CN100335499C - C-terminal amino acid lactone modified extrasin alpha-1 and its uses - Google Patents

Abstract

The present invention relates to a biological polypeptide of a thymosin alpha1 derivative. More specially, the present invention relates to thymosin alpha1 with the C terminal modified with amino acid lactone. The thymosin alpha1 derivative of the present invention has stronger bioactivity than the known natural thymosin alpha1 with strong bioactivity and has new bioactivity unpossessed by the natural thymosin alpha-1, for example, the thymosin alpha1 derivative can resist hematopoietic inhibition caused by chemotherapeutic medicines or radiation therapy. Thus, the present invention is suitable for antiviral therapy, antitumor therapy, chemotherapy and radiotherapeutic adjunctive therapy. The present invention also relates to encoding nucleic acid of the thymosin alpha1 derivative, a medical composition containing the thymosin alpha1 derivative and an application thereof.

Description

C-terminal amino acid lactone modified thymosin α1 and its application

technical field

The invention relates to biological polypeptide thymosin α1 derivatives. More specifically, the present invention relates to thymosin α1 whose C-terminus is modified with an amino acid lactone. The thymosin α1 derivatives described in the present invention not only have stronger biological activity than the known biological activity of natural thymosin α1, but also have new biological activities that natural thymosin α1 does not possess, such as anti-chemotherapy drugs or Inhibition of hematopoietic function caused by radiotherapy, so it is more suitable for adjuvant treatment of antiviral therapy, antitumor therapy, chemotherapy and radiotherapy. The present invention also relates to the encoding nucleic acid of the thymosin α1 derivative, the pharmaceutical composition containing the thymosin α1 derivative and the use thereof.

Background technique

Thymosin-α1 (Thymosinα1) was first discovered in thymus tissue by Goldstein et al. in 1977. In the body, it is produced by enzymatic processing of the precursor thymosin-alpha 1 (prothymosin) consisting of 111 amino acids. Mature thymosin-α1 consists of 28 amino acids, with a molecular weight of 3108 Daltons and an isoelectric point of 4.2. Thymosin-α1 is not only present in thymocytes, T-lymphocytes and thymus tissue, but also in liver, kidney, heart, lung, spleen and other organs.

Thymosin α1 acts on the early and late stages of thymocyte maturation, increases the expression of Thy_1, 2 and Lyt_1, 2, 3 on the surface of T cells, and promotes the maturation of TH cells. Thymosin α1 can also regulate the level of terminal deoxynucleotidyl transferase (TDT) in thymocytes in vitro, stimulate macrophage inhibitory factor (Microphage Inhibition Factor, MIF), interferon (Interferon, IFN), interleukin- 2 (Interleukin-2, IL-2) and its receptor expression, enhance human NK cell activity. It shows that thymosin α1 can regulate the immune function of the body, promote the expression of cytokines and their receptors, enhance the activity of immune cells, and exert its antiviral effect from many aspects. The absolute number of thymosin α1 active cells decreased with age, and the existence of thymosin α1 could not be detected after the age of 40. According to radioimmunology (RadioimmuneAssay, RIA) determination, abnormal thymosin α1 levels in serum are related to certain diseases, and some reports indicate that low levels of thymosin α1 lead to thymus-dependent immunodeficiency diseases. At the same time, the interaction between thymosin α1 and neuroendocrine is also very interesting. The researchers found that there are high concentrations of thymosin α1-like peptides in the hypothalamus and pituitary extracts. Thymosin α1 can affect ACTH at the level of hypothalamus and/or pituitary. , Pr1, TSH and LH secretion, indicating that it may be a neuroendocrine regulator.

As a biological response modifier (BRM), thymosin α1 can significantly improve the immune function of the body. In recent years, it has been widely used as an immune enhancer in the clinical treatment of various immunodeficiency diseases, autoimmune diseases, tumors and viruses and other microbial infections. When combined with other biological response regulators, such as IL-2, IFN-α, thymus factor The curative effect is better when they act synergistically, especially for the treatment of chronic viral hepatitis, the effect is more significant.

In addition, due to the small molecule of thymosin α1, natural single-molecule expression products cannot be obtained in prokaryotic expression systems such as Escherichia coli. At present, thymosin α1 is mainly prepared by chemical synthesis, but its problem is that the cost of use is too high.

