CN103864916A - Human thymosin beta4 triploid protein, encoding gene and isolation and purification method of protein - Google Patents

Abstract

The invention relates to a human thymosin beta4 triploid protein, an encoding gene and an isolation and purification method of the protein. An amino acid sequence of the related human thymosin beta4 triploid protein is shown in SEQ ID NO:1; the related encoding gene sequence is shown in SEQ ID NO:2; and the related isolation and purification method is isolation and purification by adopting an affinity column. The human thymosin beta4 tandem triploid protein disclosed by the invention is subjected to rose wreath experiment detection, so that the immune response performance of T-lymphocyte can be obviously improved. A fur growth experiment in mice proves that a hair removal agent can be obviously promoted to process fur growth of the mice by the human thymosin beta4 tandem triploids.

Description

Human thymosin β 4 triploid albumen, encoding gene and separation purification method thereof

Technical field

The invention belongs to genetic engineering technique research field, be specifically related to one and utilize the genetic engineering technique Restruction human thymosin β 4 triploid production method of connecting.

Background technology

Thymus gland is immune organ important in human immune system, can secrete tens of kinds of thymosins (thymosins) to regulate the immunologic function of body, because it extracts and gain the name the earliest from calf thymus.

Tens of kinds of thymosins of cognition of the mankind are thymosin ɑ, β, γ three major types because iso-electric point is different by difference, and each class comprises multiple hypotype.β family thymosin (thymosin-β) is the one group thymosin of iso-electric point between pI5.0-7.0, and size is 40-44 amino-acid residue, the about 5kD of relative molecular mass, sequential structure high conservative.The β family thymus gland of finding in different plant species have kind more than ten, in human body, be mainly thymosin-β 4, thymosin-β 10 and thymosin-β 15, play a significant role maintaining in a lot of physiology such as the running balance of Actin muscle and tumor invasion and transfer, apoptosis, inflammatory reaction, vasculogenesis and wound healing etc., pathologic process.

Oneself becomes the immunostimulant that all circles extremely pay close attention to thymosin class preparation.The thymosin class preparation of application is all by the polypeptide quasi-molecule mixture extracting in ox or porcine thymus clinically; there is more stable immunity words property; be applicable to clinically immunodeficient disease or the caused a series of diseases of immunity function imbalance, if thymic aplasia, severe Combination immunodeficiency disease, ataxia telangiectasia, leprosy, severe infection, recurrent aphtha etc. are with the low patient of cellular immune function.Also can be used for viral hepatitis, malignant tumour and anti-ageing.

There is more stable clinical efficacy although extract the thymosin class preparation of preparation from animal tissues, the risk that also exists potential pathogen to pollute.And, the mixture of multiple peptide quasi-molecule owing to extracting product, its action effect there are differences between different batches, may be also to cause existing Thymopeptide common adverse reactions as the immanent cause generating heat, urticaria, fash, giddy etc. appear in a few patients.

Natural human thymosin β 4, only has 44 amino-acid residues, and relative molecular mass is less than normal, only has 5kD left and right.Although prior art has the genetic engineering technique of employing to produce it, expression amount and the stability in host cell fail to solve well always.Therefore, conventionally select technically, by its and other protein fusion expression, after preliminary purification, to recycle proteolytic enzyme and cut out from fusion rotein.Processing step and the cost of separation and purification are increased.

Summary of the invention

For defect or the deficiency of prior art, one of object of the present invention is for providing a kind of human thymosin β 4 triploid albumen.

For this reason, the aminoacid sequence of human thymosin β 4 triploid albumen provided by the invention is as shown in SED ID NO:1.

For defect or the deficiency of prior art, object of the present invention two for the triploid encoding gene of a kind of human thymosin β 4 is provided.

For this reason, the sequence of human thymosin β 4 triploid encoding genes provided by the invention is as shown in SED ID NO:2.

For defect or the deficiency of prior art, object of the present invention three for a kind of separation purification method of human thymosin β 4 triploid albumen is provided.

For this reason, separation purification method provided by the invention comprises: adopt affinity column to carry out separation and purification.

Preferably, described separation purification method comprises:

The first step, thick purifying: by resuspended to abduction delivering human thymosin β 4 triploid albumen thalline waters or PBS, boil rear centrifugal collection supernatant liquor;

Second step, utilizes affinity column to carry out purifies and separates to supernatant liquor, and column material used is the column material of specific binding affinity tag.

