CN1724663A - Method of preparing natural human thymosin a1 using series expression mode - Google Patents
Method of preparing natural human thymosin a1 using series expression mode Download PDFInfo
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- CN1724663A CN1724663A CN 200510050069 CN200510050069A CN1724663A CN 1724663 A CN1724663 A CN 1724663A CN 200510050069 CN200510050069 CN 200510050069 CN 200510050069 A CN200510050069 A CN 200510050069A CN 1724663 A CN1724663 A CN 1724663A
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- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 20
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- IALQAMYQJBZNSK-WHFBIAKZSA-N Gly-Ser-Asn Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O IALQAMYQJBZNSK-WHFBIAKZSA-N 0.000 claims 1
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- NZVYCXVTEHPMHE-ZSUJOUNUSA-N thymalfasin Chemical compound CC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NZVYCXVTEHPMHE-ZSUJOUNUSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method to manufacture biology polypeptide, especially a method to make natural human thymosin by gene series express method. It contains compounding two polynucleotide section, compounding double enzyme cut, T alpha 1 gene three times connecting in series, constructing high efficiency expression, constructing engineering fungus, expressing T alpha 1 polypeptide six series bodies in engineering fungus, purifying and cracking. The invention could abundantly express aim albumen, and it has simple technology, easy to operate and low cost.
Description
Technical field
The present invention relates to a kind of preparation method of biological polypeptide, particularly prepare the method for natural human thymosin with gene polyphone phraseology.
Background technology
The polypeptide that thymosin (Thymosin-α 1, T α 1) is made up of 28 amino-acid residues, iso-electric point is 4.2, and no secondary structure does not have disulfide linkage, and unique modification is that N-is terminated acetylated.Its aminoacid sequence is N-Ac-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-C, is a kind of at the lymphocytic immunostimulant of T.It can make T cell maturation and differentiation, can impel sophisticated T cell, the various lymphokines of NK emiocytosis such as interleukin-2, gamma-interferon, also can promote the generation of interleukin-2 acceptor simultaneously, impel the generation of the interleukin-2 acceptor (IL-2) of high-affinity.
T α 1 is mainly used in immune deficiency and immunosuppression class disease as a kind of immunostimulant.After some virus of patient infection was as HIV, HBV or cancer and process radiotherapy or chemotherapy, its immunity system tended to be suppressed gradually even destroy, and can not bring into play the normal killer effect to virus or cancer cells.T α 1 can recover the lymphocytic function of T, promote the propagation of mature T cells, promote the gathering of lymphocyte around cause of disease tissue and cancer cells, promote the generation of lymphokine and lymphokine acceptor, so just strengthened the effect of immunocyte greatly, improved resistivity virus and cancer.
The application of T α 1 in medical treatment mainly contains the following aspects: (1) treatment for cancer has confirmed malignant melanoma, lung cancer, leukemia, squamous cell cancer, straight colorectal carcinoma effective.(2) treatment of viral hepatitis HBV.(3) treatment of hiv virus (HIV).T α 1 also is applied to radiation and chemotherapy patient's immunity system and recovers.
T α 1 not only has good curing and mitigation to diseases common, multiple, high mortality such as multiple malignant tumour, viral hepatitis, HIV, and also the application at neonatal immunity, the elderly's aspects such as health care has very big potentiality.
At present, domestic to prepare thymus gland α 1 method commonly used be to extract from the thymus gland of animal, and the shortcoming of this method is a complicated operation, and the purity of extracting T α 1 is not high, and the source is subjected to the thing quantity limitation.External prepare the method that thymosin uses always be chemical synthesis, and the shortcoming of this method is that cost is higher and cause environmental pollution easily.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, utilize genetic engineering technique, aminoacid sequence according to T α 1, derive and synthetic its coding nucleotide sequence, with the polyphone mode at expression in escherichia coli, after the inclusion body separation and purification, utilize hydroxylamine cleavage, obtain highly purified natural human thymosin.
