CN1532289A - Method for preparing N-end acetylation modified thymosin alpha with recombined E. coli - Google Patents
Method for preparing N-end acetylation modified thymosin alpha with recombined E. coli Download PDFInfo
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Abstract
The present invention discloses the preparation process of thynosin-alpha with acetylation modified N-end by using recombinant colibacillus. The preparation process includes obtaining thynosin-alpha gene, constructing recombinant expression vector containing thynosin-alpha, transforming prokaryotic cell, culturing prokaryotic cell and expressing thynosin-alpha, and separating and purifying thynosin-alpha with acetylation modified N-end. The present invention also discloses the process of utilizing recombinant colibacillus in preparing thynosin-alpha antigen with acetylation modified N-end and then cutting and separating to prepare thynosin-alpha-1 antigen with acetylation modified N-end, thynosin-alpha-2 antigen with acetylation modified N-end and similar matter. The thynosin-alpha with acetylation modified N-end has the functions of regulating immunity, cell proliferation, etc. and has wide application foreground in antivirus and antitumor.
Description
Technical field
The present invention relates to the method that a kind of using gene engineering means prepare N-acetylated protein matter, specifically prepare the method for the extrasin alpha of the terminated acetylated modification of N-with recombination bacillus coli.
Background technology
Extrasin alpha is the immunoloregulation polypeptide that gang has same or analogous N-terminal sequence, comprises prophymosin-alpha, thymosin, thymosin 1 etc.The extrasin alpha of different plant species has very high conservative property, and for simplicity, the present invention only mentions human thymosin alpha, but other vertebrate extrasin alphas include interior.
The acidic protein that human thymosin alpha-source (proT) is made up of 109 amino acid, the content of its L-glutamic acid and aspartic acid is greater than 30%, iso-electric point 3.55, can combine with histone, tissue distribution is extensive, except that thymus gland, spleen, lymph, liver, kidney, testis, ovary, brain etc. are organized also and can be detected its mRNA.Prophymosin-alpha has immunostimulatory activity, but enhancing body is antiviral, the ability of tumour and fungi etc.It is the precursor of thymosin and thymosin 1, and its 28 of N-end is identical with thymosin 1 with thymosin respectively with 35 amino acid.But the immunostimulatory activity of prophymosin-alpha points out its rest part except that 28 peptides of N-end also to play an important role apparently higher than thymosin.
Weakness such as the terminated acetylated thymosin of N-goes on the market, and trade(brand)name " Zadaxin " is used for the treatment of viral hepatitis etc., and it is to adopt the method for artificial chemosynthesis to obtain, and the method for artificial chemosynthesis has the cost height, and yield is low.
110 amino acid of cDNA coding of human thymosin alpha-source are mature peptide behind the initial methionine, do not contain signal peptide.The prophymosin-alpha of natural origin and thymosin, α 11 have not only cut initial methionine(Met), and are the terminated acetylated modification of N-, and the terminated acetylated modification of N-has vital role to improving its body internal stability.
The terminated acetylated modification of N-is the acetylation modification of aminoterminal amino of the peptide chain of finger protein or polypeptide, is the ubiquitous a kind of rhetorical function of eukaryotic cell.And as procaryotic intestinal bacteria, then very rare to the N-acetylation modification of recombinant protein or polypeptide.Thymosin and prophymosin-alpha all obtain to express intestinal bacteria, and have obtained purifying, but there is no report (the Wetzel et a1.Production of biologically active N of the terminated acetylated modified outcome of N-
a-Desacetylthymosin α 1 inEscherichia coli through espression of a chemical synthesized gene.Biochemistry1980,19,6096-6104; Evstafieva AG et al.Overproduction in Escherichia coli; purification and properties of human prothymosin alpha.Euro J Biochem231:639-643; 1995.), do not see that useful recombination bacillus coli prepares the report of the extrasin alpha of N-acetylation modification yet.
Summary of the invention
In order to reduce the cost of the terminated acetylated modification extrasin alpha of preparation N-, improve its yield, the invention discloses a kind of method for preparing the terminated acetylated modification extrasin alpha of N-with recombination bacillus coli.
The inventive method may further comprise the steps:
It at first is the preparation of extrasin alpha gene.The gene of coding extrasin alpha can be used PCR method (Science.239:487-491 such as Saiki, 1988), method such as the method for RT-PCR method, synthetic or structure screening cDNA library obtains, as pcr template or be used for the mRNA in construction cDNA library or cDNA can derive from any tissue that contains corresponding mRNA or cDNA, cell, and library etc., can obtain from people embryo thymus gland, also can obtain with artificial synthetic method.The codon that can select for use the host to have a preference for during synthetic, the expression that so often can improve product.If need, the known method in available this area is suddenlyd change, lacks, inserts and is connected with other polynucleotide polynucleotide etc.