Compared with prior art, beneficial effect of the present invention is:

(1) human thymosin β 4 triploid protein structures of the present invention are single polypeptide chain structure, the people source Zadaxin aminoacid sequence that comprises long 44 amino-acid residues of 3 copies, corresponding encoding gene is that nucleotide sequence length is 3 times of this polypeptide chain total number of atnino acid objects, i.e. the long nucleotide sequence of 474bp.The 5kD that its relative molecular mass has from monoploid is increased to 15kD left and right, is more prone to separation and purification than existing monoploid human thymosin β 4, improves the yield of separation and purification.

(2) aminoacid sequence of human thymosin β 4 triploid albumen of the present invention comprises according to three copy human thymosin β 4 sequences of 5 ' → 3 ' direction series connection restructuring and is beneficial to the correct folding flexible joint aminoacid sequence of each copy recombination human thymus peptide beta 4 β 4, i.e. GGGSGGGS.Utilize this flexible joint sequence to be formed by glycine and the Serine of little side chain, in polypeptide chain folding process, tend to form β-pleated sheet structure or irregular curling, the target protein that is conducive to its both wings carries out natural folding process separately, tends to form the protein molecule that approaches natural structure state.Namely be conducive to form activated recombinant protein.

(3) aminoacid sequence of the aminoacid sequence of the human thymosin β 4 of each copy that the aminoacid sequence of human thymosin β 4 triploid albumen of the present invention comprises and natural human thymosin β 4 is in full accord.

(4) the recombination human thymus peptide beta 4 β 4 of the present invention triploid of connecting has the activity of part natural human source Thymosin β4.Experimentation on animals shows, this series connection triploid recombination human thymus peptide beta 4 can promote experimental mouse hair regeneration, shows natural human Zadaxin immunocompetence.

(5) purification process of the present invention is through affinity chromatography, refining, lyophilize acquisition target protein sterling.

Accompanying drawing explanation

Fig. 1 is that in embodiment 1, the connect enzyme of triploid expression vector of recombination human thymus peptide beta 4 β 4 is cut detection, and wherein 1-4 swimming lane is respectively: 1, the NcoI of 2,3, No. 4 recombinant expression vector DNA and XhoI double digestion product; 5-8 swimming lane is respectively: 1,2,3, No. 4 recombinant expression vector DNA; No. 9 swimming lane is: prokaryotic expression carrier pET22b DNA; No. 10 swimming lane is: 125bp DNA Ladder;

Fig. 2 is embodiment 2 recombination human thymus peptide beta 4 β 4 expression effects of triploid in engineering bacteria of connecting, wherein swimming lane 1 be Marker, swimming lane 2 for induction before sample, swimming lane 3 for induction 1h, swimming lane 4 for induction 2h, swimming lane 5 for induction 3h, swimming lane 6 for induction 4h, swimming lane 7 for induction 5h, swimming lane 8 be induction 6h;

Fig. 3 be embodiment 2 recombination human thymus peptide beta 4 β 4 connect the expression of results of triploid in engineering bacteria western blotting identify, wherein swimming lane 1-6 swimming lane is respectively: the recombination human thymus peptide beta 4 β 4 triploid sample of connecting, swimming lane 7 is empty carrier transformant lysate, and swimming lane 8 is Marker;

Fig. 4 is the embodiment 2 recombination human thymus peptide beta 4 β 4 triploid separation and purification results of connecting, and M swimming lane is that Marker, A swimming lane are that purification of samples, B swimming lane are not that thick purification of samples, C, D, E swimming lane are respectively after His-tag affinity chromatography concentrated successively purification result;

Fig. 5 is the recombination human thymus peptide beta 4 β 4 of the embodiment 2 purifying three times of uv-absorbing figure that connect;

Fig. 6 is that the rosettes experiment of embodiment 2 detects the recombination human thymus peptide beta 4 β 4 triploid immunologic competence of connecting;

Fig. 7 is that the recombination human thymus peptide beta 4 β 4 of embodiment 2 triploid of connecting is processed 2 weeks promoter actions to experimental mouse hair growth;

Fig. 8 is that the HE staining examine recombination human thymus peptide beta 4 β 4 of embodiment 2 triploid of connecting is processed the effect to experimental mouse hair follicle cell in 2 weeks.

