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
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- xho
- double digestion
- thymosin
- bgl
- bamh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 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
- 108010046075 Thymosin Proteins 0.000 title claims abstract description 16
- 102000007501 Thymosin Human genes 0.000 title claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 20
- 230000029087 digestion Effects 0.000 claims description 20
- 239000012634 fragment Substances 0.000 claims description 18
- 108020004707 nucleic acids Proteins 0.000 claims description 14
- 150000007523 nucleic acids Chemical class 0.000 claims description 14
- 102000039446 nucleic acids Human genes 0.000 claims description 14
- 108091008146 restriction endonucleases Proteins 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 13
- 210000003000 inclusion body Anatomy 0.000 claims description 9
- 102000003960 Ligases Human genes 0.000 claims description 8
- 108090000364 Ligases Proteins 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 239000004471 Glycine Substances 0.000 claims description 7
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 5
- 108020004705 Codon Proteins 0.000 claims description 4
- 230000002650 habitual effect Effects 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 3
- 229930182817 methionine Natural products 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- JLXVRFDTDUGQEE-YFKPBYRVSA-N Gly-Arg Chemical compound NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N JLXVRFDTDUGQEE-YFKPBYRVSA-N 0.000 claims 1
- 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
- NFNVDJGXRFEYTK-YUMQZZPRSA-N Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O NFNVDJGXRFEYTK-YUMQZZPRSA-N 0.000 claims 1
- ZSZFTYVFQLUWBF-QXEWZRGKSA-N Val-Asp-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N ZSZFTYVFQLUWBF-QXEWZRGKSA-N 0.000 claims 1
- 108010005942 methionylglycine Proteins 0.000 claims 1
- 229920001184 polypeptide Polymers 0.000 abstract description 11
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 11
- 101150084750 1 gene Proteins 0.000 abstract description 8
- 238000005336 cracking Methods 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 108091033319 polynucleotide Proteins 0.000 abstract description 2
- 102000040430 polynucleotide Human genes 0.000 abstract description 2
- 239000002157 polynucleotide Substances 0.000 abstract description 2
- 241000233866 Fungi Species 0.000 abstract 2
- 238000013329 compounding Methods 0.000 abstract 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 238000001962 electrophoresis Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000009413 insulation Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 206010019799 Hepatitis viral Diseases 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229960001438 immunostimulant agent Drugs 0.000 description 2
- 239000003022 immunostimulating agent Substances 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000000527 lymphocytic effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 102220023256 rs387907547 Human genes 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 201000001862 viral hepatitis Diseases 0.000 description 2
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010078233 Thymalfasin Proteins 0.000 description 1
- 102400000800 Thymosin alpha-1 Human genes 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- -1 isopropyl- Chemical group 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 208000017572 squamous cell neoplasm Diseases 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 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
- 229960004231 thymalfasin Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100500696A CN100379863C (en) | 2005-06-14 | 2005-06-14 | Method of preparing natural human thymosin a1 using series expression mode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100500696A CN100379863C (en) | 2005-06-14 | 2005-06-14 | Method of preparing natural human thymosin a1 using series expression mode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1724663A true CN1724663A (en) | 2006-01-25 |
| CN100379863C CN100379863C (en) | 2008-04-09 |
Family
ID=35924293
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100500696A Active CN100379863C (en) | 2005-06-14 | 2005-06-14 | Method of preparing natural human thymosin a1 using series expression mode |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100379863C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2005
