CN100342026C - Method for preparing recombined apoptosis induction ligand relevant to human soluble tumor necrosis factor - Google Patents
Method for preparing recombined apoptosis induction ligand relevant to human soluble tumor necrosis factor Download PDFInfo
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- CN100342026C CN100342026C CNB2004100271762A CN200410027176A CN100342026C CN 100342026 C CN100342026 C CN 100342026C CN B2004100271762 A CNB2004100271762 A CN B2004100271762A CN 200410027176 A CN200410027176 A CN 200410027176A CN 100342026 C CN100342026 C CN 100342026C
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method for preparing recombination apoptosis induction ligand (TRAIL) relevant to a human soluble tumor necrosis factor, which comprises the steps that: a. an engineering bacterium rs TRAIL96-281-pET15b-BL21 (DE3) CGMCC No 0659 serves as a mycelial strain for fermentation cultivation; b. mycelium obtained from fermentation cultivation is washed and crushed by ultrasonic; c. the crushed mycelium substances are salted out; d. chromatography and purification are carried out to the salted out mycelium substances; e. the replacement of a buffer solution system is carried out to the mycelium substances obtained by chromatography and purification. Compared with the prior art, the commercial production of TRAIL protein can be realized by the preparation method, and possibility is provided for the practical application of TRAIL protein.
Description
Technical field
The present invention relates to the preparation of pharmaceutical protein material, relate in particular to the method for utilizing genetic engineering bacterium to carry out the target protein preparation, particularly the apoptosis induction ligand related preparation method of recombination human soluble tumor necrosis factor.
Background technology
Tumour is serious threat human life's common disease and a frequently-occurring disease, and patient's high lethality rate accounts for second of the full cause of the death.But, the feedback data of going through the many decades clinical application shows, though chemotherapy is first-selected at present antitumor scheme, but there are two serious problems in most of antineoplastic chemotherapy medicines: to the resistance of Normocellular strong toxic side effect and tumour cell, cause tumour patient effectively not treated.In the time of tradition chemotherapeutics killing tumor cells, " failing to differentiate between the enemy and ourselves " has the intensive toxic side effect to normal cell, and hemopoietic system and immunity system are caused very big infringement.
Tumor necrosin relative death inducing ligand (TNF-related apotosis inducing ligand, be called for short TRAIL) clone and name (Wiley SR in nineteen ninety-five by the Wiley laboratory the earliest, Schooley K, Smolak PJ, et al, Identification and characterizations of a new member of the TNF family that inducesapoptosis, Immunity, 1995,3:673-682).People's trail dna is positioned long-armed 2 districts of karyomit(e) 6 bands (3q26) No. 3, and its transcription is 1.8-2.0kb, molecular weight 32.5kDa, and theoretical iso-electric point is 7.63.Trail protein is a kind of typical transmembrane glycoprotein, and wherein cytoplasmic domain has 17 amino-acid residues, and striding the film district has 21 amino-acid residues, and extracellular region has 243 amino-acid residues.
Wide expression in the multiple in vivo tissue of TRAIL, as spleen, lung, prostate gland, liver, kidney, heart, skeletal muscle, small intestine, colon and peripheral blood lymphocyte etc., wherein expression amount is the highest in spleen, lung and prostate gland, does not reach but see Table in cerebral tissue.Can inducing apoptosis of tumour cell after trail protein and the receptors bind, and normal cell is not had toxic action.The external activity analysis revealed, various tumor cell strains comprises that large intestine, lung, mammary gland, central nervous system, kidney, malignant melanoma etc. reply the stimulation of TRAIL, and compares, and can reach 50～100% to the inhibiting rate of growth of tumour cell.Therefore, TRAIL has shown the potential potential applicability in clinical practice, and TRAIL will open up the new page of oncotherapy.
At present, most of in the world human cytokines medicines all are to adopt intestinal bacteria as host expresses, mainly are because technology maturation is expressed stable.And express usually there not to be active inclusion body form to produce as the intestinal bacteria high-density, need sex change, renaturation.Deficiency such as have in the sex change renaturation process that albumen mispairing, renaturation yield are low, complex operation and time are long has become worldwide technological puzzle.