Carry out the extrasin alpha gene then and be connected, make up recombinant expression plasmid with prokaryotic expression carrier.Construction process adopts the known molecular biology method in this area (referring to J. Sa nurse Brooker etc., " molecular cloning experiment guide " second edition, Science Press, 1995).Prokaryotic expression carrier can be selected various coli expression carriers for use, can have replication site, selection markers etc., these construction of carrier many documents open (as J. Sa nurse Brooker etc., " molecular cloning experiment guide " second edition, Science Press, 1995), also can buy (as Invitrogen life technologies, carlsbad from various companies, california 92008, USA).Preferred carrier have pBV220, pET series etc. (Guo Baoyu. the genetically engineered pharmacy. press of The 2nd Army Medical College 2000.).Expression vector can have various induction types or constitutive promoter, and preferred inducible promoter helps improving the stability of recombination bacillus coli like this.The preferred temperature sensitive promotor of inducible promoter is as P
LP
RPromotors etc., and chemically inducible promoter are as Lac, Tac, Trp etc.
Further will contain the recombinant plasmid transformed intestinal bacteria of extrasin alpha gene, carry out culture expression.Transform required nucleic acid and to host cell, remove available usual method, as preparation competent cell, electroporation etc.The success cell transformed promptly contains the cell of the recombinant plasmid of thymosin alpha protogene, can be identified by the technology that people know, and through collecting and cracking, extracts DNA as cell, and PCR method is identified then.The said intestinal bacteria of the present invention are general intestinal bacteria, can be DH α.
Intestinal bacteria after the conversion can be cultivated with shaking bottle or bio-reactor etc., cultivate preferred bio-reactor during production.Substratum should be able to provide thalli growth and product to express required composition, can comprise nitrogenous source, carbon source, pH buffering composition etc., and preferred culture medium prescription generally should obtain by test according to different Objects of Development.Cultivation can divide two stages, and the fs is mainly used in the growth of thalline (or cell), and subordinate phase is mainly used in synthetic product.Embodiment one provides a kind of preferred cultural method.
And then express the separation and purification of the extrasin alpha of back N-terminated acetylated modification.Method can comprise: (1) separating thallus, (2) bacterial cell disruption, steps such as (3) liquid chromatography (LC).The method of separating the extrasin alpha contain N-end acetylation modification from above-mentioned culture can be with the method for various albumen sepn, as the combination of technology such as extraction, precipitation, ultrafiltration, liquid chromatography (LC) and these technology.Wherein extraction can be used phenol, chloroform etc., and precipitation can be used salts such as organic solvent such as ethanol, Virahol and ammonium sulfate.Liquid chromatography (LC) can be used technology such as ion-exchange, gel exclusion, affine, hydrophobic, reversed phase chromatography, preferred anionic exchange or reversed phase chromatography.Embodiment one provides the preparation method of the prophymosin-alpha of the terminated acetylated modification of a kind of preferred N-.The thymosin of the terminated acetylated modification of N-, the also available same or analogous method preparation of α 11.
The extrasin alpha content of the terminated acetylated modification of N-that the present invention is prepared is preferably more than 50% of total extrasin alpha, more preferably greater than 90%.
The present invention also provides cutting N-end acetylation modification; with sequence in the sequence table 1 or 2 be the polypeptide or the albumen of homologous sequence; its homology is greater than 80%; be preferably greater than 90%; more preferably greater than 95%; most preferably be 100%, obtain the method for thymosin, α 11 or its analogue, the modified outcome etc. of N-end acetylation modification through separation and purification.Cutting can be with chemical process or enzyme process, preferably azanol.Cleaved products can adopt various separation methods to obtain thymosin, α 11 or its analogue, the modified outcome etc. of N-end acetylation modification, and preferable methods is a reversed phase chromatography.
Extrasin alpha is gang's polypeptide hormone, the said extrasin alpha of the present invention be meant N-end contain with sequence table in sequence 1 or sequence 2N-end 1-28 amino acids sequence homology polypeptide of sequence or protein or its modified outcome, its homology is greater than 80%, homology is preferably greater than 90%, more preferably greater than 95%, most preferably be 100%.It can be the thymosin that contains sequence 1 in the ordered list or sequence 2N-end 1-28 amino acids, or contain the thymosin 1 of 1-35 amino acids in sequence 1 in the ordered list or the sequence 2, or with sequence table in sequence 1 or sequence 2 amino acid sequence homologies greater than 90%, be preferably more than 95%, most preferably be the homologous sequence of 100% prophymosin-alpha.