Contents of the invention

In order to obtain low-cost functional thymosin α1, the inventors have studied a variety of genetic engineering production methods of thymosin α1. Since single-molecule thymosin α1 cannot be expressed in Escherichia coli, fusion or tandem expression plus chemical cleavage was used. The method is to obtain the derivatives of thymosin α1, such as adding methionine derivatives (homoserine lactone) and tryptophan derivatives (tryptophan lactone) to the C-terminus of thymosin α1. The present inventors further studied the biological activity of thymosin α1-related derivatives, and surprisingly found that when the C-terminus of thymosin α1 was modified by these two amino acid lactones, its known biological activity was improved. Enhanced, and exhibited some new biological effects. The modified thymosin α1 derivative product can be produced by using genetic engineering technology, and its production cost is greatly reduced, which is conducive to the popularization of drug use.

Accordingly, one aspect of the present invention relates to biological polypeptide thymosin α1 derivatives. More specifically, the present invention relates to thymosin α1 whose C-terminus is modified with an amino acid lactone. The thymosin α1 derivatives described in the present invention not only have stronger biological activity than the known biological activity of natural thymosin α1, but also have new biological activities that natural thymosin α1 does not possess, such as anti-chemotherapy drugs or Inhibition of hematopoietic function caused by radiotherapy, so it is more suitable for adjuvant treatment of antiviral therapy, antitumor therapy, chemotherapy and radiotherapy.

The thymosin α1 polypeptide derivative of the present invention is characterized in that it has an amino acid sequence whose C-terminus is modified by amino acid lactone as shown below:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsn-@

Wherein, @ represents the amino acid lactone modified at the C-terminus.

Preferably, the C-terminal modified amino acid lactone is selected from tryptophan lactone derived from tryptophan and homoserine lactone derived from methionine.

In addition, the present invention relates to a nucleic acid sequence encoding the above-mentioned thymosin α1 polypeptide derivative of the present invention, a vector containing the nucleic acid sequence, and a host cell containing the vector.

The thymosin α1 polypeptide derivatives of the present invention can be prepared by combining chemical synthesis of polypeptides with chemical modification, or by combining genetic engineering expression techniques with chemical cleavage techniques. The methods and their specific operations are well known to those skilled in the art. For example, see "Instrumentation for automated solid-phase peptide synthesis." Cammish, L.E., Kates, S.A. In Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Chan, W., White, P.) Oxford University Press, Oxford, for example, for the method of polypeptide chemical synthesis. 1998. For the chemical modification method of amino acids, see Chemical reagents for protein modification.2nd edition.Roger L.Lundblad.CRC press, 1991.

When the genetic engineering method is combined with the chemical cleavage technology to prepare, at first through the gene recombination expression technology well known to those skilled in the art (one of the optional reference books is "Molecular Cloning Experiment Guide", the second edition), produce the amino acid with C-terminal modification The polypeptide expression product, and then prepare the thymosin α1 polypeptide derivative of the present invention by a suitable chemical cleavage method (see Chemical reagents for protein modification. 2nd edition. Roger L. Lundblad. CRC press, 1991 for chemical cleavage technology). The main technical point of using genetic engineering technology to produce the thymosin α1 derivative is to artificially synthesize the corresponding nucleotide sequence according to the provided amino acid sequence. Then the artificially synthesized nucleotide sequence of the thymosin α1 derivative is cloned into an appropriate Escherichia coli or yeast expression vector for expression, and the expression methods can be varied. For example, it can be fused and expressed with other proteins or polypeptides, or expressed in tandem with itself, or fused with other proteins after tandem expression. After the expression product is purified, it is cleaved by a chemical cleavage method. For example, a gene expression product modified with methionine at the C-terminus is produced by cyanogen bromide cleavage to produce a thymosin α1 polypeptide derivative with a methionine derivative at the C-terminus; N-chlorosuccinimide and N-bromosuccinimide) cleave gene expression products modified with tryptophan at the C-terminus to generate thymosin α1 polypeptide derivatives with tryptophan derivatives added at the C-terminus. The methionine derivative refers to homoserine lactone formed after methionine cleavage; the tryptophan derivative refers to tryptophan lactone formed after oxidative cleavage of tryptophan.