- 2005-06-14 CN CNB2005100500696A patent/CN100379863C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100379863C (en) | 2008-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101063104A (en) | Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof | |
| CN1724663A (en) | Method of preparing natural human thymosin a1 using series expression mode | |
| CN1693466A (en) | Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method | |
| CN101798350A (en) | Fusion protein of human interferon alpha-2b and human thymosin alpha1, and preparation thereof | |
| CN1654656A (en) | FenneropenaeusChinensis Crustin gene and coded protein and cloning method therefor | |
| CN1611604A (en) | Swine alpha-interferon gene synthesis, expression vector establishment and product preparing method | |
| CN102827289B (en) | Porcine circovirus type 2 Cap protein and thymosin alpha1 fusion protein and application | |
| CN1786165A (en) | Preparation method of recombination staphylococcus aureus enterotoxin C2 | |
| CN1177055C (en) | Antibacterial peptide gene of fly and its cloning process | |
| CN1205335C (en) | Tumor death induction ligand gene, gene expression protein and its preparation method | |
| CN1287858C (en) | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method | |
| CN1306030C (en) | Human interleukin-10 gene sequenc and E coli containing the said gene sequence | |
| CN1861635A (en) | Human pancreas hyperglycemiacin relative peptide-2 analogue | |
| CN100335633C (en) | Method for preparing N-end acetylation modified thymosin alpha with recombined E. coli | |
| CN1740320A (en) | Fenneropenaeus chinensis Anti-LPS factor gene and cloning process thereof | |
| CN1778914A (en) | Expression of soluble TRAIL protein | |
| CN1258747A (en) | Preparation of 4-tandem thymosin Alpha-1 with colon bacillus | |
| CN1900119A (en) | Staphylococcus aureus enterotoxin 1 and preparation and use | |
| CN103937828A (en) | Preparation method of fusion protein of porcine interferon-alpha 1 and thymosin-alpha 1 | |
| CN1185254C (en) | First hypervariable region antigen of hepatitis C and fusion antigen | |
| CN1810830A (en) | HIV-1 yesisting polypeptide C22, its coding sequence and prepn process | |
| CN1273248A (en) | Efficient gene engineering process for preparing polypeptide medicines | |
| CN1260359C (en) | High soluble expressed tricholsanthin mutant and its preparing method | |
| CN1184232C (en) | Human tumor necrosin relative death inducing ligand mutein and its prepn process and medicine composition | |
| CN1185261C (en) | Interleukin -2/granulocyte-macrophage colony stimulating factor fusion protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI ENTS BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: ZHEJIANG UNIVERSITY Effective date: 20140731 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 310027 HANGZHOU, ZHEJIANG PROVINCE TO: 200120 PUDONG NEW AREA, SHANGHAI |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20140731 Address after: 200120, room 2, building 3377, 321, Kang Xin Road, Shanghai, Pudong New Area Patentee after: Shanghai ENTS Biotechnology Co., Ltd. Address before: 310027 Hangzhou, Zhejiang Province, Xihu District, Zhejiang Road, No. 38, No. Patentee before: Zhejiang University |
|
| ASS | Succession or assignment of patent right |
Owner name: HUNAN FANGSHENG YUCHEN BIOLOGICAL TECHNOLOGY CO., Free format text: FORMER OWNER: SHANGHAI ENTS BIOTECHNOLOGY CO., LTD. Effective date: 20150820 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20150820 Address after: 410295, building 4, building 402, building 789, Lu Song Road, hi tech Development Zone, Hunan, Changsha Patentee after: Hunan Sheng Yuchen Biotechnology Co. Ltd. Address before: 200120, room 2, building 3377, 321, Kang Xin Road, Shanghai, Pudong New Area Patentee before: Shanghai ENTS Biotechnology Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201215 Address after: 570316 No. A-8, national high tech Industrial Development Zone, Haikou City, Hainan Province Patentee after: HAINAN DOCTOR PHARMACEUTICAL Co.,Ltd. Address before: Room 402, 4 / F, complex building, 789 lushong Road, high tech Development Zone, Changsha City, Hunan Province Patentee before: HUNAN FANGSHENG YUCHEN BIOTECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20210611 Address after: No. 206, Kaiyuan Avenue, Science City, Guangzhou Economic and Technological Development Zone, Guangdong 510000 Patentee after: GUANGDONG JIDA GENETIC MEDICINE ENGINEERING RESEARCH CENTER Co.,Ltd. Address before: 570316 No. A-8, national high tech Industrial Development Zone, Haikou City, Hainan Province Patentee before: HAINAN DOCTOR PHARMACEUTICAL Co.,Ltd. |
|
| TR01 | Transfer of patent right |