CN1314708C - Human alpha 1-thymic peptide composition and its preparation - Google Patents
Human alpha 1-thymic peptide composition and its preparation Download PDFInfo
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- CN1314708C CN1314708C CNB200410018595XA CN200410018595A CN1314708C CN 1314708 C CN1314708 C CN 1314708C CN B200410018595X A CNB200410018595X A CN B200410018595XA CN 200410018595 A CN200410018595 A CN 200410018595A CN 1314708 C CN1314708 C CN 1314708C
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Abstract
The present invention relates to a composition of a human thymus peptide alpha1 and a preparation method thereof, which belongs to the technical field of a peptide or protein modified composition and preparation thereof. The composition is prepared from a human thymus peptide alpha1 and monomethoxypolyethylene glycol, and lysine epsilon-NH2 or N-terminal alpha-NH2 in the human thymus peptide alpha1 is respectively connected with a succinimide ester bond or an aldehyde group by stabile amido bond or secondary amido bond formation. Free amino groups on the protein surface of the human thymus peptide alpha1 react with monomethoxypolyethylene glycol with succinimide activated active groups or monomethoxypolyethylene glycol containing formyl groups to form a composition of monomethoxypolyethylene glycol and a human thymus peptide alpha1, namely mPEG-Talpha1, by connection. The preparation method of the present invention has the advantages of convenient operation and easy material acquisition. The composition prepared by the present invention, which has high biological stability and long half life in vivo, is helpful to increase the bioavailability of the human thymus peptide alpha1.
Description
Technical field
The present invention relates to mixture of a kind of people α 1-Zadaxin and preparation method thereof, belong to the technical field of peptide or proteinic modification mixture and preparation thereof.
Background technology
People α 1-Zadaxin (α 1-thymosin, T α 1) is produced through enzymolysis processing by the precursor α 1-Zadaxin former (prothymosin) that 111 amino acid are formed in vivo.Sophisticated people α 1 Zadaxin is made up of 28 amino acid, and molecular weight is 3.108KD, and iso-electric point is pH4.2.Mainly be present in thymocyte, T-lymphocyte and the thymic tissue in the people α 1-Zadaxin body, at organs such as liver, kidney, the heart, lung, spleens distribution is arranged also, normal concentration in human serum is approximately 540-670pg/mL, its major physiological function is for regulating the immunocompetence of body, comprise and stimulate multipotential stem cell to be divided into thymocyte, promote the differentiation and the maturation of T-cell, strengthen the function of T cell, and CD3+ and the isocellular quantity of CD4+ are raise.People α 1-Zadaxin can promote interleukin α (IL-1 α), interleukin-22 (IL-2), interleukin (IL-3), the expression of interferon alpha and γ (IFN-α, IFN-γ) and migration inhibition factor (migratory inhibitory factor).Lymphocyte is after being subjected to mitogen or antigenic stimulation, and people α 1-Zadaxin can make the IL-2 high-affinity receptor quantity of its generation increase.This stranger α 1-Zadaxin also with the regulation and control of cell cycle, angiogenic growth, cell migration, the penetrativitys of tissue repair and sperm etc. are relevant.
People α 1-Zadaxin is as a kind of wide spectrum immunomodulator, and clinical effectiveness shows that it has significant curative effect to hepatitis B and hepatitis C, and tumour and acquired immune deficiency syndrome (AIDS) are also had certain curative effect.In addition, people α 1-Zadaxin also can be used as immune assistant medicament, and with the responsibility of the weak patient of enhancing immunity function to vaccine, its application prospect is very extensive.
But people α 1-Zadaxin is a kind of bioactive micro peptide, is easy to degraded in vivo and causes biologically stable poor, and the transformation period is short in the body.Clinical data shows that the patient of personnel selection α 1-Zadaxin treatment hepatitis B needs twice subcutaneous injection 1.6mg weekly, and continue 6 months a course of treatment, and secular injection brings misery to the patient or makes the patient be unwilling to accept.Therefore press for development long people α 1-Zadaxin medicine biological half-life.
Summary of the invention
First technical problem that the present invention will solve is the mixture that proposes a kind of people α 1-Zadaxin, and this mixture is made up of people α 1-Zadaxin and mono methoxy polyethylene glycol, by the ε-NH of Methionin in the people α 1-Zadaxin
2Or the α-NH of N-terminal
2Respectively with the succinimide ester bond of mono methoxy polyethylene glycol or aldehyde radical forms stable amido linkage or secondary amine key links together.