There is polymorphism in some site on the aminoacid sequence of extrasin alpha, hold the 13rd amino acids residue as its N-, has plenty of Threonine (Goodall, G.J., Dominguez, F., and Horecker, B.L.Molecular cloning of cDNA for human prothymosin.Proc Natl Acad SciUSA. (1986), 83:8926-8928.), has plenty of Isoleucine (Rubtsov IuP, Vartapetian AB.New intronless members of human prothymosin alpha genes. (1995) Mol Biol (Mosk) 29 (6): 1320-5), they have same or analogous function, and extrasin alpha of the present invention includes these polymorphism analogues.
Among the present invention, with prophymosin-alpha (Ile13) and prophymosin-alpha (Thr13) is example, wherein prophymosin-alpha (Ile13) or proT α (Ile13) gene, the clone is from Chinese's fetal thymus cDNA, with the prophymosin-alpha aminoacid sequence basically identical of report such as Goodall, be Isoleucine (Ile) rather than Threonine (Thr) (aminoacid sequence is seen sequence 1 in the sequence table) but its N-holds the 13rd amino acids.And prophymosin-alpha (Thr13) or proT α (Thr13) are the on all four prophymosin-alphas of aminoacid sequence (aminoacid sequence is seen sequence 2 in the sequence table) with reports such as Goodall.
The N-end acetylation modification extrasin alpha of the present invention's preparation can also have various derivatives again, and these derivatives can be but be not limited to its multi-form salt, modified outcome etc.Modify again as amino, carboxyl, hydroxyl at polypeptide.Used modifier can but be not limited to polyoxyethylene glycol, dextran etc., and derivative.
The N-end acetylation modification extrasin alpha or derivatives thereof of the present invention preparation can use separately, and is preferred then for form the form use of pharmaceutical preparation with one or more acceptable carriers.This carrier generally should be compatible with extrasin alpha and can not be harmful to receptor autophosphorylation, and typical carrier is water or salt solution, or carbohydrate, or alcohols, or amino acid.Usually they need be aseptic, and do not have pyrogeneous substance.Said preparation can unitary dose form exist, and can prepare by any methods known in the art.These methods comprise holds N-acetylation modification extrasin alpha or derivatives thereof and has one or more auxiliary composition blended steps.Preferred said preparation comprises that aqueous liquid preparation and water content are lower than 10% or water-free freeze-dried preparation.These preparations can contain buffer reagent, salt, and small molecules carbohydrates etc. make the perviousness of medicine equate with perviousness in the acceptor blood or similar.Said preparation can be present in the container of dosage unit or multiple doses, as the peace bottle of sealing, in cillin bottle or the tubule.Freeze-dried preparation adds aseptic, pyrogen-free liquid carrier before using, as water for injection with liquid preparation lyophilize preparation.
The N-end acetylation modification extrasin alpha or derivatives thereof of the present invention preparation or its pharmaceutical composition can be by any known methods, comprise injection (as subcutaneous or muscle), venoclysis, transdermal, suction, method administration such as oral.Preferable methods is venoclysis or drug administration by injection.Treatment is included in and uses single dose or compound dosage in for some time.
The terminated acetylated modification extrasin alpha of N-among the present invention, or derivatives thereof or its pharmaceutical composition can be used for the immunotherapy of various diseases, and these diseases can be virus disease, tumour, mycosis, immunocompromised etc.As viral hepatitis, HIV infection, solid tumor, leukemia etc.
Description of drawings
Fig. 1. the SDS-PAGE of the human thymosin alpha-source of the escherichia coli expression of purifying
A: molecular weight standard b: prophymosin-alpha
Fig. 2. proT-A in the human thymosin alpha-source (16.18 minutes) separates with the RP-HPLC of proT-B (16.41 minutes), transverse axis be elution time (minute), the longitudinal axis is absorbancy (mAU)
The mass spectrum molecular weight analyse of Fig. 3 .proT-A and proT-B
A：proT-A B：proT-B
Quality peptide spectrum analysis after Fig. 4 .proT-A and the cutting of proT-B azanol
A：proT-A B：proT-B
Mass spectroscopy after Fig. 5 .proT-B azanol, the pancreatin cutting
The mass spectrum sequential analysis of Fig. 6 .1478.78 peptide section
Fig. 7. prophymosin-alpha (Thr
13) the mass spectrum molecular weight analyse of A, B component
A:A component B:B component
Embodiment
Prophymosin-alpha (the Ile that embodiment one N-is terminated acetylated
13) preparation
1. express prophymosin-alpha (Ile
13) the structure of recombination bacillus coli
Pfu enzyme in the experiment, restriction endonuclease, ligase enzyme, test kit, DH5 α etc. are available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing.