In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions:
The invention provides and a kind ofly prepare the method for natural human thymosin, may further comprise the steps with the polyphone phraseology:
(1) 28 amino acid that the natural human thymosin is comprised are translated into following sequence according to colibacillary habitual codon:
TCTGATGCTGCTGTAGATACTTCTTCTGAGATTACTACTAAAGACCTAAAGGAGAAGAAGGAAGTTGTCGAAGAGGCTGAGAAC
AGACTACGACGACATCTATGAAGAAGACTCTAATGATGATTTCTGGATTTCCTCT
TCTTCCTTCAACAGCTTCTCCGACTCTTG；
(2) add nucleic acid restriction endonuclease Hind III, methionine(Met), nucleic acid restriction endonuclease BamH I, N, the pairing Nucleotide of glycine in natural thymosin sequence front; Add glycine, nucleic acid restriction endonuclease Bgl II, terminator, the pairing Nucleotide of nucleic acid restriction endonuclease Xho I in natural thymosin sequence back;
(3) two cracked ends annealing, extensions obtain as next fragment gene segment:
GTC?GAC?ATG?
GGA?TCC?AAT?GGG--84bp—GGC?
AGA?TCT?TGA?
CTC?GAG
Val?Asp?Met?Gly?Ser?Asn?Gly?Tα1?Gly?Arg?Ser?StopLeu?Glu
HindIII BamH?I Bgl?II Xho?I
This segment is cloned among the pUCm-T, after the order-checking assay certificate is errorless, enlarged culturing;
(4) extract plasmid and be divided into two parts, BamH I and Xho I double digestion reclaim small segment; Bgl II and Xho I double digestion reclaim big fragment; Two fragments are connected to T α 1 two string bodies with T4 ligase;
(5) with this two strings body clone enlarged culturing, extract plasmid and be divided into two parts,, reclaim small segment with BamH I and Xho I double digestion; Bgl II and Xho I double digestion reclaim big fragment; Two fragments are connected to T α 1 four string bodies with T4ligase;
(6) extract two string physique grains,, reclaim small segment with BamH I and Xho I double digestion; Extract four string physique grains,, reclaim big fragment with Bgl II and Xho I double digestion; Two segments are connected to T α 1 six string bodies with T4 ligase;
(7) natural thymosin six string bodies are cloned between the nucleic acid restriction endonuclease Hind III, Xho I site of plasmid pET22-b (+), through enzyme cut, check order identify errorless, be transformed into e. coli bl21 (DE3), induce down with the inclusion body formal representation at IPTG (isopropyl-);
(8) the separation and purification inclusion body obtains T α 1 six strand body proteins, and under 37 ℃, pH4.5 condition, the hydroxylamine cleavage 48h through 0.2M obtains the natural human thymosin.
Compared with prior art, the invention has the beneficial effects as follows:
(1) using artificial synthetic method of the present invention according to the gene order of the synthetic T α 1 of the habitual codon of intestinal bacteria, and adopts the method for six string bodies to express, and expression product is present in the bacterium with the form of inclusion body, thus the great expression target protein;
(2) the present invention adopts the formal representation of inclusion body, only needs to use the method for centrifugation just can separate behind the broken thalline, the purifying target protein, and technology is simple, easy and simple to handle, with low cost.
(3) design has glycine between each monomer of T α 1 six string bodies, can pass through the effective cutting and separating of azanol, and make monomeric N-end be with ethanoyl, recovers the natural composition of T α 1, does not influence its biological activity.
Embodiment
Below the present invention is further described by specific embodiment 1.
