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 extrasin alpha-1 and application thereof

Technical field

The present invention relates to biological polypeptide thymosin derivative.More specifically, the present invention relates to the thymosin that C-terminal utilizes amino acid lactone to modify.Thymosin derivative described in the invention not only possesses than the strong biologic activity of the known biological activity of natural thymosin, has the not available new biologic activity of natural thymosin simultaneously, suppress as resisting chemotherapeutics or hemopoietic function that radiotherapy caused, so it is more suitable for being used for antiviral therapy, antineoplaston, chemotherapy and radiotherapeutic assisting therapy.The invention still further relates to described thymosin derivative coding nucleic acid, contain the medical composition and its use of described thymosin derivative.

Background technology

Thymosin-α 1 (Thymosin α 1) is that Goldstein etc. found in thymic tissue in 1977 first.Its precursor thymosin-α 1 former (prothymosin) of being made up of 111 amino acid produces through enzymolysis processing in vivo.Sophisticated thymosin-α 1 is made up of 28 amino acid, and molecular weight is 3108 dalton, and iso-electric point is 4.2.Thymosin-α 1 outside T-lymphocyte and the thymic tissue, also has distribution at organs such as liver, kidney, the heart, lung, spleens except being present in thymocyte.

Thymosin acts on thymocyte sophisticated early stage and late period, increases T cell surface Thy_1, and 2 and Lyt_1,2,3 expression promotes the TH cell maturation.Thymosin also can be in terminal deoxynucleotidyl transferase (TDT) level of external adjusting thymocyte, body internal stimulus macrophage inhibition factor (Microphage Inhibition Factor, MIF), Interferon, rabbit (Interferon, IFN), interleukin II (Interleukin-2, IL-2) and receptor expression, strengthen human body NK cytoactive.Show that thymosin can regulate body's immunity, promote cytokine and expression of receptor thereof, the enhancing immunity cytoactive is brought into play its antiviral effect from many aspects.Thymosin active cells absolute number descends with age growth, and detection later on was less than the existence of thymosin in 40 years old.(RadioimmuneAssay RIA) measures, and improper thymosin level is relevant with some disease in the serum, has some reports to show the low-level immune deficiency disorder that causes thymus gland to rely on of thymosin according to radioimmunology.Simultaneously, interaction between thymosin and neuroendocrine is also very noticeable, the investigator finds to exist high density thymosin sample peptide in hypothalamus and hypophysis extract, thymosin can show that it may be a kind of neuroendocrine conditioning agent in hypothalamus and/or hypophysis level affects ACTH, Pr1, TSH and LH secretion.

As a kind of biological response modifier (BRM), thymosin can obviously improve body's immunological function.Extensively be used for infected by microbes such as the various immunodeficient diseases of clinical treatment, autoimmune disease, tumour and virus in recent years as immunostimulant, and when itself and other biological reaction control agent, as IL-2, IFN-α, better efficacy during synergy such as thymic factor, especially for the treatment chronic viral hepatitis, effect is more remarkable.

In addition, because the thymosin molecule is very little, in prokaryotic expression systems such as intestinal bacteria, can not obtain natural unit molecule expression product.Thymosin mainly prepares by chemosynthesis at present, and the problem of its existence is to use cost too high.

Summary of the invention

In order to obtain functional cheaply thymosin, the inventor has studied the several genes engineering mode of production of thymosin, because the unit molecule thymosin can not be expressed in intestinal bacteria, so adopt fusion or tandem expression to add the method for chemical cracking, obtain the derivative of thymosin, as adding methionine(Met) derivative (homoserine lactone) and tryptophan derivative (tryptophane lactone) etc. at the C of thymosin end.The inventor has further studied the biologic activity of thymosin related derivatives, the result is surprisingly found out that, when the C of thymosin end was lactone modified by this two seed amino acid, its known biologic activity strengthened to some extent, and shows some new biological actions.The thymosin derivative product of this modification can utilize genetic engineering technique production, and its production cost reduces greatly, helps popularizing medication.