A kind of engineering bacteria rsTRAIL96-281-pET15b-BL21 (DE3) CGMCC No.0659 of gene recombination is disclosed in the Chinese patent application 02104519.4 " tumor necrosin relative death inducing ligand extracellular region mutation polypeptide and method for making thereof and purposes ", and mentioned and adopted this project bacterium to prepare the apoptosis induction ligand related laboratory method of soluble tumor necrosis factor, also mentioned the apoptosis induction ligand related potential application foreground in the human body antitumor drug of soluble tumor necrosis factor.
TRAIL will really be applied to pharmacy, promotes the well-being of mankind, and for tumour patient palliates the agonizing sufferings, industrialization production is the only way which must be passed.And TRAIL also is in the laboratory study stage at present, does not see the report that pilot scale and industrialization aspect are arranged.
Summary of the invention
The object of the invention is to satisfy the needs of Bio-pharmaceutical Industryization, and proposes a kind of TRAIL preparation method that can realize suitability for industrialized production.
The present invention realizes that the technical scheme that above-mentioned purpose adopts is, the apoptosis induction ligand related preparation method of a kind of recombination human soluble tumor necrosis factor is proposed, comprise that step a. selects for use engineering bacteria rsTRAIL96-281-pET15b-BL21 (DE3) CGMCC No.0659 to do bacterial strain and carries out fermentation culture, comprise that also step: b. washs and ultrasonication fermentation culture gained thalline; C. to the processing of saltouing of broken gained thalline material; D. handle gained thalline material to saltouing and carry out the chromatography purification processing; E. chromatography purification is handled gained thalline material and carry out buffer solution system replacing processing.
Compare with prior art, adopt the apoptosis induction ligand related preparation method of recombination human soluble tumor necrosis factor of the present invention, can realize the suitability for industrialized production of trail protein, for the practical application of trail protein provides possibility.
Embodiment
Below the present invention is further elaborated.
The apoptosis induction ligand related preparation method of recombination human soluble tumor necrosis factor of the present invention, comprise that step a. selects for use engineering bacteria rsTRAIL96-281-pET15b-BL21 (DE3) CGMCC No.0659 to do bacterial strain and carries out fermentation culture, comprise that also step: b. washs and ultrasonication fermentation culture gained thalline; C. to the processing of saltouing of broken gained thalline material; D. handle gained thalline material to saltouing and carry out the chromatography purification processing; E. chromatography purification is handled gained thalline material and carry out buffer solution system replacing processing.
Described step b comprises substep:
1.. in fermentation gained thalline, add TrisCl[three (methylol) the aminomethane hydrochloric acid that contains NaCl] solution, carry out centrifugal treating, obtain bacterial sediment;
2.. in bacterial sediment, add and contain EDTANa
2The TrisCl solution of (disodium ethylene diamine tetraacetate) and DTT (DTT) stirs;
3.. carry out ultrasonication, reach more than 98% until the bacterial cell disruption rate;
4.. carry out centrifugal treating, supernatant is broken gained thalline material.
Described step c comprises:
In broken gained thalline material, add (NH
4)
2SO
4(ammonium sulfate) makes (NH
4)
2SO
4Saturation ratio reaches 60%, is stirred to whole dissolvings, leaves standstill more than 30 minutes, carries out centrifugal treating, and precipitation is to saltout handles gained thalline material.
Described steps d comprises substep:
1.. add TrisCl solution in the processing gained thalline material of saltouing, centrifugal treating keeps supernatant;
2.. go up Octyl 4 Fast Flow drainage columns, collect the prick post peak;
I. gleanings is carried out molecular sieve gel chromatography column desalting treatment;
3.. go up Q-Sepharose-Fast Flow anion-exchange chromatography post, collect 20% salt concn purpose peak;
Ii. gleanings is carried out molecular sieve gel chromatography column desalting treatment;
4.. go up Source30Q anion-exchange chromatography post, collect 8% salt concn purpose peak.