The preparation work for preparing the method front of natural human thymosin with the polyphone phraseology in the specific embodiment 1 at first may further comprise the steps:
(1) 28 amino acid that the natural human thymosin is comprised are translated into following sequence according to colibacillary habitual codon:
TCTGATGCTGCTGTAGATACTTCTTCTGAGATTACTACTAAAGACCTAAAGGAGAAGAAGGAAGTTGTCGAAGAGGCTGAGAAC
AGACTACGACGACATCTATGAAGAAGACTCTAATGATGATTTCTGGATTTCCTCT
TCTTCCTTCAACAGCTTCTCCGACTCTTG；
(2) add nucleic acid restriction endonuclease Hind III, methionine(Met), nucleic acid restriction endonuclease BamH I, N, the pairing Nucleotide of glycine in natural thymosin sequence front; Add glycine, nucleic acid restriction endonuclease Bgl II, terminator, the pairing Nucleotide of nucleic acid restriction endonuclease Xho I in natural thymosin sequence back;
(3) two cracked ends annealing, extensions obtain as next fragment gene segment:
GTC?GAC?ATG?
GGA?TCC?AAT?GGG--84bp—GGC?
AGA?TCT?TGA?
CTC?GAG
Val?Asp?Met?Gly?Ser?Asn?Gly?Tα1?Gly?Arg?Ser?StopLeu?Glu
HindIII BamH?I Bgl?II Xho?I
This segment is cloned among the pUCm-T, after the order-checking assay certificate is errorless, enlarged culturing;
Abovementioned steps is corresponding following operation steps:
(1) synthetic two polynucleotide segments
Th1
GTCGACATGGGATCCAACGGTTCTGATGCTGCTGTAGATACTTCTTCTGAGAT
TACTACTAAAGAC CTAA
Th2
CTCGAGTCAAGATCTCCCGTTCTCAGCCTCTTCGACAACTTCCTTCTTCTCCT
TTAGGTCTTTAGT AGTA
The part that wherein has underscore is mutual paired base.
(2) connection of synthetic fragment, amplification
Carry out PCR by following condition, to connect, to increase T α 1 gene:
Each reagent dosage of PCR: PCR reaction conditions:
Th1 5ul 94℃ 5min
Th2 5ul 94℃ 30s
dNTP 16ul 60℃ 30s
buffer?10ul 72℃ 30s
Mgcl2 10ul repeats three and goes on foot 30 times
H2O 53ul 72℃ 5min
Taq?plus?1ul
(3) detection of synthetic fragment and clone
The outage swimming of connection piece in the step 2 is detected, reclaim rear clone in the pUMc-T carrier and order-checking detect.
Step afterwards also comprises:
(4) double digestion of plasmid
The errorless clone of order-checking assay certificate in the step 2, enlarged culturing.Extract plasmid and be divided into two parts, BamHI and Xho I double digestion, electrophoresis reclaims small segment (about 120 nucleotide residues); Bgl II and Xho I double digestion, electrophoresis reclaim big fragment (about 2800 nucleotide residues).
(5) contact the first time of T α 1 gene
Small pieces in the step 4 is got 3ul, and big segment is got 5ul, adds 1ul and connects buffer and 1ul ligase enzyme, in 4 ℃ of insulation 16h; Connecting product is T α 1 gene two string bodies.
(6) connect the conversion of product and the screening of positive colony
Connection product in the step 5 is transformed into the bacillus coli DH 5 alpha competence, and the method for cutting by bed board, cultivation, amplification, enzyme filters out positive colony, and correct positive colony is determined in order-checking at last.
(7) contact the second time of T α 1 gene
The errorless clone of order-checking assay certificate in the step 6, enlarged culturing.Extract plasmid and be divided into two parts, BamHI and Xho I double digestion, electrophoresis reclaims small segment (about 240 nucleotide residues); Bgl II and Xho I double digestion, electrophoresis reclaim big fragment (about 3000 nucleotide residues); Get small pieces 3ul, get big segment 5ul, add 1ul and connect buffer and 1ul ligase enzyme.In 4 ℃ of insulation 16h, connecting product is T α 1 gene four string bodies.This connection product is transformed into the bacillus coli DH 5 alpha competence, and the method for cutting by bed board, cultivation, amplification, enzyme filters out positive colony, and correct positive colony is determined in order-checking at last.