Therefore, one aspect of the present invention relates to biological polypeptide thymosin derivative.More specifically, the present invention relates to the thymosin that C-terminal utilizes amino acid lactone to modify.Thymosin derivative described in the invention not only possesses than the strong biologic activity of the known biological activity of natural thymosin, has the not available new biologic activity of natural thymosin simultaneously, suppress as resisting chemotherapeutics or hemopoietic function that radiotherapy caused, so it is more suitable for being used for antiviral therapy, antineoplaston, chemotherapy and radiotherapeutic assisting therapy.

Thymosin polypeptide derivative of the present invention is characterized in that having C-terminal as follows by amino acid lactone modified aminoacid sequence:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsn-@

Wherein, @ represents the amino acid lactone that C is terminal modified.

Preferably, the terminal modified amino acid lactone of C is selected from by tryptophane deutero-tryptophane lactone with by methionine(Met) deutero-homoserine lactone.

In addition, the nucleotide sequence of the thymosin polypeptide derivative of the invention described above that the present invention relates to encode contains the carrier of this nucleotide sequence, contains the host cell of this carrier.

Thymosin polypeptide derivative of the present invention can prepare by the synthetic method in conjunction with chemically modified of chemiluminescent polypeptide, or by the method preparation of gene engineering expression technology in conjunction with the chemical cracking technology.Described method and concrete operations thereof are that those skilled in the art are well-known.For example chemiluminescent polypeptide synthetic method is referring to " Instrumentation for automated solid-phasepeptide synthesis. " Cammish, L.E., Kates, S.A.In Fmoc Solid PhasePeptide Synthesis:A Practical Approach (Chan, W., White, P.) Oxford University Press, Oxford, 1998.Amino acid whose chemical modification method is referring to Chemical reagents for protein modification.2nd edition.Roger L.Lundblad.CRC press, 1991.

When preparing in conjunction with the chemical cracking technology for gene engineering method, at first (one of optional book of reference is " a molecular cloning experiment guide " by the well-known dna recombinant expression technology of those skilled in the art, second edition), generation has the terminal modified amino acid whose expression of polypeptides product of C, prepare thymosin polypeptide derivative of the present invention (the chemical cracking technology is referring to Chemical reagents for protein modification.2nd edition.Roger L.Lundblad.CRC press, 1991) by suitable chemical cracking method then.The technical essential of utilizing genetic engineering technique to produce this thymosin derivative is: according to the aminoacid sequence that provides, and the corresponding nucleotide sequence of synthetic.Then this thymosin derivative nucleotide sequence of synthetic is cloned in suitable intestinal bacteria or the Yeast expression carrier and expresses, its phraseology can be diversified.As: can carry out amalgamation and expression with other albumen or polypeptide, also self can be carried out tandem expression, or after the series connection again with other protein fusion expression etc.After expression product carried out purifying, utilize the cracking of chemical cracking method.For example, there is the gene expression product of methionine(Met) to produce the thymosin polypeptide derivative that the C end adds the methionine(Met) derivative by bromize fluoride crack C is terminal modified; There is the gene expression product of tryptophane to produce the thymosin polypeptide derivative that the C end adds tryptophan derivative by various tryptophane cleavage methods (as BNPS-skatole, N-chlorosuccinimide and N-bromo-succinimide) cracking C is terminal modified.Described methionine(Met) derivative is meant the homoserine lactone that forms after the methionine(Met) cracking; Described tryptophan derivative is meant the tryptophane lactone that forms behind the tryptophane oxicracking.

The present invention relates on the other hand and contains aforementioned polypeptides, nucleotide sequence, carrier or host cell as activeconstituents, and contains the pharmaceutical composition of pharmaceutically useful ancillary component alternatively.Described activeconstituents is present in this pharmaceutical composition with the physiology significant quantity.Described pharmaceutical composition can exist to be used for human medicament forms at present, for example lyophilized injectable powder, aqueous injection, vein drops, oral tablet, sprays, paste etc. can prepare these medicament forms and can be according to different way of administration administration well known by persons skilled in the art according to common method.That the suitable pathways of administration for example comprises is oral, rectum, through mucous membrane or enterally administering; Parenteral transmits, comprise in intramuscular, subcutaneous, the sheath and intrathecal injection, directly in the ventricle, in the intravenously, intraperitoneal, nose or intraocular injection.Can part rather than systemic fashion administration.This activeconstituents can mix the ancillary component that is generally used for pharmaceutical composition and make pharmaceutical composition.Described ancillary component is medicine acceptable carrier, thinner, auxiliary agent, vehicle, stablizer, sanitas, permeate agent, isotonic agent and/or release control agent etc. for example.The activeconstituents of invention can use separately or with other compounds for example therapeutic compound combined utilization, for example interleukin-or Interferon, rabbit etc.