Another aspect of the present invention relates to a pharmaceutical composition containing the above-mentioned polypeptide, nucleic acid sequence, vector or host cell as an active ingredient, and optionally a pharmaceutically acceptable auxiliary ingredient. The active ingredient is present in the pharmaceutical composition in a physiologically effective amount. The pharmaceutical composition can exist in the form of medicines currently used in human beings, such as freeze-dried powder injections, aqueous injections, intravenous drops, oral tablets, sprays, ointments, etc. These pharmaceutical forms can be prepared according to common methods and can be Administration is carried out according to different routes of administration known to those skilled in the art. Suitable routes of administration include, for example, oral, rectal, transmucosal or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intrathecal and intrathecal injections, direct intraventricular, intravenous, intraperitoneal, intranasal or ocular Injection. Administration can be local rather than systemic. The active ingredient can be mixed with auxiliary ingredients usually used in pharmaceutical compositions to prepare pharmaceutical compositions. The auxiliary components are, for example, pharmaceutically acceptable carriers, diluents, adjuvants, excipients, stabilizers, preservatives, osmotic agents, isotonic agents and/or release control agents and the like. The active ingredients of the invention may be used alone or in combination with other compounds such as therapeutic compounds, eg interleukins or interferons and the like.

Another aspect of the present invention relates to the application of the above-mentioned polypeptide, nucleic acid sequence, vector or host cell in the preparation of medicaments.

Thymosin α1 polypeptide derivatives of the present invention have the same or even stronger biological activity than natural thymosin α1 in terms of immunological effects, so they can be applied to the adjuvant treatment of immune-related diseases, including viral diseases, such as hepatitis B , Hepatitis C, AIDS, Influenza, and various other viral diseases; Immunocompromised or disordered diseases, such as tumors, recurrent colds, chronic diarrhea, etc.

Thymosin α1 polypeptide derivatives of the present invention also have biological activities different from those of thymosin α1, especially in terms of hematopoietic inhibition caused by chemotherapeutic agents and radiotherapy, and have a significant protective effect. Therefore, this product can also It is suitable as an adjuvant drug for chemotherapy and radiotherapy to protect the patient's hematopoietic function.

Unless otherwise clearly stated, various terms, technical or scientific terms used in the present application documents have broad meanings known to those skilled in the art.

The present invention will be described in further detail below using specific examples.

Example 1 Thymosin α1 polypeptide modified by methionine derivatives at the C-terminus

The sequence of the thymosin α1 polypeptide whose C-terminus is modified by a methionine derivative is as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsn-homoserine lactone

1. Preparation method

The thymosin α1 polypeptide whose C-terminus is modified with homoserine lactone can be produced by using techniques known in the art.

1) Peptide chemical synthesis combined with chemical modification

(1) Polypeptide solid-phase synthesis is a mature technology. As long as the amino acid sequence is given, many companies can carry out large-scale synthesis according to the given amino acid sequence. According to the Fmoc solid-phase synthesis method (Fmoc: 9-fluorenylmethoxycarbonyl), on Applied Biosystems company's 433 type peptide synthesizer, the chemically synthesized amino acids are as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnMet

(2) The above-mentioned synthetic product was modified with cyanogen bromide according to the experimental method of the aforementioned "Chemical reagents for protein modification" to methionine. Dissolve the protein in 70% formic acid at 2mg/ml (in a pressure-resistant container that can be sealed), and then add solid cyanogen bromide (operation) at a ratio of 10g/ml. After 36 hours of cracking in the dark at room temperature, slowly 10N NaOH was added to adjust the solution to pH 7.0.

(3) Purification of modified thymosin α1 by reversed-phase chromatography

The purification medium is Source-30RPC, and the column volume is about 350ml. Load the sample at a flow rate of 20ml/min, dilute the sample obtained in the above steps 10 times with water, and load about 3000ml each time. After loading the sample, wash the chromatography column about 400ml with solution A (20mM Tris-Cl (pH=6.8), 200mM sodium chloride) at a flow rate of 25ml/min, and then use 5% to 15% solution B (20mM Tris-Cl (pH=6.8), 200mM sodium chloride, 50% ethanol) to elute the impurity protein, the elution volume is 600ml, then use 20%～60% B to elute thymosin α1, collect the peak of the target protein which is the modified thymus Protein α1, the product recovery rate is 80-90%, and the target protein identification is carried out according to the following method.