Another technical problem that the present invention will solve provides a kind of preparation method of mixture of people α 1-Zadaxin.The present invention makes the problems referred to above be resolved by the following technical programs.To have the people α 1-Zadaxin of free amine group and have through the mono methoxy polyethylene glycol of succinimide activatory active group or contain the reacting of mono methoxy polyethylene glycol of aldehyde radical at protein surface, both connect to mono methoxy polyethylene glycol-people α 1-Zadaxin mixture, and promptly mPEG-T α 1.
Now describe technical scheme of the present invention in detail.A kind of preparation method of mixture of people α 1-Zadaxin is characterized in that the concrete operations step is as follows:
The first step, the people α 1-Zadaxin that takes by weighing 1 part of weight is dissolved in the 100mM of 500 parts of weight, in the pH7.4 phosphate buffered saline buffer, makes it dissolving, and mixed with 200mM phosphate buffered saline buffer or the boric acid-borate buffer solution of the pH6.0-9.0 of 500 parts of weight;
Second step, the PEG modifier that in the first step solution, adds 1.7-133.0 part weight, evenly mixed, people α 1-Zadaxin to be finished and polyethyleneglycol modified dose mol ratio are 1: 1～10 in the solution after mixed, described polyethyleneglycol modified dose is SPA-mPEG, NHS-mPEG, ALD-mPEG, and its molecular weight is 5KD～40KD;
In the 3rd step, place 4 ℃～40 ℃ to react 0.5～24 hour down the mixed uniform reaction solution of previous step;
In the 4th step, reaction is put-20 ℃ of termination reactions after finishing, and gets the 20ul reaction solution and carries out SDS-PAGE electrophoretic examinations degree of modification;
The 5th step, getting the modification reaction miscellany uses the 20mM citric acid-sodium citrate damping fluid of pH3.2 to dilute 10 times, last Sepharose FFCM chromatography column, 20mM citric acid-sodium citrate damping fluid with the pH3.2 that contains 0～1MNaCl carries out gradient elution, flow velocity is 0.1～1ml/min, detects absorption peak with 280nm place uv-absorbing;
In the 6th step, collect each absorption peak elute soln, the polymorphism of the polyethyleneglycol modified people α of SDS-PAGE electrophoretic examinations 1-Zadaxin mixture;
The 7th step, with the single-point absorption peak solution molecular weight cut-off of collecting is that the MilliporeAmicon Ultra-15 ultrafiltration pipe of 1KD-5KD concentrates, lyophilize, obtain the sample of the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture of 0.2 part of weight-2.1 part weight single-point modification, its molecular weight is between 8.108KD-43.108KD.
The 7th step, described sample had the structure that the present invention proposes, and promptly was made up of people α 1-Zadaxin and mono methoxy polyethylene glycol, by the ε-NH of Methionin in the people α 1-Zadaxin
2Or the α-NH of N-terminal
2Respectively with the succinimide ester bond of mono methoxy polyethylene glycol or aldehyde radical forms stable amido linkage or secondary amine key links together.
People α 1-Zadaxin in the above-mentioned preparation process has the branch of chemosynthesis or genetically engineered preparation, the former can be available from any little peptide Synesis Company, and the latter sees that the inventor's application number and title are respectively 200410018543.2 and be entitled as the patent application of " efficiently expressing genetic engineering bacterium and the structure and the application of people α 1-Zadaxin ".Be used to modify the polyoxyethylene glycol of people α 1-Zadaxin, promptly PEG for through activatory mono methoxy PEG (mPEG), can buy from SHEARWATER company.These mPEG are mono methoxy polyethylene glycol-propionic acid succinimide ester (SPA-mPEG), divide dendritic mono methoxy polyethylene glycol-succinimide ester (NHS-mPEG), propionic aldehyde mono methoxy polyethylene glycol (ALD-mPEG) that molecular weight is between 5KD～40KD.