(8) polyphone for the third time of T α 1 gene
Correct clone's plasmid in the extraction step 6, BamH I and Xho I double digestion, electrophoresis reclaims small segment (about 240 nucleotide residues); Correct clone's plasmid in the extraction step 7, Bgl II and Xho I double digestion, electrophoresis reclaim big fragment (about 3200 nucleotide residues); Get small pieces 3ul, get big segment 5ul, add 1ul and connect buffer and 1ul ligase enzyme.In 4 ℃ of insulation 16h, connecting product is T α 1 gene six string bodies.This connection product is transformed into the bacillus coli DH 5 alpha competence, and the method for cutting by bed board, cultivation, amplification, enzyme filters out positive colony, and correct positive colony is determined in order-checking at last.
(9) efficiently express the structure of recon
Correct clone's plasmid in the extraction step 8, Hind III and Xho I double digestion, electrophoresis reclaims between the Hind III and Xho I site that inserts pET22-b (+) plasmid in the back.
(10) structure of engineering bacteria
With the expression plasmid transformed into escherichia coli BL21 (DE3) that builds in the step 9, the method for cutting by bed board, cultivation, amplification, enzyme filters out positive colony, and order-checking at last determines that correct positive colony is pET22-b (+)-T α 1/BL21.
(11) T α 1 polypeptide six expression of string body in engineering bacteria
With engineering bacteria pET22-b (+)-T α 1/BL21 that filters out in the step 10, cultivate 2h to logarithmic phase for 37 ℃, under the inducing of 3mmol/L IPTG, cultivated 3 hours for 37 ℃, the centrifugation thalline, add 2xSDS-PAGE and go up sample buffer95 ℃ of processing 5min, carry out SDS-PAGE, dyeing, the surface sweeping analysis of decolouring back, go out to have at 23kD and significantly induce band, account for 10% of bacterial protein.
(12) purifying of T α 1 polypeptide six string bodies
Engineering bacteria pET22-b (+)-T α 1/BL21 behind enlarged culturing and abduction delivering, centrifugation; Precipitation is suspended in the cell lysis buffer solution the broken 15min of excusing from death under the power of 200w; Broken back solution centrifugal reclaims precipitation; With inclusion body lavation buffer solution washed twice; Add the solubilization of inclusion bodies damping fluid and place shaking table to spend the night, obtain T α 1 polypeptide six string bodies in the 200rpm effect.
(13) cracking of T α 1 polypeptide six string bodies
In T α 1 polypeptide six string liquid solutions (concentration is 1mg/ml), add azanol and make final concentration, transfer pH to 4.5,, obtain T α 1 polypeptide through look general detection and separation in 25 ℃ of reaction 48h to 0.2M.
The sequence of product polypeptide and natural T α 1 are consistent to be:
N-Ac-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-C
Specific embodiment 2:
Remove (13) step cracking condition and make final concentration to 1.0M for adding azanol, transfer pH to 6.0, outside 37 ℃ of reaction 60h, other is identical with specific embodiment 1.
Specific embodiment 3:
Remove (13) step cracking condition and make final concentration to 2.0M for adding azanol, transfer pH to 7.0, outside 45 ℃ of reaction 72h, other is identical with specific embodiment 1.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (1)
1, a kind ofly prepare the method for natural human thymosin, it is characterized in that may further comprise the steps with the polyphone phraseology:
(1) 28 amino acid that the natural human thymosin is comprised are translated into following sequence according to colibacillary habitual codon:
TCTGATGCTGCTGTAGATACTTCTTCTGAGATTACTACTAAAGACCTAAAGGAGAAGAAGGAAGTTGTCGAAGAGGCTGAGAAC
AGACTACGACGACATCTATGAAGAAGACTCTAATGATGATTTCTGGATTTCCTCT
TCTTCCTTCAACAGCTTCTCCGACTCTTG；
(2) add nucleic acid restriction endonuclease Hind III, methionine(Met), nucleic acid restriction endonuclease BamH I, N, the pairing Nucleotide of glycine in natural thymosin sequence front; Add glycine, nucleic acid restriction endonuclease Bgl II, terminator, the pairing Nucleotide of nucleic acid restriction endonuclease Xho I in natural thymosin sequence back;
(3) two cracked ends annealing, extensions obtain as next fragment gene segment:
GTC?GAC?ATG?