Further aspect of the present invention relates to the application in the preparation medicine of aforementioned polypeptides, nucleotide sequence, carrier or host cell.

Thymosin polypeptide derivative of the present invention has identical even stronger biologic activity with natural thymosin aspect the immunological role, therefore go for the assisting therapy of immune related diseases, comprise virus disease, as hepatitis B, hepatitis C, AIDS, influenza and other various virus diseases; Immunologic hypofunction or dysfunctional disease are as tumour, repeated cold, chronic diarrhoea etc.

Thymosin polypeptide derivative of the present invention also has the biologic activity different with thymosin; particularly aspect the antagonism hemopoietic function inhibition that chemotheraping preparation and radiation treatment caused; has significant provide protection; therefore; this product can also be applicable to the adjuvant drug of chemotherapy and radiation, provides protection to patient's hemopoietic function.

Unless expressly stated otherwise,, various terms, technology or the scientific words that uses in the present specification has and well known to a person skilled in the art implication widely.

Utilize specific embodiment that the present invention is described in further detail below.

The thymosin polypeptide that embodiment 1C end is modified for the methionine(Met) derivative

The C end is as follows for the sequence of the thymosin polypeptide that the methionine(Met) derivative is modified:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLy sLysGluValValGluGluAlaGluAsn-homoserine lactone

1. preparation method

The thymosin polypeptide that this C end is modified for homoserine lactone can utilize technology known in the art to produce.

1) chemiluminescent polypeptide is synthetic in conjunction with chemical modification method

(1) the polypeptide solid phase synthesis is a mature technology, as long as provide aminoacid sequence, numerous companies can carry out synthetic on a large scale according to the aminoacid sequence that provides.According to Fmoc solid-phase synthesis (Fmoc:9-fluorenylmethyloxycarbonyl), on Applied Biosystems company 433 type Peptide synthesizers, the amino acid of chemosynthesis is as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnMet

(2) above-mentioned synthetic product is modified methionine(Met) according to the experimental technique of aforementioned " Chemical reagents for proteinmodification " with cyanogen bromide.Protein is dissolved in according to 2mg/ml in 70% the formic acid (in pressure vessel that can be airtight), the ratio according to 10g/ml adds solid brominated cyanogen (operation) again, and the lucifuge cracking is after 36 hours under the room temperature, and the NaOH regulator solution that slowly adds 10N is to pH7.0.

(3) thymosin after the reverse-phase chromatography purifying is modified

Purification media is Source-30RPC, the about 350ml of column volume.According to sample on the flow velocity of 20ml/min, the samples with water dilution that above-mentioned steps obtains is gone up sample for 10 times, go up the about 3000ml of sample at every turn.Use A liquid (20mM Tris-Cl (pH=6.8) after the completion of the sample, 200mM sodium-chlor) wash the about 400ml of chromatography column according to the flow velocity of 25ml/min, secondly with 5%～15%B liquid (20mM Tris-Cl (pH=6.8), 200mM sodium-chlor, 50% ethanol) the wash-out foreign protein, elution volume is 600ml, use 20%～60%B wash-out thymosin then, collect the thymosin after the target protein peak is modification, the product rate of recovery is 80-90%, carries out target protein as follows and identifies.

(4) ion exchange chromatography is further purified the thymosin after the modification

Purification media is Source-30Q, the about 300ml of column volume.Behind one times of the recombination human thymosin α 1 usefulness distilled water diluting of collecting in the step (3) according to the about 2500ml of sample on the flow velocity of 20ml/min.Wash chromatography column 400ml with A liquid (20mM Tris-Cl (pH=6.8)) according to the flow velocity of 20ml/min after the completion of the sample, use 10%～40%B liquid (20mM Tris-Cl (pH=6.8) then, 1M sodium-chlor) the about 400ml of wash-out, collect the target protein peak, the product rate of recovery is 85-95%, carries out target protein as follows and identifies.