Advantage of the present invention is that the free amine group that utilizes people α 1-Zadaxin surface to exist is the epsilon-amino of Methionin or the α-NH of N-terminal
2React under relatively mild physiological status with the activation succinimide ester bond or the aldehyde radical of mono methoxy polyethylene glycol, form amido linkage or secondary amine key, thereby make the character of the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture that obtains very stable, be difficult for being hydrolyzed separation; Because the modifier mPEG that uses is the linear nontoxic macromolecular compound of high-hydrophilic, after itself and people α 1-Zadaxin stable bond, can form a kind of barrier at people α 1-Zadaxin molecular surface, make people α 1-Zadaxin be difficult for, thereby improve its biologically stable by intravital proteasome degradation; Simultaneously owing to prolong, thereby improved the bioavailability of people α 1-Zadaxin through the people α 1-Zadaxin transformation period in vivo that PEG modifies.Produce the mixture of people α 1-Zadaxin with present method, method is easy, and raw material is easy to get, and production cost is lower relatively.
Embodiment
Below in conjunction with specific embodiment, be described in further detail the present invention.Used PEG, chemical reagent etc. in specification sheets and following examples, and the experimental technique of unreceipted actual conditions, condition is carried out routinely, or is undertaken by the condition that goods supplier is advised.
Embodiment 1 PEG modifies one of preparation method of people α 1-Zadaxin mixture.In the present embodiment, the PEG modifier that is used to modify is derivative-SPA-mPEG of PEG, and its molecular weight is 5KD.
The first step takes by weighing the 100mM that 1.0mg people α 1-Zadaxin is dissolved in 0.5ml, in the pH7.4 phosphate buffered saline buffer, makes it dissolving, and mixed with 200mM phosphate buffered saline buffer or the boric acid-borate buffer solution of the pH6.0-9.0 of 0.5ml;
In second step, adding the 1.7mg molecular weight in the first step solution is the monomethoxypolyethylglycol glycol derivative (SPA-mPEG) of 5KD, evenly mixed, and making the people α 1-Zadaxin to be finished in the solution after mixing and the mol ratio of polyethyleneglycol derivative is 1: 1;
In the 3rd step, second miscellany that goes on foot gained is placed 4 ℃～40 ℃ reaction 0.5～24h down;
In the 4th step, reaction is put-20 ℃ of termination reactions after finishing, and gets the 20ul reaction solution and carries out SDS-PAGE electrophoretic examinations degree of modification.
The 5th step, getting the modification reaction miscellany uses the 20mM citric acid-sodium citrate damping fluid of pH3.2 to dilute 10 times, last CM Sepharose FF chromatography column, 20mM citric acid-sodium citrate damping fluid with the pH3.2 that contains 0～1MNaCl carries out gradient elution, flow velocity is 0.1～1ml/min, detects absorption peak with 280nm place uv-absorbing;
The 6th step, collect each absorption peak elute soln, SDS-PAGE electrophoretic examinations PEG modifies the polymorphism of people α 1-Zadaxin mixture;
The 7th step, with the absorption peak solution molecular weight cut-off of collecting is that the MilliporeAmicon Ultra-15 ultrafiltration pipe of 1KD-5KD concentrates, lyophilize, obtaining the pure molecular weight of 0.2mg is the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture of the single-point modification of 8.108KD.
In above-mentioned second step, molecular weight is that the add-on of the monomethoxypolyethylglycol glycol derivative (SPA-mPEG) of 5KD can be respectively 8.5mg and 17mg, and mol ratio was respectively 1: 5 and 1: 10; In above-mentioned the 7th step, can obtain respectively 0.6 and the molecular weight of 0.4mg be mono methoxy polyethylene glycol-people α 1-Zadaxin mixture that the single-point of 8.108KD is modified.
The preparation method's of embodiment 2 PEG modification people α 1-Zadaxin mixture two.In the present embodiment, the PEG modifier that is used to modify is derivative-NHS-mPEG of PEG, and its molecular weight is 10KD.
The first step takes by weighing the 100mM that 1.0mg people α 1-Zadaxin is dissolved in 0.5ml, in the pH7.4 phosphate buffered saline buffer, makes it dissolving, and mixed with 200mM phosphate buffered saline buffer or the boric acid-borate buffer solution of the pH6.0-9.0 of 0.5ml;
In second step, adding the 3.3mg molecular weight in the first step solution is the monomethoxypolyethylglycol glycol derivative (NHS-mPEG) of 10KD, evenly mixed, and making the people α 1-Zadaxin to be finished in the solution after mixing and the mol ratio of polyethyleneglycol derivative is 1: 1;
The 3rd step to the 6th step is undertaken by cycle and taking corresponding operation among the embodiment one.