Embodiment

The present invention considers by human thymosin β 4 encoding gene arranged in series together, utilizes the encoding gene of oligomerization flexible joint (GGGSGGGS) to be separated by between each repeating unit.According to the related data of protein folding research, glycine and Serine are rich in flexible joint, and this two seed amino acids residue alternately occurs, tends to form β-pleated sheet structure structure in polypeptide chain.Be conducive to aminoacid sequence folding activated higher structure of tool that produces on primary structure basis of its both wings.According to this cognition, contriver has designed the human thymosin Beta-4 gene sequence that codon process is optimized, has built its prokaryotic expression carrier and express engineering bacteria.Existing experimental result shows, the human thymosin β 4 treble histone of connecting can be crossed and express in intestinal bacteria, and expression process is simple; Separation purification method is relatively simple; The series connection triploid of expressing has corresponding biologic activity.

The production method of human thymosin β 4 triploid albumen of the present invention comprises:

(1) for the structure of expressing, Restruction human thymosin β 4 connects triploid engineering bacteria; In building process, introduce corresponding affinity tag, be beneficial to later separation purifying; Existing affinity tag is all for the present invention, for example: nickel post affinity is histidine-tagged, Trx, maltose binding protein etc.;

(2) fermentation culture of engineering bacteria;

(3) connect triploid induction and expression of recombination human thymus peptide beta 4 β 4;

(4) the recombination human thymus peptide beta 4 β 4 triploid separation and purification of connecting, through thick pure after, utilize affinity column to further separate purifying:

The first step, thick purifying: by 1.5 times of water or PBS(the mass ratio thalline of collecting after abduction delivering for) thoroughly resuspended, after boiling in order to the enrichment restructuring Zadaxin triploid of connecting, centrifugal collection supernatant liquor;

Second step, utilizes affinity column to carry out purifies and separates to supernatant liquor: column material used is the main material of the above-mentioned affinity tag of specific binding.With phosphoric acid buffer, as elutriant, wash-out target protein from affinity column material is prepared into target protein preparation after desalination, cryoconcentration, lyophilize.

Wherein engineering bacteria construction process comprises:

(1) the codon optimized recombination human thymus peptide beta 4 β 4 of the synthetic triploid coding gene sequence of connecting, utilizes gene recombination technology to be cloned under nucleic acid restriction endonuclease and the effect of nucleic acid ligase order and specifies in cloning vector.

(2) nucleic acid restriction endonuclease with under the effect of nucleic acid ligase order, will comprise 3 copy recombination human thymus peptide beta 4 β 4 and connect that triploid coding gene sequence cuts out from cloning vector and be connected restructuring with expression vector;

(3) can be any expression vector being suitable at prokaryotic cell prokaryocyte efficiently expressing exogenous gene for the connect expression vector of triploid Expression product of recombination human thymus peptide beta 4 β 4.

(4) the recombination human thymus peptide beta 4 β 4 triploid carrier DNA of connecting that carries of restructuring is imported to suitable host cell.

(5) importing the recombination human thymus peptide beta 4 β 4 triploid host cell of connecting is intestinal bacteria.

The present invention is described in further detail for the embodiment providing by contriver below.

In following examples, material therefor and reagent are:

The gene order that comprises 3 copy human thymosin β 4: entrust Dalian precious biotinylated biomolecule Engineering Co., Ltd synthetic;

PUC18: Dalian precious biotinylated biomolecule Engineering Co., Ltd provides;

Intestinal bacteria: E.coli Top10 is for vector construction, BL21(DE3) for genetic expression;

Restructuring has the E.coli intestinal bacteria of goal gene carrier: BL21(DE3);

Plasmid extraction kit: purchased from Beijing Zhou Dingguo Bioisystech Co., Ltd;

Gel reclaims test kit: purchased from Beijing Zhou Dingguo Bioisystech Co., Ltd;

Host cell: BL21(DE3);

Affinity column: Ni post;

Phosphoric acid buffer: sodium-chlor 8g, Repone K 0.2g, Sodium phosphate dibasic 1.42g, potassium primary phosphate 0.27 are dissolved in 1 liter of deionized water;

Be the phosphoric acid buffer of 1.7% imidazoles containing mass percent.

embodiment 1

This embodiment is the connect structure of triploid engineering bacteria of recombination human thymus peptide beta 4 β 4.

