kind of strong disease treatment medicine.
After tested, Ala-T β 4 activity of the present invention's production are greater than chemosynthesis T β 4.
Four. description of drawings
Fig. 1 is the fermentation expression SDS-PAGE electrophoretogram of Ala-T β 4.
Fig. 2 is the SDS-PAGE electrophoretogram behind the purifying of Ala-T β 4.
Fig. 3 is to rat model injection T β 4 and the Ala-T β 4 back results that influence to the rat heart wall thickness.
Fig. 4 is to rat model injection T β 4 and the Ala-T β 4 back results that influence to rat heart wall fibrosis.
Five. embodiment
1. the acquisition of the structure of extrasin beta 4 precursor protein expression plasmid and engineering bacteria:
At first by the synthetic complete genome sequence that amplifies the extrasin beta 4 precursor protein of full gene:
GGA?TCC?CCT?CGA?GCT?TCT?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?GTC?GAC
With the gst gene is template, and pcr amplification obtains the product about 500bp, and through order-checking plasmid clone, gene sequencing, it is standby to obtain implementation sequence.
The purpose fragment reclaims small segment respectively behind BamH I, EcoR I, XhoI double digestion, plasmid PGM reclaims big fragment behind XhoI and EcoRI double digestion, 18 ℃ in T4 ligase enzyme system three sections target gene fragment connect the recombinant plasmid called after PGMT β 4 that obtains through screening.Use CaCl then
2Method transformed into escherichia coli BL21, be coated on the LB flat board that contains the 50ug/ml kantlex, selecting positive bacteria drops on when being cultured to OD600 for 0.6-0.8 among the LK and to add IPTG 0.1mM and induce 3-4 hour centrifugal collection thalline, occur the protein band about a 30KD behind the broken bacterium of 8M urea during the 15%SDS-PAGE electrophoresis, expression amount is 40%.Monoclonal antibody immunity trace calibrating through extrasin beta 4 shows positive reaction.The engineering bacteria called after PGMT β 4/BL21 that efficiently expresses thymosin Ala-T β 4 precursor proteins that is obtained.
2. fermentation
1). shake bottle and express: the PGMT β 4/BL21 that contains extrasin beta 4 precursor protein gene, in the LB substratum that contains 50 μ g/ml kantlex, shake bottle and spend the night (37 ℃, 200rpm), contain in the LB substratum of 50 μ g/ml kantlex by inoculation in 1: 30 again, cultivate after 3 hours for 37 ℃, add 0.1mM IPTG and induced 4 hours.Collect thalline through the SDS-PAGE electrophoretic analysis, find to contain extrasin beta 4 precursor protein (30KD) based on solubility expression, expression amount accounts for about 60% of bacterial protein.
2) zymotechnique:
A. substratum:
A) seed liquor substratum (LK):
Tryptones: 10 grams per liters, yeast powder: 5 grams per liters, sodium-chlor: 10 grams per liters, kantlex: 50 mcg/ml.
B) breeding base (15 liters):
Sodium phosphate dibasic: 315 grams, potassium primary phosphate: 100 grams, sodium-chlor: 10.05 grams,
Ammonium chloride: 50 grams, Tryptones: 90 grams.
Above composition is sterilized in jar together; After sterilizing separately, following composition adds fermentor tank.
Sal epsom: 15 grams, glucose: 450 grams, feed supplement (500 grams per liter): glucose
B. fermenting process:
A) seed culture:
Draw from the plate identified and to get bacterial classification, 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.
B) fermenting process:
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 2 hours with 2.5 fast streams after 5 hours, adds 800 milliliters of feed supplements approximately, adds 1 gram IPTG and induces (three hours).
3. chromatography:
1) affinity chromatography
Adopt GSH-Agrose chromatography media (Sigma company), balance liquid adopts 25mM Tris-HCl, and pH8 is after the last sample balance, split Glutathione Sepharose chromatography column on the centrifugal supernatant of bacterium, wash fusion rotein with the elutriant that contains 10mM reduced glutathion (GSH) after the balance.