(4) Further purification of modified thymosin α1 by ion exchange chromatography

The purification medium is Source-30Q, and the column volume is about 300ml. Dilute the recombinant human thymosin α1 collected in step (3) by one time with distilled water, and load about 2500ml of sample at a flow rate of 20ml/min. After loading the sample, wash 400ml of the column with solution A (20mM Tris-Cl (pH=6.8)) at a flow rate of 20ml/min, and then use 10% to 40% solution B (20mM Tris-Cl (pH=6.8) to 1M sodium chloride) to elute about 400ml, collect the target protein peak, the product recovery rate is 85-95%, carry out target protein identification according to the following method.

(5) Gel filtration method to obtain the final purified modified thymosin α1

The purification medium is Sephacryl S-100HR with a column bed volume of 2000ml, and the recombinant human thymosin α1 collected in step (4) is loaded at 0.1% to 5% of the column bed volume, and the flow rate is 3 to 8ml/min, and the protein peak is collected. The protein peak is the purified recombinant human thymosin α1, the product recovery rate is 80-90%, and the target protein is identified according to the following method. Equilibrium and washing buffer formulations are: 25mM phosphate buffer (pH6.0-7.5), 125mM sodium chloride.

(6) Identification method of thymosin α1 protein

a. Identification of molecular weight by protein electrophoresis

Methods: Refer to the second edition of "Molecular Cloning Experiment Guide", polyacrylamide gel electrophoresis of proteins, Science Press. The gel crosslinking degree is 29:1 (acrylamide:bisacrylamide), and the gel concentration is 18%. Using the thymosin α1 standard (purchased from Sigma) as a reference, the electrophoresis results showed that the purified target protein had the same molecular weight as the thymosin α1 standard.

b. N-terminal sequence determination

The N-terminal sequence of the product was determined using standard protein N-terminal sequencing technology on an Applied Biosystems ABI Proeise 491 protein sequencer. The N-terminal sequence determination results are as follows:

The sequence of 15 amino acid residues at the N-terminus is as follows: Ser, Asp, Ala, Ala, Val, Asp, Thr, Ser, Ser, Glu, ILe, Thr, Thr, Lys, Asp. Consistent with the expected results.

2) Genetic engineering method combined with chemical cracking technology

(1) Artificial gene sequence synthesis

For this product, the designed amino acid sequence is as follows:

SerAspAlaAlaValAspThrSerSerGlulleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnMet

According to the above amino acid sequence, using the principle of degeneracy of the genetic code, a variety of sequences that meet the requirements can be designed. Here, the following sequences are used according to the gene expression principle and vector expression scheme of Escherichia coli:

ATG TCTGATGCTGCTGTTGATACTTCTTCTG GA

ATG TCTGATGCTGCTGTTGATACTTCTTCTG

AGATTACTACTAAAGATCTTAAGGAGAAGAAGGAAGTT

CG GTCGAAGAGGCTGAGA AC ATG

CG

The underlined sequence is the thymosin α1 sequence, and the double underlined sequence represents the restriction endonuclease cutting sites, which are BamHI and EcoRI respectively.

According to the above sequence, the following two nucleotide fragments were artificially synthesized on the ABI 3900 desktop high-throughput DNA synthesizer according to the β-acetonitrile phosphite chemical synthesis method:

Fragment 1:

GAGGATCCATGTCTGATGCTGCTGTTGATACTTCTTCTG

AGATTACTACTAAAAGATCTTAA

Fragment 2:

CGGAATTCCATGTTTCTCAGCCTCTTCGACAACTTCCTTC

TTCTCCTTAAGATCTTTAGTAG

(2) For gene cloning, refer to the second edition of "Molecular Cloning Experiment Guide".

Add 20 pmol each of the above two fragments, and anneal the fragments in 50 μL of a restriction enzyme (such as PstI) buffer with a final concentration of 100 mM NaCl. Place the sample tube in a water bath at 95°C, cool it down to room temperature naturally, and complete the annealing. Then add 5 μL of 10mMol/L dNTP, 5u of T4 DNA polymerase, 37°C for 1 hour, and synthesize double-stranded DNA. Then inactivate T4 DNA polymerase at 80°C for 30 minutes. Finally, 10u of two restriction enzymes, EcoRI and BamHI, were added respectively, and incubated at 37°C for 1 hour. Agarose gel electrophoresis, with reference to DNA molecular weight standards, according to the fragment length, recovery of target gene fragments. The commonly used Escherichia coli GST fusion expression vector pGEX-4T1 (purchased from Phamacia Company) was digested with the same two enzymes, and a large fragment was recovered by agarose gel electrophoresis. The vector fragment was connected with the target gene segment to obtain the recombinant vector, which was transformed into Escherichia coli DH5α (purchased from Clontech Company), the plasmid was extracted, positive clones were screened by restriction enzyme digestion (EcoRI and BamHI), and finally the correctness of the target gene segment was identified by DNA sequencing. sex.