Get the human fetal thymus gland of miscarrying age in April, prepare test kit with total RNA, the method that provides by test kit prepares total RNA, uses the RT-PCR test kit, and the method that provides by test kit becomes cDNA with the mRNA reverse transcription, is template with this cDNA, and prophymosin-alpha (Ile therefrom increases
13) cDNA.Used primer Prot1 and Prot2 are synthetic with the oligonucleotide synthesizer.
Prot1：cggaattcatgtctgatgcagctgtagataccagctccgaaatcaccatcaaggactta
Prot2：cgggatccctagtcatccacgtcggtcttctg
PCR method is:
Add 1 μ l cDNA in the 100 μ l reaction systems, the Prot1 of 20 μ mol/L, each 3 μ l of Prot2 primer, the dNTP of 2mmol/L, 10 μ l, 10X reaction buffer 10 μ l, pfu archaeal dna polymerase 5U, (dNTP, reaction buffer, pfu archaeal dna polymerase are Shanghai biotechnology Services Co., Ltd product).With 9600 PCR instrument of Perk-Elmer company, the PCR condition is 94 ℃ of sex change 1 minute, anneals 30 seconds for 52 ℃, and 72 ℃ were extended 1.5 minutes, and circulated 35 times.Show the band of an expection big or small (0.3kb) by the gel electrophoresis analysis reactant, the PCR product reclaims the purification kit purifying with the PCR product and reclaims.Cut with BamHI and EcoR I enzyme, behind the agarose gel electrophoresis purifying, be cloned into pBV220 plasmid (Zhang Zhiqing, Hou Yunde, Li Yuying etc. with the BamHI/EcoRI enzymolysis, use PRPL tandem promoter and Cits857 regulatory gene and efficiently express human gama-interferon, the virus journal, 1988,4 (2): 97), make up pBV220-proT (Ile
13), transformed into escherichia coli DH5 α is with the LB plate screening positive colony that contains penbritin.Enzyme is cut and is identified that correctly dna sequencing the results are shown in sequence 1 in the sequence table.
2. express prophymosin-alpha (Ile
13) the cultivation of recombination bacillus coli
In the single colony inoculation LB of picking DH5 α (pBV-proT) substratum, 30 ℃ of 150r/min of shaking table cultivate 10-12h, by 5% (v/v) inoculum size above-mentioned activated spawn is met 10 500ml that 100mlLB is housed and shake bottle, 30 ℃ of 150r/min of shaking table cultivated 5 hours, intensification inducing culture 8 hours, centrifugal receipts bacterium.
3.N-terminated acetylated prophymosin-alpha (Ile
13) purifying
(10mmol/LTris-HCl's 4g thalline pH8.0) suspends, and breaks bacterium 10min (9s/ time with carrying out ultrasonic bacteria breaking instrument (cole pamer company) with the 40ml buffer A, interval 9s), 10000r/min, 4 ℃ of centrifugal 20min (Backman JA-2 type whizzer) get supernatant, add the saturated phenol of 10ml, thorough mixing, 4 ℃ of centrifugal 10min, water intaking phase, after adding 40ml buffer A dilution, be loaded into the flow velocity of 4ml/min and use buffer A equilibrated DEAE-Sepharose FF (Φ 1.6 * 15cm) posts in advance.After the buffer A balance, (pH8.0) gradient elution is collected effluent liquid for 1mol/LNaCl, 10mmol/LTris-HCl, and SDS-PAGE analyzes with buffer B.Engineering bacteria carrying out ultrasonic bacteria breaking supernatant, take out through phenol and can remove most of foreign protein (Evstafieva A.G., Chichkova N.V., Makarova T, N, .etal.Overproduction in Escherichia coli, purification and properties of humanprothymosin alpha.Euro J Biochem 231:639-643,1995),, obtain electrophoretically pure sample (seeing accompanying drawing 1) then through the anion-exchange chromatography purifying, this sample separates (employing HP1050 high pressure liquid chromatograph with RP-HPLC, C18 post (4.6 * 250mm, Dalian chemical physics institute of the Chinese Academy of Sciences)), A liquid is the pure water that contains 0.1%TFA; B liquid is the trifluoroacetic acid aqueous solution (Deneb friend company) that contains 0.1%TFA, and gradient is carefully taken off: 0 → 30min, A100% → 10%, B 0% → 90%, find to exist two kinds of component (see figure 2)s, called after proT-A (retention time is 16.2min), and proT-B (retention time is 16.4min) respectively.ProT-A and proT-B freeze-drying.Mass spectrum molecular weight analyse and further peptide spectrum analysis show that wherein the sample of proT-B is the prophymosin-alpha of N-acetylation modification.
