CN1661019A - Method for producing recombined endothelium chalone in bacillus colis and application - Google Patents
Method for producing recombined endothelium chalone in bacillus colis and application Download PDFInfo
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Abstract
A process for producing soluble bioactive recombinant human endothelium inhibin in colibacillus and separating and purifying it is disclosed. The resultant recombinant human endothelium inhibin can specifically and dosage-dependently suppress the reproduction of endothelium cells and the generation of new blood vessel.
Description
One, technical field:
The invention belongs to the genetically engineered biological technical field.
Two, background technology:
Vasculogenesis is necessary to the growth and the transfer of solid tumor.In order to promote vasculogenesis, tumor tissues generates a series of angiogenesis factor (as bF6F), simultaneously many tumours also can produce anti-angiogenic proteins, one of them Endostatin (endostatin), by doctor Folkman study group of Harvard University from mouse hemangioendothelioma clone conditioned medium, separate first and obtain (O ' Reilly, M.S., Boehm, T., Folkman, J.Cell 88:277-285,1997).Endostatin is the C-end fragment of collagen XVIII, and molecular weight is 20kDa, can specificity suppresses the propagation and the migration of endotheliocyte, reduces glioma blood vessel and blood flow, effectively suppresses the various primary tumo(u)rs of mouse.People attempt to produce the Endostatin of biologically active, but the reorganization endostatin protein easily is gathered into active, the insoluble inclusion body of lifeless matter in Bacillus coli cells matter.In external change renaturation process, the productive rate of the active Endostatin of solubility is very low, and since contain many to disulfide linkage and non-effectively folding, most of sedimentary albumen precipitate once more (O ' Reilly, M.S., Boehm, T., Folkman, J.Cell 88:277-285,1997; You, W.K., So, S.H., Lee, H., Park, S.Y., Yoon, M.R., Chang, S.L., Hong, Y.K., Chung, S.L.Experimental and Molecular Medicine 31:197-202,1999).Once be reported in expression activity Endostatin (Dhanabal, M., Ramchandran, R., Volk, R. in the yeast, Stillman, I.E., Lombardo, M., Iruela-Arispe, M.L., Simons, M.Sukhatme, V.P.Cancer Res.59:189-197,1999).In the mouse model administration, need a large amount of Endostatins (20mg/kg), just can make tumor regression.Producing the Endostatin of biologically active efficiently, in a large number, at low cost, is its important bottleneck as the development and application of antitumor drug, also is a difficult point in the present Endostatin research.The separation and purification of recombinant human endostatin at present mainly relies on heparin-affinity chromatography medium to carry out purifying, obtains highly purified recombinant human endostatin, and its production process is time-consuming, separation and purification is expensive.
Three, summary of the invention:
This patent invention is based upon on the basis of the Chinese invention patent application 200410065733.X method and the application thereof of production solubility recombinant protein " a kind of in intestinal bacteria " and the supporting result of use the best of Chinese invention patent application 200410065733.X.
The problem that patent of the present invention need solve is to set up suitable, easy, the technology production solubility, that bioactive recombinant human endostatin is arranged and method, the corresponding soluble recombining human endostatin of foundation purifying process in intestinal bacteria at an easy rate, for recombinant human endostatin is used for the treatment of tumour as the medicine of angiogenesis inhibiting and metastases and other angiogenesis-associated diseases is set up method and technology extensive, cheap production.The present invention has important Practical significance for recombinant human endostatin as the R and D for the treatment of tumour and other angiogenesis-associated diseases medicine.
Purpose of the present invention can reach by following measure:
With the human endostatin gene clone in colibacillus expression plasmid, transformed into escherichia coli, the protein that will have a chaperone activity at low temperatures with the human endostatin coexpression, low temperature and molecular chaperones all can promote the output of soluble human Endostatin respectively.When the coexpression under low temperature (35 ℃～16 ℃) with human endostatin or angiostatin expression strain, the coexpression molecular chaperones can prevent more effectively that the human endostatin inclusion body from forming.For example, in the time of 25 ℃, when human endostatin and molecular chaperones DnaK-DnaJ-GrpE and GroEL/ES coexpression, the productive rate of soluble human Endostatin is about 36mg/L, but every liter of nutrient solution at least purifying obtain the human endostatin of 16mg.The soluble recombining human endostatin dissolves, dialyses through ammonium sulfate precipitation, and ion exchange chromatography and gel permeation chromatography are able to purifying.The propagation that the human endostatin of purifying can specificity, dose-dependently ground suppresses endotheliocyte on chick chorioallantoic membrane (CAM), has the effect that new vessel generates that suppresses.