(3) For gene expression, refer to the second edition of "Molecular Cloning Experiment Guide".

A positive clone strain containing the correct recombinant vector was obtained through DNA sequencing identification, and the gene expression of the strain was induced according to the IPTG induction method.

(4) Product purification, refer to the second edition of "Molecular Cloning Experiment Guide".

A. Centrifuge to collect the above induced E. coli strains, weigh the total amount of bacterial pellets, and suspend according to the ratio of adding 10ml lysis buffer (20mM Tris-Cl, pH=6.8, 5mM EDTA (pH=8.0)) for every 1g of bacterial pellets Bacteria, stirred evenly, kept at 80°C for 30 minutes, cooled rapidly to room temperature; then centrifuged at 7000 rpm for 20 minutes, collected the supernatant, added mercaptoethanol to a final concentration of 0.1%, and set aside.

B. Purification by ion exchange chromatography: the purification medium is Q-Sepharose fastflow, and the column volume is about 350ml. Samples were loaded at a flow rate of 20ml/min, and about 3500ml of the product obtained in A was loaded each time. After loading the sample, wash the chromatography column about 400ml with solution A (20mM Tris-Cl (pH=6.8), 0.1% mercaptoethanol) at a flow rate of 25ml/min, and then use 15% to 40% solution B (20mM Tris-Cl (pH=6.8), 0.1% mercaptoethanol, 1M sodium chloride) elution, the elution volume is 600ml, collects target protein peak, uses the identification method of above-mentioned thymosin α1 protein identification.

C. Cyanogen bromide cleavage and purification: the method and steps are the same as (2)-(6) in combination with chemical modification of polypeptide chemical synthesis, cleavage of Escherichia coli GST, and obtaining the above-mentioned thymosin α1 modified by homoserine lactone at the C-terminus .

2. Activity Assay

1), [3H]-TdR (thymidine) incorporation method:

The principle is: [ ³ H]-TdR is converted into [ ³ H]dTTP in the cell, and the latter is incorporated into DNA when the cell proliferates and synthesizes DNA, and its incorporation amount is proportional to the amount of DNA synthesis and the number of proliferating cells. The cell proliferation rate can be calculated by measuring the incorporation of [ ³ H]-TdR.

The mice were killed by necking, and the spleen was taken out for grinding, and the mononuclear cells were separated with lymphocyte separation medium, washed twice with serum-free culture medium, resuspended in 1640 culture medium containing 10% calf serum, counted cells, 96 wells Add 100 μl of cell suspension to each well of the plate, then add ConA (final concentration is 5 μg/ml), incubate in a _CO2 incubator at 37°C for 6 hours, add different concentrations of C-terminal homoserine or lactone-modified thymosin α1 samples , adjust the volume to 200 μl, continue to culture for 72 hours, add [ ³ H]-TdR 18.5×10 ³ GBq and culture for 6 hours, collect the cells on glass fiber filter paper, measure the cpm value after drying, and calculate the proliferation rate according to the following formula:

Table 1 Concentration (μg/ml) cpm(X±SD) value-added rate natural thymosin alpha 1 50.00 92806.22±4751.80 86.86 12.50 84640.02±2715.81 70.42 1.94 61171.38±5097.59 23.17 C-terminal homoserine lactone-modified thymosin α1 50.00 117077.07±4870.02 135.73 12.50 84478.09±2217.02 70.09 1.94 64557.58±7900.56 30.00 blank control 49665.74±3904.44 0

2), de-E receptor method:

Reagents and methods:

(1) Hank's solution: 0.3% potassium dihydrogen phosphate (KH ₂ PO ₄ ) solution, 0.76% sodium dihydrogen phosphate (Na ₂ HPO ₄ ) solution, 2% potassium chloride solution and 20% sodium chloride The solution is mixed in the ratio of 20:20:20:40 in turn, add 1g of glucose, dissolve and mix, dilute to 1000ml with water, and adjust the pH to 7.2-7.3 with 4% sodium carbonate solution (preparation before use).

(2) Alfred's solution: Accurately weigh 0.420g of sodium chloride, 0.055g of citric acid, 0.766g of sodium citrate, and 2.05g of glucose, add water to dissolve to 100ml, and boil for 60min to sterilize.