(5) gel filtration method obtains the modification thymosin of final purifying

Purification media is Sephacryl S-100HR column volume 2000ml, the recombination human thymosin α 1 that collects in the step (4) press column volume 0.1%～5% on sample, flow velocity 3～8ml/min, collect protein peak, maximum protein peak is the good recombination human thymosin α 1 of purifying, the product rate of recovery is 80-90%, carries out target protein as follows and identifies.Balance and stream wash buffer prescription are: 25mM phosphate buffered saline buffer (pH6.0～7.5), 125mM sodium-chlor.

(6) the proteinic authentication method of thymosin

A. protein electrophorese is identified molecular weight

Method: with reference to " molecular cloning experiment guide " second edition, proteinic polyacrylamide gel electrophoresis, Science Press.Gel degree of crosslinking 29: 1 (acrylamide: bisacrylamide), gel strength 18%.As reference, electrophoresis result shows that the target protein matter that purifying obtains has identical molecular weight with the thymosin standard substance with thymosin standard substance (available from Sigma company).

The b.N end sequence is measured

Product N-terminal sequencing adopts standard protein N-terminal sequencing technologies, carries out on the ABI Proeise of Applied Biosystems company 491 type protein sequencers, and the result is as follows for the N-terminal sequencing:

15 amino acid residue sequences of N-terminal are followed successively by: Ser, and Asp, Ala, Ala, Val, Asp, Thr, Ser, Ser, Glu, ILe, Thr, Thr, Lys, Asp. is consistent with expected results.

2) gene engineering method is in conjunction with the chemical cracking technology

(1) the artificial gene sequence is synthetic

At this product, the aminoacid sequence of its design is as follows:

SerAspAlaAlaValAspThrSerSerGlulleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnMet

According to above-mentioned aminoacid sequence, utilize the degeneracy principle of genetic code, can design multiple satisfactory sequence.Use following sequence according to colibacillary genetic expression principle and vector expression scheme herein:

GA

ATG TCTGATGCTGCTGTTGATACTTCTTCTG

AGATTACTACTAAAGATCTTAAGGAGAAGAAGGAAGTT

GTCGAAGAGGCTGAGA ACATG

CG

Wherein the underscore sequence is the thymosin sequence, and on behalf of restriction enzyme, the double underline sequence cut the site, is respectively BamHI and EcoRI.

According to above-mentioned sequence, press β-acetonitrile phosphorous acid amination synthesis method on ABI 3900 desk-top high-throughput dna synthesizers, following two nucleotide fragments of synthetic:

Fragment 1:

GAGGATCCATGTCTGATGCTGCTGTTGATACTTCTTCTG

AGATTACTACTAAAGATCTTAA

Fragment 2:

CGGAATTCCATGTTCTCAGCCTCTTCGACAACTTCCTTC

TTCTCCTTAAGATCTTTAGTAG

(2) gene clone, reference＜molecular cloning experiment guide〉second edition.

With above-mentioned two fragments, respectively add 20pmol, contain in the restriction enzyme that final concentration is 100mM NaCl (as the PstI) damping fluid at 50 μ L, carry out fragment annealing.Sample hose is placed 95 ℃ water-bath, naturally cool to room temperature, finish annealing.The dNTP that adds 5 μ L 10mMol/L then, the T4 archaeal dna polymerase of 5u, 37 ℃, 1 hour, synthetic dsdna.Again under 80 ℃, 30 minutes deactivation T4 archaeal dna polymerases.EcoRI and two kinds of restriction enzymes of BamHI of adding 10u at last respectively, 37 ℃ are incubated 1 hour.Agarose gel electrophoresis with reference to dna molecular amount standard, according to fragment length, reclaims target gene fragment.To use intestinal bacteria GST fusion expression vector pGEX-4T1 (available from Phamacia company) always and digest with two kinds of same enzymes, agarose gel electrophoresis reclaims big fragment.Carrier segments is connected with target gene fragment, obtain recombinant vectors,, extract plasmid its transformed into escherichia coli DH5 α (available from Clontech company), enzyme is cut (EcoRI and BamHI) screening positive clone, and last dna sequencing is identified the exactness of target gene fragment.