The invention provides the method for a kind of feasible ground, recombinant human endostatin production solubility easily, biologically active, set up easy, economic purifying process correspondingly.The recombinant human endostatin of explained hereafter of the present invention can be used in preparation treatment tumour and the diseases related medicine of vasculogenesis.
Compare with existing recombinant human endostatin production technique, characteristic of the present invention and innovation part are:
(1) the present invention set up one easy, inexpensive, in Escherichia coli system, improve the method for soluble recombined human Endostatin output efficiently.Utilize molecular chaperones coexpression and low-temperature epitaxy to unite the method for use, can nearly all change script into almost whole soluble recombinant human endostatins at an easy rate with the recombinant human endostatin of inclusion body formal representation.
(2) present, the separation purification method of human endostatin commonly used mainly is to depend on heparin affinity chromatography, and heparin affinity chromatography medium price is comparatively expensive.Among the present invention, for the soluble recombining human endostatin, we have used very, and the purification step of ammonium sulfate precipitation, dialysis, ion exchange chromatography and gel permeation chromatography traditional and commonly used has just reached the purpose that obtains the recombinant human endostatin of purifying, method is easy, inexpensive, has good suitability, it is convenient to promote, and is suitable for producing.
Four, description of drawings:
Fig. 1. the recombinant human endostatin of purifying
1a.SDS-PAGE analytical results;
1b.Western blot analytical results.
Fig. 2. recombinant human endostatin suppresses the vascular endothelial cell proliferation experiment that b-FGF stimulates.
Black: NIH3T3 cell; White: vascular endothelial cell.
Fig. 3. recombinant human endostatin suppresses the chick chorioallantoic membrane new vessel and generates activity experiment.
Five, embodiment:
Embodiment: the clone of recombinant human endostatin gene, expression, separation and purification and character are identified:
1. the expression of recombinant human endostatin: with recombinant human endostatin expression plasmid pET23a-hE and molecular chaperones expression plasmid pG-TF2 (coexpression molecular chaperones TF and GroEL/ES) and pG-KJE8 (coexpression molecular chaperones DnaK-DnaJ-GrpE and GroEL/ES) cotransformation e. coli bl21 (DE3) bacterial strain (Novagen company).TF and GroEL/ES are by 20ng/ml tsiklomitsin abduction delivering, and DnaK-DnaJ-GrpE is by 0.3mg/ml L-arabinose abduction delivering, and 1mM IPTG is used for inducing recombinant human endostatin to express.Express thalline through the ultrasonication cell, the centrifugal 10min of 8000rpm collects thalline.
2. the separation and purification of recombinant human endostatin: express thalline for 1 liter and suspend, ice-bath ultrasonic 20 minutes (5 seconds ultrasonic, and 5 seconds at interval) with the ultrasonic liquid of 50ml (20mMTris-Cl, pH7.5,2mM EDTA).Mixture 12000rpm after ultrasonic, 4 ℃ were descended centrifugal 30 minutes.To express supernatant liquor concentrates with 20% ammonium sulfate precipitation, 4 ℃ of dialysed overnight of sedimentary albumen, dialyzate A (10mMT Tris-HCl, 50mM NaCl, pH7.4), changed liquid once in 6-8 hour, and changed liquid altogether 3 times, sample directly carries out SP-Sepharose Fast Flow (Amersham Pharmacia Biotech) chromatography.Dialyzate A is all used in the washing of post bed balance, uses 0.6MNaCl, Tris-HCl, pH7.4 and 1M NaCl, Tris-HCl, pH7.4 stepwise elution, elutriant is mixed, and dialyzate A dialysis back freeze-drying concentrates, and Sephardex G75 gel (Amersham Pharmacia Biotech) chromatography is further purified.
3. recombinant protein expression analysis: harvested cell and carrying out ultrasonic bacteria breaking, cleer and peaceful precipitation in the centrifugation, the 15%SDS-PAGE electrophoretic analysis, SDS-PAGE scanning (UVPWhite/Ultraviolet transilluminator) with coomassie brilliant blue staining, analytical results and according to quantitative molecular weight standard calculates the productive rate (using the software Grab-it 2.5 and the Gelwork of UVP company) of recombinant human endostatin.Analyze definite recombinant human endostatin purified product with western blot, use PVDF westernblotting film (Roche), the anti-human endostatin antibody of mouse polyclone (Santa Cruz) in the experiment.