Described step e comprises:
1.. chromatography purification is handled gained thalline material carry out the processing of molecular sieve gel chromatography;
2.. the damping fluid that chromatography purification is handled in the gained thalline material is converted to Na
2HPO
4-NaH
2PO
4(Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC) solution.
Employing the inventive method is prepared, and finally can obtain purity and reach solubility activated protein more than 95%, and this trail protein can be applicable to prepare antitumor medicine.
Below provide specific embodiment of the whole process of preparation method of the present invention:
Washing: engineering bacteria rsTRAIL96-281-pET15b-BL21 (DE3) the CGMCC No.0659 thalline (being called for short the TRAIL thalline) that fermentation culture obtains is put into Centrifuge Cup, and each Centrifuge Cup adds 80g thalline and 800-900ml solution A, stirs with glass stick; Mixing liquid is carried out centrifugal, the whizzer model is BECKMAN J6-HC, and parameter of noncentricity is: 4000rpm, and 30min, 15 ℃, keep centrifuged deposit, weigh; Washed thalline is positioned in-50 ℃ of refrigerator-freezers preserves;
Ultrasonication: the TRAIL thalline of preserving in the weighing 200g-50 ℃ refrigerator-freezer, add the 1000ml solution B, stir 10min with tissue mashing machine and make its mixing; With a container pack into trash ice and less water, the beaker that fills thalline is put into wherein; Ultrasound probe is immersed mixed solution, apart from beaker bottom 1cm, the beginning ultrasonication, ultrasound intensity 800W 60s/ time, ultrasonic 30 times altogether, controlled temperature≤13 ℃, each ultrasonic back stirring makes it even; Broken mixed solution is centrifugal, and condition is: 14000rpm, and 40min, 15 ℃, keep supernatant, abandon precipitation, survey volume;
The processing of saltouing: foundation: 0-25% (saturation ratio) 14.4g (NH
4)
2SO
4/ 100ml supernatant liquor 25%-60%22.7g (NH
4)
2SO
4/ 100ml supernatant liquor
The first step 0-25% is in proportion with load weighted (NH
4)
2SO
4Solid joins in the ultrasonication supernatant liquor, is stirred to whole dissolvings, and room temperature leaves standstill 30min.Mixed solution is centrifugal, centrifugal condition 12000rpm, 30min, 15 ℃, keep supernatant, abandon precipitation, survey volume;
The second step 25%-60% is in proportion with load weighted (NH
4)
2SO
4Solid joins in the supernatant liquor of the centrifugal back of 25% saturation ratio, is stirred to whole dissolvings, and room temperature leaves standstill 30min.Mixed solution is centrifugal, centrifugal condition, 12000rpm, 30min, 15 ℃, keep precipitation, abandon supernatant;
Chromatography purification is handled:
Dissolving to 60% salt precipitation: measure 1000ml solution C and 40ml solution D, pour the beaker mixing into, change 60% salt precipitation over to beaker, be stirred to dissolving.Mixed solution is centrifugal, centrifugal condition 12000rpm, 30min, 15 ℃, abandon precipitation, keep supernatant;
Last Octyl 4 Fast Flow drainage columns: specification: BPG 100/12CV (column volume)=900ml; With solution C balance pillar, flow velocity 30ml/min, balance 4-5 column volume; With the preparation sample of saltouing on the 30ml/min; With 30ml/min, continue drip washing with solution C; Collection penetrates sample (UV:0.5-0.5, the ultraviolet monitor model is: 8823A-UV, the New Technique Application Inst., Beijing City provides, and is as follows);
Through the desalination of Sephadex G25 chromatography column: specification: Φ 100/30 CV=2400ml; With 40ml/min, with solution D liquid balance pillar, balance 1-1.5 column volume; Go up hydrophobic sample with 40ml/min; Collect first peak; To covering 1 column volume, end operation;
Last Q-Sepharose-Fast Flow anion-exchange chromatography post: specification: XK50/10, CV=200ml; With 40ml/min, walk 1 column volume with solution E earlier, use solution D balance pillar again, 5-8 column volume; Go up G25 desalination sample with 40ml/min; With 40ml/min, with 2 column volumes of solution D drip washing; With 40ml/min, earlier with 2 column volumes of 10% solution E wash-out, use 20% solution E wash-out again, collect 20% elution peak sample (UV:0.3-0.3);