(3) Separation liquid: lymphocyte separation liquid.

(4) Sheep blood: extract 5ml of venous blood from sheep, add it to 5ml of Astoria's solution, and store it in the refrigerator (semi-sterile operation).

(5) Fixative solution: 25% glutaraldehyde solution, 3.5% sodium bicarbonate solution and Hank's solution were mixed sequentially at a ratio of 1:1:38.

(6) Staining solution: Take 2ml of Giemsa staining solution (stock solution), add 6ml of Hank's solution, shake well, centrifuge (1500rpm) for 10min, and take the supernatant for use.

(7) Preparation of de-E thymocytes: take fresh porcine thymus, remove fat and cut it into pieces, add an appropriate amount of Hank's solution to make it turbid, filter through a 100-mesh sieve, add the filtrate to the separation liquid that already has 1/3 of the filtrate volume In a centrifuge tube, centrifuge at 2000rpm for 20min, carefully suck out the white thymocytes in the middle layer, put them into another centrifuge tube, add an appropriate amount of Hank's solution to wash, shake and shake well, centrifuge at 1500rpm for 3min, discard the supernatant, and repeat three times at 45°C Keep warm in a constant temperature water bath for 30 minutes (shaking every 5 minutes). Centrifuge at 1500rpm for 3min, discard the supernatant, then add an appropriate amount of Hank's solution to the original volume, mix well and place in a constant temperature water bath at 45°C for 30min, take it out and centrifuge at 1500rpm for 3min, discard the supernatant. Wash with Hank's solution three times (the operation is the same as before), and finally dilute and count appropriately with Hank's solution, so that the final concentration is 3-5×10 ⁶ cells/ml.

(8) Preparation of sheep erythrocyte suspension: take an appropriate amount of sheep blood, wash with appropriate amount of Hank's solution three times (operation is the same as before), discard the supernatant, properly dilute with Hank's solution and count, so that the final concentration is 2～7×10 ⁷ cells/ml.

(9) Determination: take this product and use appropriate amount of Hank's solution to make a solution containing 10.1 mg of thymosin α per 1 ml as the test solution. Take 6 small test tubes, add 0.1ml of Hank’s solution to 3 tubes as a control tube, add 0.1ml of test solution to each of 3 tubes as a measurement tube, add 0.2ml of E thymocytes to each tube, keep warm at 37°C for 1 hour, then add sheep red blood cells 0.2ml, shake well, centrifuge at 500rpm for 3min, put in 4℃ refrigerator overnight. Take it out the next day, discard the supernatant, add 1 drop of fixative solution to each tube, shake gently, let it stand for 10 minutes, add 2 drops of staining solution and shake it well, start counting after standing for 15 minutes, light blue in the microscope field of view The larger cells are lymphocytes, a total of 200, count the number of E rosettes (lymphocytes combined with 3 or more sheep red blood cells), and take the average value, which is the E rosette of the sample or control tube number.

The sample viability was calculated according to the formula:

Sample activity = (the number of rosettes in test tube E - the number of rosettes in control tube E)/200*100%

The result is as follows:

Adding different concentrations of purified recombinant protein and chemically synthesized thymosin α1, each concentration was repeated 3 times, the results showed that both C-terminal homoserine or lactone-modified thymosin α1 and chemically synthesized products could promote the flowering rate of T cells The increase, and the flowering rate is related to the concentration of thymosin α1 (Table 2). In terms of promoting the flower formation rate, C-terminal homoserine or lactone-modified thymosin α1 was slightly enhanced compared with chemically synthesized products. The results are shown in the table below:

Table 2 sample name Concentration (mg/ml) Flower formation rate Absolute value added relative value added blank 0 14.5% - - C-terminal homoserine lactone-modified thymosin α1 1 18.5% 4% 27.6% 0.1 36.6% 22.1% 152.4% 0.01 30.5% 16.0% 110.3% 0.001 17.4% 2.9% 20.0% natural thymosin alpha 1 1 16.4% 1.9% 13.1% 0.1 35.2% 20.7% 142.7% 0.01 28.2% 14.7% 101.4% 0.001 15.8% 1.3% 8.9%

3) Effect of C-terminal homoserine lactone-modified thymosin α1 on hematopoietic function in mice

Animal grouping and administration: Select Kunming mice (purchased from the Animal Center of the Fourth Military Medical University), with a weight difference of less than 2g, of the same sex (male), and randomly divide them into normal control group, chemically synthesized thymosin α1 control group and C-terminal Modified thymosin α1 test group, cyclophosphamide immunosuppression control group, cyclophosphamide + chemically synthesized thymosin α1 control group and cyclophosphamide + C-terminal modified thymosin α1 test group, 8-15 rats in each group. :

Administration method: Subcutaneous injection of CTX 50mg/kg, once every other day, 3 times in total, modified thymosin α1 and chemically synthesized natural thymosin α1, once a day, 14 times in total.