2) enzymolysis
The elutriant of previous step adds 37 ℃ of enzymes of zymoplasm (5NIHU/mL) and cut 2 hours.
3) anion-exchange chromatography
Adopt the Source30Q chromatography media, balance liquid is 20mM PB, pH7, and the samples with water dilution that previous step obtains is gone up sample for 1 times, adopts 20mM PB after the balance, pH7, the gradient elution of 0-1M NaCl is collected the target protein elution peak.
4. detect
The extrasin beta 4 that obtains detects by SDS-PAGE purity detecting and reversed-phase HPLC, and purity is greater than 98% (see figure 2).
5.Ala-T the treatment effectiveness evaluation of β 4 and 4 pairs of rat heart ischemia models of T β
1), ill Animal Model Making and test method
In order to estimate the drug effectiveness of Ala-T β 4 and 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 the wall of the chest before the tincture of iodine, the alcohol disinfecting rat; Shop aseptic operation towel exposes operative site behind the complete drying, undergos surgery under the perfectly sterile state.Cut the preceding wall of the chest 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 gentamicin 3mg/kg/ days 3 days, is protected from infection totally.
2), utilize relatively left ventricle antetheca thickness of ultrasonic cardiogram
After setting up the rat heart ischemia model, injection negative control thing (physiological saline), positive control (T β 4), substances (Ala-T β 4).Utilized cardiac ultrasonic cardiogram (Live 3D Echo 7500) 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), physiological saline group 0.2594 ± 0.0225,4 group 0.2378 ± 0.018 of T β, 4 group 0.2432 ± 0.01 of Ala-T β, there was no significant difference between each group.
The 14th day physiology salt solution group 0.1306 ± 0.0128 of administration, 4 group 0.1215 ± 0.013 of T β, 4 group 0.1299 ± 0.011 of Ala-T β, there was no significant difference between each group.
Administration the 28th day, physiological saline group 0.1207 ± 0.0151,4 group 0.1233 ± 0.0071 of T β, 4 group 0.1288 ± 0.012 of Ala-T β, there was no significant difference between each group.
Administration 56 days, physiological saline group 0.1265 ± 0.0125,4 group 0.1232 ± 0.0057 of T β, 4 group 0.1508 ± 0.0078 of Ala-T β, 4 groups of heart left ventricle of Ala-T β antetheca thickness increases (p＜0.05).
These results show, Ala-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 the histology picture relatively at the ventricle fibrosis
Tested the 56th day, it is dirty to core, and is as the criterion with corpora mammillaria 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 Cybemetics) method between each group.
The ratio that myocardial fibrosis partly accounts for full myocardium of left ventricle is physiological saline group 28.13 ± 2.1%, 4 group 22.91 ± 2.3% of T β (P≤0.05), 4 group 16.86 ± 1.8% of Ala-T β (P≤0.0005); Between 4 groups of 4 groups of physiological saline group and T β, the Ala-T β significant difference is arranged, especially Ala-T β has significant differences for 4 groups.These results show that Ala-T β 4 can effectively reduce myocardial damage and myocardial fibrosis (see figure 4) that myocardial infarction causes.
Sequence table
＜110〉Beijing Nuo Silande Bioisystech Co., Ltd
＜120〉high-activity extrasin beta 4 derivative of gene engineering method production
<140>
<141>
<160>1
<210>1
<211>44
<212>PRT
<213>Ala-Tβ4
<400>1
Ala?Ser?Asp?Lys?Pro?Asp?Met?Ala?Glu?Ile?Glu?Lys?Phe?Asp?Lys
1 5 10 15
Ser?Lys?Leu?Lys?Lys?Thr?Glu?Thr?Gln?Glu?Lys?Asn?Pro?Leu?Pro
20 25 30
Ser?Lys?Glu?Thr?Ile?Glu?Gln?Glu?Lys?Gln?Ala?Gly?Glu?Ser
35 40 44
Claims (8)
1. the mode with amalgamation and expression is produced the method for extrasin beta 4 and with the 44 peptide extrasin beta 4 derivatives that it is produced, it is characterized by the albumen of one section energy excess stably express, existence in the intestinal bacteria kytoplasm and 43 amino acid whose extrasin beta 4s merge precursor protein that the back forms can the excess stably express in the intestinal bacteria kytoplasm; This precursor fusion rotein through fermentation, enzyme cuts, obtain highly active maturation protein 44 peptide extrasin beta 4 derivative aminoacid sequences behind the purifying is: Ala-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. 44 peptide extrasin beta 4 derivatives in the use claim 1 are used for the treatment of the reparation of skin histology damage as major ingredient.