4. the cell of reorganization Endostatin is surveyed and is lived: cultivate BCE cell (bovine capillary endothelial cell) or NIH 3T3 cell to sufficient amount in the DMEM substratum of the people's recombinant bfgf that adds 20% calf serum, microbiotic and 3ng/ml, add 0.05% tryptic digestion, cell is resuspended in the DMEM substratum that contains 20% calf serum.About 12500 cell/0.5ml (hole) add in 24 orifice plates, 37 ℃ of (10%CO
2) cultivate after 24 hours and add the recombinant human endostatin of 25 μ l blanks and various dose respectively in each hole.Add bFGF after 30 minutes to final concentration 1ng/ml.Further cultivate 48h, trypsin digestion and cell is resuspended in PBS, and with the fixing 30min of 70% ice-cold ethanol, 7-AAD dyes, and uses the flow cytometry analysis result, and the viable count in comparative sample and the blank well can calculate the inhibiting rate of different concns.
5. the chick chorioallantoic membrane blood vessel detects the recombinant human endostatin activity experiment: get 8～9 days chicken embryos of hatching, choose the trilateral eggshell at the rich blood vessel place, needle shell membrane, recombinant human endostatin is added on the sterilization filter paper, filter paper is attached to above the chorioallantoic membrane again.Use aseptic tape seal.After the administration 3 days, open the chicken embryo, drip the mixed solution of equivalent methyl alcohol and acetone in administration place, room temperature is fixed 15 minutes, treats to cut chorioallantoic membrane after the blood coagulation, puts into water and flattens, and observes and takes pictures.
The purification step that the present invention has set up ammonium sulfate precipitation, dialysis, ion exchange chromatography and the gel permeation chromatography using tradition and use always has just reached the purifying process that obtains the recombinant human endostatin of purifying, method is easy, inexpensive, have good suitability, it is convenient to promote, and is suitable for producing.
Under 25 ℃, utilize low temperature and molecular chaperones coexpression can produce about 36mg soluble recombining human endostatin, can obtain 16mg at least by purifying by above-mentioned purification step, it through the SDS-PAGE purity assay 98% recombinant human endostatin, its molecular weight can be discerned by anti-human endostatin antibody for the 20kDa of expection.Recombinant human endostatin can suppress the propagation of BCE cell, and is dose-dependence (seeing accompanying drawing 2), its ED
50=0.5 μ g/ml does not then suppress the activity of cell proliferation for other cell (as NIH3T3 etc.).Recombinant human endostatin has obvious suppression chick chorioallantoic membrane new vessel and generates active and tangible blood vessel restraining effect, and 4 μ g recombinant human endostatins cause blood vessel to be suppressed fully, cause chicken embryo death.
Claims (4)
1. method of in intestinal bacteria, producing the soluble recombined human Endostatin, it is characterized in that with the recombinant human endostatin expression strain at low temperatures with the molecular chaperones coexpression.
2. the method for a separation and purification soluble recombined human Endostatin is characterized in that the soluble recombining human endostatin is carried out purifying through ammonium sulfate precipitation, dissolving, dialysis, ion exchange chromatography and gel permeation chromatography.
3. the application of recombinant human endostatin in the medicine of preparation treatment tumour and angiogenesis-associated diseases of producing according to the described method of claim 1.
4. the application of recombinant human endostatin in the medicine of preparation treatment tumour and angiogenesis-associated diseases of producing according to the described method of claim 2.
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| CN200510038122.0A CN1661019A (en) | 2005-01-18 | 2005-01-18 | Method for producing recombined endothelium chalone in bacillus colis and application |
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| CN200510038122.0A CN1661019A (en) | 2005-01-18 | 2005-01-18 | Method for producing recombined endothelium chalone in bacillus colis and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105218660A (en) * | 2014-07-03 | 2016-01-06 | 苏州方舟基因药业有限公司 | The novel purification renaturation method of Recombinant Endostatin and antitumor application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218660A (en) * | 2014-07-03 | 2016-01-06 | 苏州方舟基因药业有限公司 | The novel purification renaturation method of Recombinant Endostatin and antitumor application thereof |
| CN105218660B (en) * | 2014-07-03 | 2019-02-12 | 苏州方舟基因药业有限公司 | The novel purification renaturation method and its antitumor application thereof of Recombinant Endostatin |
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