Determination of red blood cell and hemoglobin content: Measure hemoglobin content and red blood cell count with a blood cell analyzer.

Table 3. Effect of C-terminal homoserine lactone-modified thymosin α1 on peripheral blood red blood cell count and hemoglobin content in mice treated with chemotherapy group Dose μg/kg Number of rats/only RBC×10 ¹² /L HBg/L Normal control natural thymosin α1 C-terminal modified thymosin α1 cyclophosphamide cyclophosphamide + natural thymosin α1 cyclophosphamide + C-terminal modified thymosin α1 -32032050mg/kg×3320320 9111311815 8.17±0.688.25±0.648.61±0.357.65±0.637.69±0.858.26±0.54 ^* 117.6±10.3117.8±9.4124.2±4.9113.7±10.9114.0±12.6119.5±8.5 ^*

^* P<0.05 cyclophosphamide + C-terminal modified thymosin α1 compared with cyclophosphamide + natural thymosin α1;

P<0.01 Cyclophosphamide + C-terminal modified thymosin α1 compared with cyclophosphamide group.

The results showed that the C-terminal modified thymosin α1 could resist the hematopoietic inhibitory effect caused by cyclophosphamide, and there was a significant statistical difference, while the natural thymosin α1 had no such effect.

Example 2 Thymosin α1 polypeptide modified by tryptophan derivatives at the C-terminus

1. Preparation method

The thymosin α1 polypeptide whose C-terminus is modified by tryptophan lactone can be produced by techniques known in the art.

1) Peptide chemical synthesis combined with chemical modification

Same as Example 1, the difference is that the last amino acid in the synthetic amino acid sequence is not methionine but tryptophan. The amino acid sequence is as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnTrp

The above synthetic product was modified with N-chlorosuccinimide to tryptophan according to the experimental method of the aforementioned "Chemical reagents for protein modification". The synthetic product was dissolved in 50% acetic acid at 2 mg/ml (in a pressure-resistant container that can be sealed), and then N-chlorosuccinimide was added to a final concentration of 0.1M. After cleavage at room temperature for 12 hours, slowly added The solution was adjusted to pH 7.0 with 10N NaOH.

Since the product is very close to homoserine lactone-modified thymosin α1 polypeptide in physical and chemical properties, the product can be purified by the same purification method.

2) Genetic engineering method combined with chemical cracking technology

This product can be obtained by using the same scheme and purification technical route for the production of homoserine lactone-modified thymosin α1, the difference lies in the following two points:

A. The last amino acid of the amino acid sequence is different, resulting in the design of the gene sequence is different

Same, the amino acid sequence is as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnTrp

Its gene sequence is as follows:

TGG TCTGATGCTGCTGTTGATACTTCTTCTGAGA GA

TGG TCTGATGCTGCTGTTGATACTTCTTCTGAGA

TTACTACTAAAGATCTTAAGGAGAAGAAGGAAGTTGTCGAAG

CG AGGCTGAGA AC TGG

CG

where ATG (methionine) is replaced by the codon TGG (tryptophan).

According to the above-mentioned sequence design, as in Example 1, the synthetic fragment is as follows:

Fragment 1:

GAGGATCCTGGTCTGATGCTGCTGTTGATACTTCTTCTG

AGATTACTACTAAAAGATCTTAA

Fragment 2:

CGGAATTCCCAGTTCTCAGCCTCTTCGACAACTTCCTTC

TTCTCCTTAAGATCTTTAGTAG

B. The cleavage method is different. Tryptophan cleavage adopts the experimental method of "Chemical reagents for protein modification", uses N-chlorosuccinimide to cleavage, cleavages E. coli GST, and obtains the above-mentioned C-terminal as tryptophan lactone Modified thymosin alpha 1 polypeptide.