(3) genetic expression, reference＜molecular cloning experiment guide〉second edition.

Identify the positive colony bacterial strain that obtains to contain the correct recombinant vectors of insertion by dna sequencing,, induce the genetic expression of this bacterial strain according to the IPTG induction method.

(4) product purification, reference＜molecular cloning experiment guide〉second edition.

A. the centrifugal coli strain of collecting above-mentioned abduction delivering, the bacterial precipitation total amount of weighing, add 10ml lysis buffer (20mM Tris-Cl according to every 1g bacterial precipitation, pH=6.8,5mM EDTA (pH=8.0)) ratio suspension bacterium, stir, 80 ℃ of constant temperature 30min are cooled to room temperature rapidly; 7000 rev/mins of centrifugal 20min collect supernatant then, add mercaptoethanol to final concentration and are 0.1%, and are standby.

B. ion exchange chromatography purifying: purification media is Q-Sepharose fastflow, the about 350ml of column volume.According to sample on the flow velocity of 20ml/min, go up the product that obtains among the about 3500ml A of sample at every turn.Use A liquid (20mM Tris-Cl (pH=6.8) after the completion of the sample, 0.1% mercaptoethanol) washes the about 400ml of chromatography column according to the flow velocity of 25ml/min, use 15%～40%B liquid (20mM Tris-Cl (pH=6.8) then, 0.1% mercaptoethanol, 1M sodium-chlor) wash-out, elution volume is 600ml, collects the target protein peak, identifies with the proteinic authentication method of above-mentioned thymosin.

C. bromize fluoride crack and purifying: method combines (2)-(6) of chemical modification method with the chemosynthesis of step homopolypeptide, intestinal bacteria GST is fallen in cracking, and obtains the thymosin of above-mentioned C end for the homoserine lactone modification.

2. determination of activity

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

Its principle is: [ ³H]-TdR in cell, be converted into [ ³H] dTTP, the latter mixes among the DNA when the cell proliferation synthetic DNA, and its incorporation is directly proportional with DNA synthetic amount and proliferative cell number, measure [ ³H]-the TdR incorporation can calculate cell proliferation rate.

Mouse is craned one to put to death to get and isolates monocyte with lymphocyte separation medium after spleen grinds, with twice of the nutrient solution washing that does not contain serum, be resuspended in the RPMI-1640 that contains 10% calf serum, the cell numeration, every hole adds 100 μ l cell suspensions in 96 orifice plates, add ConA (final concentration is 5 μ g/ml) again, CO ₂Cultivated 6 hours for 37 ℃ in the incubator, add the C end homoserine or the lactone modified thymosin sample of different concns, adjusted volume to 200 μ l continues to cultivate 72 hours, add [ ³H]-TdR 18.5 * 10 ³GBq cultivated 6 hours, and collecting cell dries the back and measures the cpm value on glass fiber filter paper, calculates proliferation rate according to following formula:

Table 1

	Concentration (μ g/ml)	cpm(X±SD)	Appreciation rate
				Natural thymosin	50.00	92806.22±4751.80	86.86
12.50	84640.02±2715.81	70.42
			1.94		61171.38±5097.59	23.17
The thymosin that C end homoserine lactone is modified	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		49665.74±3904.44		0

2), take off the E receptor method:

Reagent and method:

(1) Hank ' s liquid: with 0.3% potassium primary phosphate (KH ₂PO ₄) solution, 0.76% SODIUM PHOSPHATE, MONOBASIC (Na ₂HPO ₄) solution, 2% Klorvess Liquid and 20% sodium chloride solution are successively by 20: 20: 20: 40 mixed, add glucose 1g, and dissolving and mixing are diluted with water to 1000ml, and the sodium carbonate solution with 4% is transferred PH to 7.2-7.3 (facing the time spent configuration).