3. 44 peptide extrasin beta 4 derivatives in the use claim 1 are used for the treatment of the reparation of damage to cardiac tissue as major ingredient.
4. 44 peptide extrasin beta 4 derivatives in the use claim 1 are used for the treatment of coronary heart disease as major ingredient.
5. 44 peptide extrasin beta 4 derivatives in the use claim 1 are used for the reparation of corneal injury as major ingredient.
6. the expression plasmid selection markers that adopts in the technology described in the claim 1 is a kalamycin resistance gene.
7. the albumen of a section energy excess stably express, existence in the intestinal bacteria kytoplasm adopting in the technology described in the claim 1 is a section or complete sequence of natural Glutathione S transferase (GST).
8. the precursor protein in the claim 1 is that the host bacterium carries out solubility expression with engineered method with intestinal bacteria or yeast.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510083894 CN1896096A (en) | 2005-07-15 | 2005-07-15 | Production of high-activity extrasin beta 4 derivative by gene engineering |
| US11/995,817 US7816321B2 (en) | 2005-07-15 | 2006-07-14 | Thymosin β4 derivatives and use thereof |
| JP2008520699A JP5180074B2 (en) | 2005-07-15 | 2006-07-14 | Thymosin β4 derivative and method of using the same |
| KR1020087003496A KR100984635B1 (en) | 2005-07-15 | 2006-07-14 | Thymosin ?4 derivatives and use thereof |
| CN200680025339A CN100582121C (en) | 2005-07-15 | 2006-07-14 | Thymosin beta 4 derivatives and use thereof |
| EP06761426A EP1908779B1 (en) | 2005-07-15 | 2006-07-14 | Thymosin beta 4 derivatives and use thereof |
| PL06761426T PL1908779T3 (en) | 2005-07-15 | 2006-07-14 | Thymosin beta 4 derivatives and use thereof |
| PCT/CN2006/001679 WO2007009359A1 (en) | 2005-07-15 | 2006-07-14 | Thymosin beta 4 derivatives and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510083894 CN1896096A (en) | 2005-07-15 | 2005-07-15 | Production of high-activity extrasin beta 4 derivative by gene engineering |
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| CN1896096A true CN1896096A (en) | 2007-01-17 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110215513A (en) * | 2018-11-15 | 2019-09-10 | 北京诺思兰德生物技术股份有限公司 | Purposes of the modified extrasin beta 4 in terms of therapeutic radiation enteritis |
| CN111386337A (en) * | 2017-11-24 | 2020-07-07 | Gtreebnt科技有限公司 | Composition for promoting goblet cell proliferation or mucin secretion comprising thymosin β 4 or a derivative thereof as an active ingredient |
-
2005
- 2005-07-15 CN CN 200510083894 patent/CN1896096A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111386337A (en) * | 2017-11-24 | 2020-07-07 | Gtreebnt科技有限公司 | Composition for promoting goblet cell proliferation or mucin secretion comprising thymosin β 4 or a derivative thereof as an active ingredient |
| CN110215513A (en) * | 2018-11-15 | 2019-09-10 | 北京诺思兰德生物技术股份有限公司 | Purposes of the modified extrasin beta 4 in terms of therapeutic radiation enteritis |
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