GGA?TCC?AAT GGG?--84bp-GGC
AGA?TCT?TGA
CTC?GAG
Val Asp Met Gly Ser Asn Gly Tα1 Gly Arg SerStop Leu Glu
Hind?III BamH?I Bgl?II Xho?I
This segment is cloned among the pUCm-T, after the order-checking assay certificate is errorless, enlarged culturing;
(4) extract plasmid and be divided into two parts, BamH I and Xho I double digestion reclaim small segment; Bgl II and Xho I double digestion reclaim big fragment; Two fragments are connected to T α 1 two string bodies with T4 ligase;
(5) with this two strings body clone enlarged culturing, extract plasmid and be divided into two parts,, reclaim small segment with BamH I and Xho I double digestion; Bgl II and Xho I double digestion reclaim big fragment; Two fragments are connected to T α 1 four string bodies with T4 ligase;
(6) extract two string physique grains,, reclaim small segment with BamH I and Xho I double digestion; Extract four string physique grains,, reclaim big fragment with Bgl II and Xho I double digestion; Two segments are connected to T α 1 six string bodies with T4 ligase;
(7) natural thymosin six string bodies are cloned between the nucleic acid restriction endonuclease Hind III, Xho I site of plasmid pET22-b (+), through enzyme cut, check order identify errorless, be transformed into e. coli bl21 (DE3), induce down with the inclusion body formal representation at IPTG;
(8) the separation and purification inclusion body obtains T α 1 six strand body proteins, and under 25～45 ℃, pH4.5～7.0 conditions, the hydroxylamine cleavage 48～72h through 0.2～2.0M obtains the natural human extrasin alpha.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101544693B (en) * | 2008-12-11 | 2012-06-06 | 中国人民解放军第四军医大学 | Recombined extrasin alpha 1 two-strand body protein and preparation method thereof |
| CN102676534A (en) * | 2012-04-12 | 2012-09-19 | 上海育臣生物工程技术有限公司 | Method for preparing thymosin polypeptide by using interin |
| CN103789321A (en) * | 2014-02-24 | 2014-05-14 | 东北制药集团股份有限公司 | Method for preparing recombinant human thymic peptide alpha1 |
| CN110964117A (en) * | 2019-10-21 | 2020-04-07 | 哈尔滨医科大学 | Polyethylene glycol modified human thymosin β 4 two-string protein and preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN100335633C (en) * | 2003-03-20 | 2007-09-05 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing N-end acetylation modified thymosin alpha with recombined E. coli |
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2005
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101544693B (en) * | 2008-12-11 | 2012-06-06 | 中国人民解放军第四军医大学 | Recombined extrasin alpha 1 two-strand body protein and preparation method thereof |
| CN102676534A (en) * | 2012-04-12 | 2012-09-19 | 上海育臣生物工程技术有限公司 | Method for preparing thymosin polypeptide by using interin |
| CN102676534B (en) * | 2012-04-12 | 2014-07-16 | 上海育臣生物工程技术有限公司 | Method for preparing thymosin polypeptide by using interin |
| CN103789321A (en) * | 2014-02-24 | 2014-05-14 | 东北制药集团股份有限公司 | Method for preparing recombinant human thymic peptide alpha1 |
| CN103789321B (en) * | 2014-02-24 | 2016-06-08 | 东北制药集团股份有限公司 | A kind of preparation method of rhthymosin ��1 |
| CN110964117A (en) * | 2019-10-21 | 2020-04-07 | 哈尔滨医科大学 | Polyethylene glycol modified human thymosin β 4 two-string protein and preparation method and application thereof |
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| CN100379863C (en) | 2008-04-09 |
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