2. Activity Assay

Tryptophan lactone-modified thymosin α1 activity assay method is completely consistent with embodiment 1, and various activity assays are carried out simultaneously with homoserine lactone-modified thymosin α1 activity assay, and the results are basically consistent between the two, and there is no Significant difference. The result is as follows:

1), [ ³ H]-TdR (thymidine) incorporation method:

Table 4 Concentration (μg/ml) cpm(X±SD) value-added rate natural thymosin alpha 1 50.00 92806.22±4751.80 86.86 12.50 84640.02±2715.81 70.42 1.94 61171.38±5097.59 23.17 C-terminal tryptophan lactone-modified thymosin α1 50.00 118053.07±4940.01 137.70 12.50 84532.09±2217.02 70.02 1.94 65063.58±5306.56 31.00 blank control 49665.74±3904.44 0

2), de-E receptor method:

table 5 sample name Concentration (mg/ml) Flower formation rate Absolute value added relative value added blank 0 14.5% - - C-terminal tryptophan lactone-modified thymosin α1 1 19.2% 4.5% 28.7% 0.1 36.8% 22.3% 153.1% 0.01 31.5% 16.6% 117.3% 0.001 17.2% 2.8% 19.9% natural thymosin alpha 1 1 16.4% 1.9% 13.1% 0.1 35.2% 20.7% 142.7% 0.01 28.2% 14.7% 101.4% 0.001 15.8% 1.3% 8.9%

3) Effect of C-terminal tryptophan lactone-modified thymosin α1 on hematopoietic function in mice

Table 6. Effect of C-terminal tryptophan lactone-modified thymosin α1 on peripheral blood red blood cell count and hemoglobin content in mice during chemotherapy group Dose μg/kg Number of rats/only RBC×10 ¹² /L HBg/L Normal control natural thymosin α1 C-terminal modified thymosin α1 cyclophosphamide cyclophosphamide + natural thymosin α1 cyclophosphamide + C-terminal modified thymosin α1 -32032050mg/kg×3320320 9111311815 8.17±0.688.25±0.648.55±0.457.65±0.637.69±0.858.33±0.56 ^* 117.6±10.3117.8±9.4123.1±4.9113.7±10.9114.0±12.6119.9±8.6 ^*

^* P<0.05 "Cyclophosphamide + C-terminal modified thymosin α1 group" vs "cyclophosphamide + natural thymosin α1 group";

^* P<0.01 "Cyclophosphamide + C-terminal modified thymosin α1 group" compared with cyclophosphamide group.

The results showed that the C-terminal modified thymosin α1 could resist the hematopoietic inhibitory effect caused by cyclophosphamide, and the difference was statistically significant, while the natural thymosin α1 had no such effect.

Claims (12)

1. Thymosin α1 polypeptide derivative is characterized in that having the amino acid sequence that C-terminus is modified by amino acid lactone as shown below: Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn-@ Wherein, @ represents the amino acid lactone modified at the C-terminus. 2. The polypeptide derivative according to claim 1, characterized in that said C-terminal modified amino acid lactone is a tryptophan lactone derived from tryptophan. 3. The polypeptide derivative according to claim 1, characterized in that the amino acid lactone modified at the C-terminus is homoserine lactone derived from methionine. 4. A nucleic acid sequence encoding a polypeptide derivative according to any one of claims 1-3. 5. A vector comprising the nucleic acid sequence of claim 4. 6. A host cell comprising the vector of claim 5. 7. A pharmaceutical composition, which contains the polypeptide derivative according to one of claims 1-3, the nucleic acid sequence according to claim 4, the carrier according to claim 5 or the host cell according to claim 6 as an active ingredient, and optionally contains a drug Auxiliary ingredients used. 8. The use of the polypeptide derivative according to any one of claims 1-3, the nucleic acid sequence according to claim 4, the vector according to claim 5 or the host cell according to claim 6 in the preparation of medicaments. 9. The application according to claim 8, wherein said medicine is a medicine for adjuvant treatment of immune-related diseases or adjuvant treatment of chemotherapy or radiotherapy. 10. The application according to claim 8, wherein said medicine is a medicine for adjuvant treatment of viral diseases or immunocompromised or disordered diseases, or adjuvant treatment of chemotherapy or radiotherapy. 11. The use according to claim 10, wherein said viral diseases include hepatitis B, hepatitis C, AIDS, and influenza. 12. The application according to claim 10, wherein said immunocompromised or disordered diseases include tumors, recurrent colds, and chronic diarrhea.