4. the peptide spectrum analysis and the structural identification of prophymosin-alpha
(instrument is the electron spray(ES) quadrupole polyphone mass spectrum that Britain Micromass company produces with the flight mass spectrum analysis respectively for proT-A and proT-B; Q-TOF2); its molecular weight is respectively 11953.75 (proT-A) (Fig. 3 A) and 11995.75 (proT-B) (Fig. 3 B); pressing aminoacid sequence calculates; remove terminal methionine(Met); and the prophymosin-alpha molecular weight of unmodified is 11954; consistent with proT-A; illustrate that proT-A is for removing terminal methionine(Met) but the prophymosin-alpha of unmodified; and proT-B molecular weight ratio proT-A is big by 42, and the increase of the molecular weight that produces with acetylation modification is consistent.The modification but many sites of prophymosin-alpha can be acetylation needs further to determine its decorating site.A plurality of pancreatin cleavage sites are arranged on the prophymosin-alpha aminoacid sequence, but the experiment discovery is difficult to be cut into each peptide section with pancreatin.Supposition may be relevant with its space conformation.Thereby use azanol instead and cut.Prophymosin-alpha A, B component are dissolved in the oxammonium hydrochloride of 2mol/L respectively, transfer pH to 9.0 with 4.5mol/L LiOH again, 45 ℃ of water bath heat preservation 4h, and freeze-drying after the reversed-phase HPLC desalination is used for Q-TOF2 quality peptide spectrum analysis (Fig. 4).The results are shown in Table 1; except that molecular weight greater than 5000 the peptide section undetermined; it is 716.3 and 1445.6 peptide section that B component and A component all have molecular weight; (T29-35 represents the peptide section that N holds the 29th to 35 amino-acid residue to constitute corresponding to the T29-35 of prophymosin-alpha; as follows) and T29-42; it is 3093.5 and 3775.9 peptide section that molecular weight is arranged in the A component quality peptide spectrum; T1-28 and T1-35 corresponding to prophymosin-alpha; and have only molecular weight in the B component quality peptide spectrum is 3135.6 and 3817.9 peptide section; T1-28 and T1-35 molecular weight than component A is big by 42 respectively; thereby the B component is the prophymosin-alpha of acetylation modification, and its acetylation modification site is in 28 peptides of its N end.In order further to determine the site of acetylation modification in the B component, with this component azanol cleaved products, cut with pancreatin again, after enzyme is cut the product desalination, do the polyphone mass spectroscopy.Find that in the first step mass spectrum molecular weight is 1478.78 peptide section (Fig. 5); bigger by 42 than T1-14 peptide segment molecule amount; show that this may be the T1-14 of acetylation modification; in order accurately to determine decorating site; with second stage mass spectrum to this peptide section check order (Fig. 6); sequencing result is consistent with the T1-14 sequence of prophymosin-alpha, and decorating site is the Serine of N-end.Explanation is the human thymosin alpha-source of the terminated acetylated modification of N-from the proT-B of recombination bacillus coli preparation.
The piecewise analysis of two kinds of component azanol cuttings of table 1 prophymosin-alpha peptide
Peptide theoretical value ProT-A ProT-B
Measured value difference measured value difference
T1-28 3093.3 3093.5 0.2 3135.6 42.3
T1-35 3775.0 3775.9 0.9 3817.9 42.0
T29-35 714.7 716.3 1.6 716.3 1.6
T29-42 1443.4 1445.6 2.2 1445.6 2.2
Thymosin (the Ile that embodiment two N-are terminated acetylated
13) preparation of analogue
The terminated acetylated prophymosin-alpha (Ile of N-with embodiment two preparations
13), be dissolved in the oxammonium hydrochloride of 2mol/L, transfer pH to 9.0 with 4.5mol/L LiOH again, 45 ℃ of water bath heat preservation 4h, product separates (employing HP1050 high pressure liquid chromatograph, C18 post (4.6 * 250mm with RP-HPLC, Dalian chemical physics institute of the Chinese Academy of Sciences), A liquid is the pure water that contains 0.1%TFA; B liquid is the trifluoroacetic acid aqueous solution (Deneb friend company) that contains 0.1%TFA; gradient is carefully taken off: 0 → 50min; A100% → 10%; B 0% → 90%.); collect each elution peak, after the freeze-drying, use the Q-TOF2 mass spectroscopy respectively; get molecular weight and be about 3136 peptide section, be the terminated acetylated thymosin (Ile of N-
13) analogue.The N-that it has sequence 1 in the sequence table holds the identical sequence of 28 amino acids residues, and the modification that has been acetylation of N-end, and two carboxyls of the terminal aspartic acid of its C-are also by hydroxylamination.