The 7th step, with the absorption peak solution molecular weight cut-off of collecting is that the MilliporeAmicon Ultra-15 ultrafiltration pipe of 1KD-5KD concentrates, lyophilize, obtaining the pure molecular weight of 0.4mg is the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture of the single-point modification of 13.108KD.
In above-mentioned second step, molecular weight is that the add-on of the monomethoxypolyethylglycol glycol derivative (NHS-mPEG) of 10KD can be respectively 16.5mg and 33mg, and mol ratio was respectively 1: 5 and 1: 10; In above-mentioned the 7th step, can obtain respectively 0.9 and the molecular weight of 0.7mg be mono methoxy polyethylene glycol-people α 1-Zadaxin mixture that the single-point of 13.108KD is modified.
The preparation method's of embodiment 3 PEG modification people α 1-Zadaxin mixture three.In the present embodiment, the PEG modifier that is used to modify is derivative-SPA-mPEG of PEG, and its molecular weight is 20KD.
The first step takes by weighing the 100mM that 1.0mg people α 1-Zadaxin is dissolved in 0.5ml, in the pH7.4 phosphate buffered saline buffer, makes it dissolving, and mixed with 200mM phosphate buffered saline buffer or the boric acid-borate buffer solution of the pH8.4 of 0.5ml;
In second step, adding the 6.8mg molecular weight in the first step solution is the monomethoxypolyethylglycol glycol derivative (SPA-mPEG) of 20KD, evenly mixed, and making the people α 1-Zadaxin to be finished in the solution after mixing and the mol ratio of polyethyleneglycol derivative is 1: 1;
The 3rd step to the 6th step is undertaken by cycle and taking corresponding operation among the embodiment one.
The 7th step, with the absorption peak solution molecular weight cut-off of collecting is that the MilliporeAmicon Ultra-15 ultrafiltration pipe of 1KD-5KD concentrates, lyophilize, obtaining the pure molecular weight of 0.8mg is the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture of the single-point modification of 23.108KD.
In above-mentioned second step, molecular weight is that the add-on of the monomethoxypolyethylglycol glycol derivative (SPA-mPEG) of 20KD can be respectively 34.0mg and 68.0mg, and mol ratio was respectively 1: 5 and 1: 10; In above-mentioned the 7th step, can obtain respectively 1.5 and the molecular weight of 1.2mg be mono methoxy polyethylene glycol-people α 1-Zadaxin mixture that the single-point of 23.108KD is modified.
The preparation method's of embodiment 4PEG modification people α 1-Zadaxin mixture four.In the present embodiment, the PEG modifier that is used to modify is derivative-ALD-mPEG of PEG, and its molecular weight is 40KD.
The first step takes by weighing the 100mM that 1.0mg people α 1-Zadaxin is dissolved in 0.5ml, in the pH7.4 phosphate buffered saline buffer, makes it dissolving, and mixed with 200mM phosphate buffered saline buffer or the boric acid-borate buffer solution of the pH8.4 of 0.5ml;
In second step, adding the 13.3mg molecular weight in the first step solution is the monomethoxypolyethylglycol glycol derivative (ALD-mPEG) of 40KD, evenly mixed, and making the people α 1-Zadaxin to be finished in the solution after mixing and the mol ratio of polyethyleneglycol derivative is 1: 1;
The 3rd step to the 6th step is undertaken by cycle and taking corresponding operation among the embodiment one.
The 7th step, with the absorption peak solution molecular weight cut-off of collecting is that the MilliporeAmicon Ultra-15 ultrafiltration pipe of 1KD-5KD concentrates, lyophilize, obtaining the pure molecular weight of 1.4mg is the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture of the single-point modification of 43.108KD.
In above-mentioned second step, molecular weight is that the add-on of the monomethoxypolyethylglycol glycol derivative (SPA-mPEG) of 20KD can be respectively 66.5mg and 133.0mg, and mol ratio was respectively 1: 5 and 1: 10; In above-mentioned the 7th step, can obtain respectively 2.1 and the molecular weight of 1.7mg be mono methoxy polyethylene glycol-people α 1-Zadaxin mixture that the single-point of 43.108KD is modified.