Through the desalination of Sephadex G25 chromatography column: specification: Φ 100/21CV=1650ml; With 40ml/min, with solution D liquid balance pillar, balance 1-1.5 column volume; Go up the 20% elution peak sample that Q-Sepharose-Fast Flow anion-exchange chromatography obtains with 40ml/min; Collect first peak; To covering 1 column volume end operation;
Last Source30Q anion-exchange chromatography post: specification: XK26/11 CV=55ml; With 20ml/min, with 1 column volume of solution E balance, use solution D balance pillar again, 8-10 column volume earlier; Go up 1/2 of G25 desalination sample with 20ml/min; With 20ml/min, with 2 column volumes of solution D drip washing; Set salt gradient 8%, collect sample (UV:0.3-0.3); With 1 column volume of solution E wash-out, to baseline, repeat aforesaid operations with the solution D balance, carry out the wash-out second time.
Cross Superdex 75 gel chromatographies: specification: XK50/85, CV=1700ml; With 2 column volumes of F liquid balance; Go up Source30Q sample, last sample volume≤50ml with 4.0ml/min; Collect first peak, measurement volumes; Obtain target protein and be accredited as a band through SDS-PAGE (sodium laurylsulfonate-polyacrylamide gel), the monomer molecule amount is 21kDa, and iso-electric point is 7.2, and HPLC shows a main peak.
Through above-mentioned preparation process, adopt the TRAIL thalline of 200g, can obtain the above TRAIL solubility activated protein of 1000mg, this trail protein has been removed pyrogen, can be applicable to prepare the human body antitumor medicine.
The definition of the relevant solution that above-mentioned preparation process is mentioned:
A：30mM?Tris·Cl，0.9％NaCl，pH?8.0；
B：50mM?Tris·Cl，5mM?EDTANa
2，1mM?DTT，pH?8.5
C：30mM?Tris·Cl，1.1M(NH
4)
2SO
4
D：30mM?Tris·Cl，pH?8.5；
E：30mM?Tris·Cl，1M?NaCl
F：100mM?Na
2HPO
4-NaH
2PO
4，pH?7.3。
The most preferred embodiment of the above is intended to specify the present invention's mentality of designing: by selecting specific engineering bacteria and specific chromatography purification process combination for use, to reach purification TRAIL solubility activated protein, and removed pyrogen and exchange buffering liquid system, make prepared product can be applicable to prepare the human body antitumor medicine.The present invention's enforcement, be not limited to the disclosed mode of above most preferred embodiment, all mentalities of designing based on the present invention are simply deduced and are replaced, the apoptosis induction ligand related preparation embodiment of the concrete recombination human soluble tumor necrosis factor that obtains all belongs to concrete enforcement of the present invention.
Claims (3)
1, the relevant inducing ligand preparation method that dies that transfers of a kind of recombination human soluble tumor necrosis factor, may further comprise the steps: a, select for use engineering bacteria rsTRAIL96-281-pET15b-BL21 (DE3) CGMCC No.0659 to do bacterial strain to carry out fermentation culture, it is characterized in that, also comprise step:
B, fermentation culture gained thalline is washed and ultrasonication;
C, to the processing of saltouing of broken gained thalline material;
D, handle gained thalline material to saltouing and carry out chromatography purification and handle, may further comprise the steps:
1.. in the processing gained thalline material of saltouing, add TrisCl solution, carry out centrifugal treating, keep supernatant;
2.. go up drainage column, collect the prick post peak;
3.. gleanings is carried out molecular sieve gel chromatography column desalting treatment;
4.. the last first anion-exchange chromatography post, collect 20% salt concn purpose peak;
5.. gleanings is carried out molecular sieve gel chromatography column desalting treatment;
6.. the last second anion-exchange chromatography post, collect 8% salt concn purpose peak;
E, chromatography purification is handled gained thalline material carry out buffer solution system and change and handle, may further comprise the steps:
1.. chromatography purification is handled gained thalline material carry out the processing of molecular sieve gel chromatography;
2.. the buffer exchange that chromatography purification is handled in the gained thalline material is Na
2HPO
4-NaH
2PO
4Solution.