Thymosin 1 (the Ile that embodiment three N-are terminated acetylated
13) preparation of analogue
The terminated acetylated prophymosin-alpha (Thr of N-with embodiment two preparations
13), be dissolved in the oxammonium hydrochloride of 2mol/L, transfer pH to 9.0 with 4.5mol/L LiOH again, 45 ℃ of water bath heat preservation 4h, product separates (employing HP1050 high pressure liquid chromatograph, C18 post (4.6 * 250mm with RP-HPLC, Dalian chemical physics institute of the Chinese Academy of Sciences), A liquid is the pure water that contains 0.1%TFA; B liquid is the trifluoroacetic acid aqueous solution (Deneb friend company) that contains 0.1%TFA; gradient is carefully taken off: 0 → 50min; A100% → 10%; B 0% → 90%.); collect each elution peak, after the freeze-drying, use the Q-TOF2 mass spectroscopy respectively; get molecular weight and be about 3818 peptide section, be the terminated acetylated thymosin of N-1 (Ile
13) analogue.The N-that it has sequence 1 in the sequence table holds the identical sequence of 35 amino acids residues, and the modification that has been acetylation of N-end, and two carboxyls of the terminal aspartic acid of its C-are also by hydroxylamination.
Prophymosin-alpha (the Thr that embodiment four N-are terminated acetylated
13) purifying
Because there is polymorphism in 13 amino acids residues of human thymosin alpha-source, what have is Threonine, and what have is Isoleucine, and front embodiment discloses the terminated acetylated prophymosin-alpha (Ile of N-
13) the preparation method, present embodiment provides N-terminated acetylated prophymosin-alpha (Thr
13) the preparation method.We change 13 amino acids into Isoleucine by Threonine by the method for positional mutation.Used mutant primer Prot 3 and Prot 4 usefulness oligonucleotide synthesizers are synthetic.
Prot3：atgaattcatgtctgatgcagctgtagataccagctccgaaatcaccaccaaggactta
Prot4：cgggatccctagtcatccacgtcggtcttctg
PCR method is: the pBV220-proT (Ile that adds 1 μ l 50ng/ μ l in the 100 μ l reaction systems
13), the Prot3 of 20 μ mol/L, each 3 μ l of Prot4 primer, the dNTP of 2mmol/L, 10 μ l, 10X reaction buffer 10 μ l, pfu archaeal dna polymerase 5U, (dNTP, reaction buffer, pfu archaeal dna polymerase are Shanghai biotechnology Services Co., Ltd product).With 9600 PCR instrument of Perk-Elmer company, the PCR condition is 94 ℃ of sex change 1 minute, anneals 30 seconds for 52 ℃, and 72 ℃ were extended 1.5 minutes, and circulated 35 times.Show the band of an expection big or small (0.3kb) by the gel electrophoresis analysis reactant, the PCR product reclaims the purification kit purifying with the PCR product and reclaims.Cut with BamH I and EcoR I enzyme, behind the agarose gel electrophoresis purifying, be cloned into on the pBV220 plasmid of BamHI/EcoRI enzymolysis (the pfu enzyme in the experiment, restriction endonuclease, ligase enzyme, pUC19 plasmid, test kit etc. are available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing), form pBV-proT.BamHI-EcoR I district dna sequencing result shows that this PCR product sequence is identical with the hproT sequence, has just added BamH I site and EcoR I site respectively at its 5 '-end and 3 '-end.And before 3 '-end EcoR I site, added terminator codon.
Sequencing result shows that sudden change is correct.Prophymosin-alpha (the Thr that expresses
13) separate A, B component with embodiment one identical method, mass spectroscopy (Fig. 7), the result shows that wherein the B component is the prophymosin-alpha (Thr of N-acetylation modification
13) component.Adopt the method identical to prepare the terminated acetylated thymosin (Thr of N-with embodiment two, embodiment three
13) and thymosin 1 (Thr
13).