(2) A Shi liquid: precision takes by weighing sodium-chlor 0.420g, Citric Acid 0.055g, and Sodium Citrate 0.766g, glucose 2.05g is dissolved in water to 100ml, boils the 60min sterilization.

(3) parting liquid: lymphocyte separation medium.

(4) sheep blood: extract sheep venous blood 5ml, add in the 5ml A Shi liquid, refrigerator is preserved (half aseptic technique).

(5) stationary liquid: with 25% glutaraldehyde solution, 3.5% sodium hydrogen carbonate solution and Hank ' s liquid are pressed 1: 1: 38 mixed successively.

(6) staining fluid: get Ji's nurse Sa dye liquor (stoste) 2ml, add Hank ' s liquid 6ml, shake up, centrifugal (1500rpm) 10min, it is stand-by to get supernatant.

(7) take off purchasing of E thymocyte: get fresh pig thymus gland, degrease also shreds, adding an amount of Hank ' s liquid makes and is muddy, filter through 100 eye mesh screens, filtered liquid adds in the centrifuge tube of the parting liquid that has had 1/3 amount of filtrate, the centrifugal 20min of 2000rpm, the white thymocyte in careful sucking-off middle layer, put into another centrifuge tube, add an amount of Hank ' s liquid washing, concussion shakes up, the centrifugal 3min of 1500rpm, abandoning supernatant, 45 ℃ of waters bath with thermostatic control insulation 30min (jolting once) after three times repeatedly every 5min.The centrifugal 3min of 1500rpm, abandoning supernatant adds an amount of Hank ' s liquid again to original volume, and the rearmounted 45 ℃ of waters bath with thermostatic control insulation of mixing 30min takes out the centrifugal 3min of back 1500rpm, abandoning supernatant.Use Hank ' s liquid washing three times (operation is the same), with suitable dilution of Hank ' s liquid and counting, making final concn is 3～5 * 10 at last ⁶Individual cell/ml.

(8) preparation of sheep red blood corpuscle suspension: get an amount of sheep blood, use an amount of Hank ' s liquid washing three times (operation is the same), abandoning supernatant is used Hank ' s liquid suitably dilution and counting, and making final concn is 2～7 * 10 ⁷Individual cell/ml.

(9) measure: get this product and use an amount of Hank ' s liquid to be made into every 1ml to contain the solution of thymosin 0.1mg as test liquid.Get 6 small test tubes, 3 Hank ' s liquid add 0.1ml fully and do control tube, and 3 respectively add test liquid 0.1ml and do the mensuration pipe, every pipe adds E thymocyte 0.2ml, and 37 ℃ of insulations added sheep red blood corpuscle 0.2ml after 1 hour, shake up, the centrifugal 3min of 500rpm puts into 4 ℃ of refrigerator overnight.Take out next day, abandoning supernatant, every pipe adds 1 of stationary liquid, shakes up gently, leave standstill 10min, add 2 of staining fluids and also shake up, begin counting after leaving standstill 15min, pale blue is lymphocyte than maxicell in the field of microscope, several 200 altogether, statistics E Rose footing (in conjunction with 3 or 3 erythrocytic lymphocytes of above sheep) is wherein averaged, and is the E Rose footing of sample or control tube.

Be calculated as follows the sample vigor:

Sample vigor=(for test tube E Rose footing-control tube E Rose footing)/200*100%

The result is as follows:

Add the purification of recombinant proteins of different concns and the thymosin of chemosynthesis, each concentration repeats 3 times, the result shows: the raising of the knot flower rate that C end homoserine or lactone modified thymosin and synthetic all can promote the T cell, and the concentration relevant (table 2) of knot flower rate and thymosin.Aspect promotion knot flower rate, C end homoserine or lactone modified thymosin are compared with synthetic, slightly strengthen.The results are shown in following table:

Table 2

The sample title	Concentration (mg/ml)	Knot flower rate	Absolute increased value	Relative increased value
					Blank	0	14.5％	-	-
The thymosin that C end homoserine lactone is modified	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	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), the thymosin of C end homoserine lactone modification is to the influence of mouse hemopoietic function