Embodiment described above is intended to set forth preferred forms of the present invention rather than limits the present invention in any form.Those skilled in the art, all drop in the scope of patent application right requirement of the present invention in conjunction with the various changes that the general knowledge of this area is done according to enlightenment of the present invention.
Claims (2)
1. the mixture of a people α 1-Zadaxin is characterized in that, this mixture is made up of people α 1-Zadaxin and mono methoxy polyethylene glycol, by the ε-NH of Methionin in the people α 1-Zadaxin
2Or the α-NH of N-terminal
2Respectively with the succinimide ester bond of mono methoxy polyethylene glycol or aldehyde radical forms stable amido linkage or secondary amine key links together.
2. the preparation method of the mixture of the described people α of claim 1 1-Zadaxin is characterized in that the concrete operations step is as follows:
The first step, the people α 1-Zadaxin that takes by weighing 1 part of weight is dissolved in the 100mM of 500 parts of weight, in the pH7.4 phosphate buffered saline buffer, makes it dissolving, and mixed with 200mM phosphate buffered saline buffer or the boric acid-borate buffer solution of the pH6.0-9.0 of 500 parts of weight;
Second step, the PEG modifier that in the first step solution, adds 1.7-133.0 part weight, evenly mixed, people α 1-Zadaxin to be finished and polyethyleneglycol modified dose mol ratio are 1: 1～10 in the solution after mixed, described polyethyleneglycol modified dose is SPA-mPEG, NHS-mPEG, ALD-mPEG, and its molecular weight is 5KD～40KD;
In the 3rd step, place 4 ℃～40 ℃ to react 0.5～24 hour down the mixed uniform reaction solution of previous step;
In the 4th step, reaction is put-20 ℃ of termination reactions after finishing, and gets the 20ul reaction solution and carries out SDS-PAGE electrophoretic examinations degree of modification;
The 5th step, getting the modification reaction miscellany uses the 20mM citric acid-sodium citrate damping fluid of pH3.2 to dilute 10 times, last Sepharose FF CM chromatography column, 20mM citric acid-sodium citrate damping fluid with the pH3.2 that contains 0～1MNaCl carries out gradient elution, flow velocity is 0.1～1ml/min, detects absorption peak with 280nm place uv-absorbing;
In the 6th step, collect each absorption peak elute soln, the polymorphism of the polyethyleneglycol modified people α of SDS-PAGE electrophoretic examinations 1-Zadaxin mixture;
The 7th step, with the single-point absorption peak solution molecular weight cut-off of collecting is that the MilliporeAmicon Ultra-15 ultrafiltration pipe of 1KD-5KD concentrates, lyophilize, obtain the sample of the mono methoxy polyethylene glycol-people α 1-Zadaxin mixture of 0.2 part of weight-2.1 part weight single-point modification, its molecular weight is between 8.108KD-43.108KD.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410018595XA CN1314708C (en) | 2004-05-24 | 2004-05-24 | Human alpha 1-thymic peptide composition and its preparation |
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|---|---|---|---|
| CNB200410018595XA CN1314708C (en) | 2004-05-24 | 2004-05-24 | Human alpha 1-thymic peptide composition and its preparation |
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| CN1583793A CN1583793A (en) | 2005-02-23 |
| CN1314708C true CN1314708C (en) | 2007-05-09 |
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| CNB200410018595XA Expired - Fee Related CN1314708C (en) | 2004-05-24 | 2004-05-24 | Human alpha 1-thymic peptide composition and its preparation |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003037272A2 (en) * | 2001-11-01 | 2003-05-08 | Sciclone Pharmaceuticals, Inc. | Thymosin alpha 1 peptide/polymer conjugates |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003037272A2 (en) * | 2001-11-01 | 2003-05-08 | Sciclone Pharmaceuticals, Inc. | Thymosin alpha 1 peptide/polymer conjugates |
Non-Patent Citations (1)
| Title |
|---|
| 多肽和蛋白质的聚乙二醇化修饰方法 王良友等，有机化学，第23卷第11期 2003 * |
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