Sequence table
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉prepare the method for the extrasin alpha of the terminated acetylated modification of N-with recombination bacillus coli
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>109
<212>PRT
<213>
<400>1
Ser?Asp?Ala?Ala?Val?Asp?Thr?Ser?Ser?Glu?Ile?Thr?Ile?Lys?Asp?Leu
1 5 10 15
Lys?Glu?Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Asn?Gly?Arg?Asp?Ala
20 25 30
Pro?Ala?Asn?Gly?Asn?Ala?Asn?Glu?Glu?Asn?Gly?Glu?Gln?Glu?Ala?Asp
35 40 45
Asn?Glu?Val?Asp?Glu?Glu?Glu?Glu?Glu?Gly?Gly?Glu?Glu?Glu?Glu?Glu
50 55 60
Glu?Glu?Glu?Gly?Asp?Gly?Glu?Glu?Glu?Asp?Gly?Asp?Glu?Asp?Glu?Glu
65 70 75 80
Ala?Glu?Ser?Ala?Thr?Gly?Lys?Arg?Ala?Ala?Glu?Asp?Asp?Glu?Asp?Asp
85 90 95
Asp?Val?Asp?Thr?Lys?Lys?Gln?Lys?Thr?Asp?Glu?Asp?Asp
100 105
<210>2
<211>109
<212>PRT
<213>
<400>2
Ser?Asp?Ala?Ala?Val?Asp?Thr?Ser?Ser?Glu?Ile?Thr?Thr?Lys?Asp?Leu
1 5 10 15
Lys?Glu?Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Asn?Gly?Arg?Asp?Ala
20 25 30
Pro?Ala?Asn?Gly?Asn?Ala?Asn?Glu?Glu?Asn?Gly?Glu?Gln?Glu?Ala?Asp
35 40 45
Asn?Glu?Val?Asp?Glu?Glu?Glu?Glu?Glu?Gly?Gly?Glu?Glu?Glu?Glu?Glu
50 55 60
Glu?Glu?Glu?Gly?Asp?Gly?Glu?Glu?Glu?Asp?Gly?Asp?Glu?Asp?Glu?Glu
65 70 75 80
Ala?Glu?Ser?Ala?Thr?Gly?Lys?Arg?Ala?Ala?Glu?Asp?Asp?Glu?Asp?Asp
85 90 95
Asp?Val?Asp?Thr?Lys?Lys?Gln?Lys?Thr?Asp?Glu?Asp?Asp
100 105
Claims (19)
1. one kind prepares the method for the extrasin alpha of the terminated acetylated modification of N-with recombination bacillus coli, may further comprise the steps:
(1) preparation of extrasin alpha gene;
(2) the extrasin alpha gene is connected with expression vector, makes up recombinant expression plasmid;
(3) contain the recombinant plasmid transformed intestinal bacteria of extrasin alpha gene, carry out culture expression;
(4) from culture, separate the component of the extrasin alpha contain the terminated acetylated modification of N-.
2. one kind prepares the method for the thymosin of the terminated acetylated modification of N-or its analogue, derivative with recombination bacillus coli, may further comprise the steps:
(1) preparation of thymosin alpha protogene;
(2) thymosin alpha protogene is connected with expression vector, makes up recombinant expression plasmid;
(3) contain the recombinant plasmid transformed intestinal bacteria of thymosin alpha protogene, carry out culture expression;
(4) from culture, separate the component of the prophymosin-alpha contain the terminated acetylated modification of N-;
(5) prophymosin-alpha of the terminated acetylated modification of N-cutting, the thymosin of separation preparation N-terminal acetylation modification or its analogue, derivative.
3. one kind prepares the method for the thymosin 1 of the terminated acetylated modification of N-or its analogue, derivative with recombination bacillus coli, may further comprise the steps:
(1) preparation of thymosin alpha protogene;
(2) thymosin alpha protogene is connected with expression vector, makes up recombinant expression plasmid;
(3) contain the recombinant plasmid transformed intestinal bacteria of thymosin alpha protogene, carry out culture expression;
(4) from culture, separate the component of the prophymosin-alpha contain the terminated acetylated modification of N-;
(5) prophymosin-alpha of the terminated acetylated modification of N-cutting, the thymosin 1 of separation preparation N-terminal acetylation modification or its analogue, derivative.
4. method according to claim 1, wherein extrasin alpha is a peptide species or protein, 28 amino-acid residue homologys of N-terminal of sequence 1 or sequence 2 are greater than 90% in 28 amino-acid residues of its N-terminal and the sequence table.
5. method according to claim 1, wherein extrasin alpha is a peptide species or protein, 28 amino-acid residues of N-terminal of sequence 1 or sequence 2 are identical in 28 amino-acid residues of its N-terminal and the sequence table.
6. according to any one the described method among the claim 1-3, wherein in extrasin alpha or prophymosin-alpha and the sequence table amino acid residue sequence homology of sequence 1 or sequence 2 greater than 90%.
7. according to any one the described method among the claim 1-3, wherein in extrasin alpha or prophymosin-alpha and the sequence table amino acid residue sequence homology of sequence 1 or sequence 2 greater than 95%.
8. according to any one the described method among the claim 1-3, wherein the amino acid residue sequence of sequence 1 or sequence 2 is identical in extrasin alpha or prophymosin-alpha and the sequence table.