Animal grouping and administration: select Kunming mouse (available from The Fourth Military Medical University's animal center), bodyweight difference is less than 2g, same sex (male), be divided into the normal control group at random, the terminal modified thymosin of chemosynthesis thymosin control group and C is organized by examination, endoxan immunosuppression control group, endoxan+chemosynthesis thymosin control group and the terminal modified thymosin of endoxan+C are organized by examination, and every group of 8-15 is only.：

Medication: CTX 50mg/kg subcutaneous injection, the next day 1 time, totally 3 times, the natural thymosin of the thymosin of modification and chemosynthesis, once a day, totally 14 times.

Erythrocyte and content of hemoglobin are measured: measure content of hemoglobin and erythrocyte counting with cellanalyzer.

The influence of the thymosin that table 3.C end homoserine lactone is modified peripheral red blood cells counting and content of hemoglobin during to the mouse chemotherapy

Group	Dosage μ g/kg	The mouse number/only	RBC×10 12/L	HBg/L
					The terminal modified thymosin α1 endoxan of the natural thymosin α1 C of the normal control endoxan+terminal modified thymosin α1 of natural thymosin α1 endoxan+C	- 320 320 50mg/kg×3 320 320	9 11 13 11 8 15	8.17±0.68 8.25±0.64 8.61±0.35 7.65±0.63 7.69±0.85 8.26±0.54 *	117.6±10.3 117.8±9.4 124.2±4.9 113.7±10.9 114.0±12.6 119.5±8.5 *

^*The terminal modified thymosin of P＜0.05 endoxan+C is compared with endoxan+natural thymosin;

The terminal modified thymosin of P＜0.01 endoxan+C is compared with the endoxan group.

The result shows that the terminal modified thymosin of C can resist the hematopoiesis restraining effect that endoxan causes, and have significant statistics difference, and natural thymosin does not have this effect.

The thymosin polypeptide that embodiment 2C end is modified for tryptophan derivative

1. preparation method

This C end can utilize technology known in the art to produce for the lactone modified thymosin polypeptide of tryptophane.

1) chemiluminescent polypeptide is synthetic in conjunction with chemical modification method

With embodiment 1, difference is that last amino acid of synthetic amino acid array is not methionine(Met) but tryptophane.Aminoacid sequence is as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnTrp

Above-mentioned synthetic product is modified tryptophane according to the experimental technique of aforementioned " Chemical reagents for proteinmodification " with N-chlorosuccinimide.Synthetic product is dissolved in according to 2mg/ml in 50% the acetate (in pressure vessel that can be airtight), and adding N-chlorosuccinimide to final concentration again is 0.1M, and cracking is after 12 hours under the room temperature, and the NaOH regulator solution that slowly adds 10N is to pH7.0.

Because this product is very approaching with the thymosin polypeptide that homoserine lactone is modified on physico-chemical property, can this purifying products be come out with same purification process.

2) gene engineering method is in conjunction with the chemical cracking technology

Same scheme and the purification technique route of modifying with aforementioned production homoserine lactone of thymosin can obtain this product, and its difference is following 2 points:

A. last amino acid difference of aminoacid sequence, and cause the gene order that designs not

With, aminoacid sequence is as follows:

SerAspAlaAlaValAspThrSerSerGluIleThrThrLysAspLeuLysGluLysLysGluValValGluGluAlaGluAsnTrp

Its gene order is as follows:

GA

TGG TCTGATGCTGCTGTTGATACTTCTTCTGAGA

TTACTACTAAAGATCTTAAGGAGAAGAAGGAAGTTGTCGAAG

AGGCTGAGA ACTGG

CG

Wherein use codon TGG (tryptophane) to replace ATG (methionine(Met)).

As follows according to above-mentioned sequences Design such as embodiment 1 synthetic fragment:

Fragment 1:

GAGGATCCTGGTCTGATGCTGCTGTTGATACTTCTTCTG

AGATTACTACTAAAGATCTTAA

Fragment 2:

CGGAATTCCCAGTTCTCAGCCTCTTCGACAACTTCCTTC

TTCTCCTTAAGATCTTTAGTAG

B. cleavage method difference, the experimental technique of " Chemical reagents for proteinmodification " is adopted in the tryptophane cracking, come cracking with N-chlorosuccinimide, intestinal bacteria GST is fallen in cracking, and obtains above-mentioned C end and be the lactone modified thymosin polypeptide of tryptophane.