2, the relevant inducing ligand preparation method that dies that transfers of recombination human soluble tumor necrosis factor according to claim 1 is characterized in that described step b comprises following substep:
1.. in fermentation gained thalline, add the TrisCl solution that contains NaCl, carry out centrifugal treating, obtain bacterial sediment;
2.. in bacterial sediment, add and contain EDTANa
2TrisCl solution with DTT stirs;
3.. carry out ultrasonication, reach more than 98% until the bacterial cell disruption rate;
4.. carry out centrifugal treating, centrifuged supernatant is broken gained thalline material.
3, the relevant inducing ligand preparation method that dies that transfers of recombination human soluble tumor necrosis factor according to claim 1 is characterized in that:
The 2. middle drainage column medium of the substep of described steps d is Octy1 4 Fast Flow;
The substep of described steps d 4. in the first anion-exchange chromatography post medium be Q-Sepharose-Fast Flow;
The substep of described steps d 6. in the second anion-exchange chromatography post medium be Source30Q.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100271762A CN100342026C (en) | 2004-05-13 | 2004-05-13 | Method for preparing recombined apoptosis induction ligand relevant to human soluble tumor necrosis factor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100271762A CN100342026C (en) | 2004-05-13 | 2004-05-13 | Method for preparing recombined apoptosis induction ligand relevant to human soluble tumor necrosis factor |
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| Publication Number | Publication Date |
|---|---|
| CN1696304A CN1696304A (en) | 2005-11-16 |
| CN100342026C true CN100342026C (en) | 2007-10-10 |
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| CN102250254A (en) * | 2010-05-19 | 2011-11-23 | 江苏先声药物研究有限公司 | Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) fusion protein, its preparation and applications |
| CN103588871B (en) * | 2012-08-16 | 2016-08-03 | 深圳新鹏生物工程有限公司 | A kind of isolation and purification method of rhTRAIL thalline |
| KR20150107742A (en) | 2012-12-11 | 2015-09-23 | 자피오텍 게엠베하 | Delphinidin for combating melanoma cells |
| CN104211808B (en) * | 2013-06-05 | 2019-03-05 | 江苏先声药业有限公司 | A kind of preparation method of tumor necrosin relative death inducing ligand fusion protein |
| EP2913050A1 (en) | 2014-02-28 | 2015-09-02 | SapioTec GmbH | Method for producing a flurane complex |
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| CN1291616A (en) * | 2000-10-02 | 2001-04-18 | 广东梅县梅雁蓝藻有限公司 | Process for separating and purifying high-purity high-activity phycocyanin |
| CN1436792A (en) * | 2002-02-08 | 2003-08-20 | 中国医学科学院基础医学研究所 | Tumor necrosis factor relative cell death inducing ligand extracellular region mutation polypeptide and its prepn and use |
| CN1473843A (en) * | 2003-07-24 | 2004-02-11 | 华东理工大学 | Industrially extracting, renaturation and purifying method for recombined TRAILinclusion body protein |
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| CN1291616A (en) * | 2000-10-02 | 2001-04-18 | 广东梅县梅雁蓝藻有限公司 | Process for separating and purifying high-purity high-activity phycocyanin |
| CN1436792A (en) * | 2002-02-08 | 2003-08-20 | 中国医学科学院基础医学研究所 | Tumor necrosis factor relative cell death inducing ligand extracellular region mutation polypeptide and its prepn and use |
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