9. according to any one the described method among the claim 1-3, wherein extrasin alpha or prophymosin-alpha are the product of randomly modifying again on the amino of the amino acid residue sequence shown in sequence 1 or the sequence 2 or the carboxyl in sequence table.
10. according to any one the described method among the claim 1-3, wherein extrasin alpha or thymosin alpha protogene adopt PCR, RT-PCR, synthetic or make up one of methods such as screening the cDNA library and obtain.
11. according to any one the described method among the claim 1-3, wherein extrasin alpha or thymosin alpha protogene are controlled by temperature sensitive promotor.
12. method according to claim 11, wherein temperature sensitive promotor is the PLPR promotor.
13. according to any one the described method among the claim 1-3, wherein prokaryotic expression carrier is pBV220.
14. according to any one the described method among the claim 1-3, wherein intestinal bacteria are DH5 α.
15. according to any one described method among the claim 1-3, wherein the separation purification method of the extrasin alpha of the terminated acetylated modification of N-or prophymosin-alpha comprises (1) separating thallus, (2) bacterial cell disruption, (3) phenol extracting, (4) ion-exchange, (5) reversed phase chromatography.
16. according to any one the described method among the claim 1-3, the extrasin alpha content of the terminated acetylated modification of N-of preparation is at least 50% of total extrasin alpha.
17. according to any one described method of claim 1-3, the extrasin alpha content of the terminated acetylated modification of N-of preparation is at least 90% of total extrasin alpha.
18. according to the method for claim 2 or 3, the method for wherein cutting the prophymosin-alpha of the terminated acetylated modification of N-is chemical method or enzyme process.
19. method according to claim 18, the method for wherein cutting the prophymosin-alpha of the terminated acetylated modification of N-are the azanol patterning methods.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100379863C (en) * | 2005-06-14 | 2008-04-09 | 浙江大学 | Method of preparing natural human thymosin a1 using series expression mode |
| WO2010054530A1 (en) * | 2008-11-13 | 2010-05-20 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing genetically engineered n-terminal acetylated thymosin alpha 1 |
| CN101979505A (en) * | 2010-10-21 | 2011-02-23 | 中国人民解放军军事医学科学院生物工程研究所 | Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide |
| CN102277327A (en) * | 2011-08-03 | 2011-12-14 | 中国人民解放军军事医学科学院生物工程研究所 | Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate |
| CN102352337A (en) * | 2011-08-03 | 2012-02-15 | 中国人民解放军军事医学科学院生物工程研究所 | Recombinant bacteria expressed by escherichia coli genome N-acetylase by control of heterogenous promoter, and use thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1059929C (en) * | 1996-05-31 | 2000-12-27 | 中国人民解放军军事医学科学院卫生学环境研究所 | Human thymosin alpha-source and preparing method thereof |
| CN1258747A (en) * | 1998-12-30 | 2000-07-05 | 中国人民解放军第四军医大学 | Preparation of 4-tandem thymosin Alpha-1 with colon bacillus |
| CN1234727C (en) * | 2000-06-01 | 2006-01-04 | 北京双鹭药业股份有限公司 | Human thymosin alpha protogene mutant synthesis, expression and use |
| CN1177051C (en) * | 2002-07-25 | 2004-11-24 | 中国人民解放军第二军医大学 | Recombinant thymin alpha-1 and its prepn |
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2003
- 2003-03-20 CN CNB031196861A patent/CN100335633C/en not_active Expired - Lifetime
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| CN100379863C (en) * | 2005-06-14 | 2008-04-09 | 浙江大学 | Method of preparing natural human thymosin a1 using series expression mode |
| WO2010054530A1 (en) * | 2008-11-13 | 2010-05-20 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing genetically engineered n-terminal acetylated thymosin alpha 1 |
| CN101736008B (en) * | 2008-11-13 | 2012-02-08 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing genetic engineering N-acetylated thymosin alpha1 |
| CN101979505A (en) * | 2010-10-21 | 2011-02-23 | 中国人民解放军军事医学科学院生物工程研究所 | Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide |
| CN101979505B (en) * | 2010-10-21 | 2012-07-04 | 中国人民解放军军事医学科学院生物工程研究所 | Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide |
| CN102277327A (en) * | 2011-08-03 | 2011-12-14 | 中国人民解放军军事医学科学院生物工程研究所 | Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate |
| CN102352337A (en) * | 2011-08-03 | 2012-02-15 | 中国人民解放军军事医学科学院生物工程研究所 | Recombinant bacteria expressed by escherichia coli genome N-acetylase by control of heterogenous promoter, and use thereof |
| CN102277327B (en) * | 2011-08-03 | 2014-12-24 | 中国人民解放军军事医学科学院生物工程研究所 | Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate |
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