2. determination of activity

Thymosin activity determination method and embodiment 1 that tryptophane is lactone modified are in full accord, and the thymosin determination of activity that various determinations of activity and homoserine lactone are modified is carried out simultaneously, and basically identical as a result between the two does not have marked difference.Its result is as follows:

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

Table 4

	Concentration (μ g/ml)	cpm(X±SD)	Appreciation rate
				Natural thymosin	50.00	92806.22±4751.80	86.86
12.50	84640.02±2715.81	70.42
			1.94		61171.38±5097.59	23.17
The lactone modified thymosin of C end tryptophane	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		49665.74±3904.44		0

2), take off the E receptor method:

Table 5

The sample title	Concentration (mg/ml)	Knot flower rate	Absolute increased value	Relative increased value
					Blank	0	14.5％	-	-
The lactone modified thymosin of C end tryptophane	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	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), C holds the influence of the lactone modified thymosin of tryptophane to the mouse hemopoietic function

The influence of peripheral red blood cells counting and content of hemoglobin when table 6.C holds the lactone modified thymosin of tryptophane to the mouse chemotherapy

Group	Dosage μ g/kg	The mouse number/only	RBC×10 12/L	HBg/L
					The terminal modified thymosin α1 endoxan of the natural thymosin α1 C of the normal control endoxan+terminal modified thymosin α1 of natural thymosin α1 endoxan+C	- 320 320 50mg/kg×3 320 320	9 11 13 11 8 15	8.17±0.68 8.25±0.64 8.55±0.45 7.65±0.63 7.69±0.85 8.33±0.56 *	117.6±10.3 117.8±9.4 123.1±4.9 113.7±10.9 114.0±12.6 119.9±8.6 *

^*P＜0.05 " the terminal modified thymosin group of endoxan+C " and " endoxan+natural thymosin group is compared ";

^*P＜0.01 " the terminal modified thymosin group of endoxan+C " is compared with the endoxan group.

The result shows that the terminal modified thymosin of C can resist the hematopoiesis restraining effect that endoxan causes, and differ to have significant statistical significance, and natural thymosin does not have this effect.

Claims (12)

1. thymosin polypeptide derivative is characterized in that having C-terminal as follows by amino acid lactone modified aminoacid sequence:

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 that C is terminal modified.

2. the polypeptide derivative of claim 1 is characterized in that the terminal modified amino acid lactone of described C is a tryptophane deutero-tryptophane lactone.

3. the polypeptide derivative of claim 1 is characterized in that the terminal modified amino acid lactone of described C is a methionine(Met) deutero-homoserine lactone.

4. the nucleotide sequence of the polypeptide derivative of one of coding claim 1-3.

5. the carrier that contains the nucleotide sequence of claim 4.

6. the host cell that contains the carrier of claim 5.

7. pharmaceutical composition, its host cell that contains the carrier of nucleotide sequence, claim 5 of polypeptide derivative, the claim 4 of one of claim 1-3 or claim 6 be as activeconstituents, and contain pharmaceutically useful ancillary component alternatively.

8. the application of the host cell of the carrier of the nucleotide sequence of the polypeptide derivative of one of claim 1-3, claim 4, claim 5 or claim 6 in the preparation medicine.

9. the application of claim 8, wherein said medicine are the medicine that is used for assisting therapy or the chemotherapy or the radiotherapeutic assisting therapy of immune related diseases.

10. the application of claim 8, wherein said medicine are the medicine that is used for assisting therapy or the chemotherapy or the radiotherapeutic assisting therapy of virus disease or immunologic hypofunction or dysfunctional disease.

11. the application of claim 10, wherein said virus disease comprises hepatitis B, hepatitis C, AIDS, influenza.

12. the application of claim 10, wherein said immunologic hypofunction or dysfunctional disease comprise tumour, repeated cold, chronic diarrhoea.