(1) entrust the synthetic gene orders that comprise 3 copy human thymosin β 4 of Dalian precious biotinylated biomolecule Engineering Co., Ltd, by synthetic sequence directed cloning in cloning vector pUC18 and the E.coli somatic cells (JM109) of this carrier of restructuring is provided;

(2) plasmid preparation: the E.coli somatic cells that the restructuring that company is provided has a goal gene carrier is cultivated 12-16h in 37 ℃, 200rpm/min, collect thalline in the centrifugal 30～60s of 12000rpm/min, adopt commercially available plasmid extraction kit to prepare high-purity plasmid DNA, DNA concentration is adjusted to 1 μ g/ μ L;

(3) nucleic acid restriction endonuclease digestion: in 50 μ L reaction systems, the high-purity plasmid DNA that adds 5 μ L nucleic acid restriction endonucleases (the each 2.5 μ L of NcoI and XhoI), 5 μ L10 × nucleic acid restriction endonuclease reaction buffers, 40 μ L previous steps to prepare, in 37 ℃ of water-baths, act on 3h, endonuclease reaction liquid is electrophoresis 1h under 1% sepharose, 80V voltage, under ultraviolet lamp, from gel, cut out gene fragment, utilize commercially available gel to reclaim test kit and reclaim gene fragment.

(4) preparation of linearizing expression vector fragment: the E.coli somatic cells that carries expression vector pET22b plasmid is cultivated to 12-16h in 37 ℃, 200rpm/min, collect thalline in the centrifugal 30～60s of 12000rpm/min, adopt commercially available plasmid extraction kit to prepare high-purity plasmid DNA, DNA concentration is adjusted to 1 μ g/ μ L; In 50 μ L reaction systems, add 5 μ L nucleic acid restriction endonucleases (the each 2.5 μ L of NcoI and XhoI), 5 μ L 10 × nucleic acid restriction endonuclease reaction buffers, 40 μ L high purity pET22b plasmid DNA, in 37 ℃ of water-baths, act on 3h, endonuclease reaction liquid is electrophoresis 1h under 1% sepharose, 80V voltage, under ultraviolet lamp, from gel, cut out linearized vector fragment, utilize commercially available gel to reclaim test kit and reclaim gene fragment.

(5) the recombination human thymus peptide beta 4 β 4 triploid encoding gene fragment of connecting is connected restructuring with the linearizing fragment of expression vector: the encoding gene fragment of the series connection triploid human thymosin β 4 that above-mentioned steps 3 is reclaimed and expression vector linearizing fragment are added to according to the ratio of mole ratio 6:4 in the 50 μ L reaction systems that contain 5 μ L T4DNA ligase enzymes, 5 μ L 10 × T4DNA ligase enzyme reaction buffers, after pressure-vaccum mixes, be distributed into 10 μ L/250 μ L PCR pipes, connect restructuring 16h in 16 ℃.

(6) preparation of Bacillus coli cells: by the escherichia coli expression bacterium BL21(DE3 of freezing preservation) be seeded to not containing overnight incubation on antibiotic LB flat board, picking mono-clonal is forwarded to fresh not containing on antibiotic LB flat board, picking mono-clonal is inoculated in 5mL and does not contain in antibiotic LB liquid nutrient medium in 37 ℃, 200rpm/min shaking table overnight incubation, overnight culture is forwarded to fresh containing in antibiotic LB liquid nutrient medium with 10% inoculum size, be cultured to OD in 37 ℃, 280rpm/min shaking table ₆₀₀reach 0.8 left and right, in 8000rpm/min, 4 ℃ of centrifugal collection thalline, thalline is resuspended in to ice-cold 0.1mol/L CaCl ₂more than processing 15min in solution, put stand-by on ice.All operations is aseptic technique.

(7) escherichia coli cloning transformation: the connection recombinant products described in above-mentioned steps 5 is added in escherichia coli expression cell suspending liquid prepared by step 6, more than ice is put 15min, processes 90s, cooled on ice 2min in 42 ℃.Add 700 μ L not containing the antibiotic LB liquid nutrient medium that is heated in advance 37 ℃, in 37 ℃, 200rpm/min shaking table cultivation 40min, centrifugal collection thalline is also resuspended in 100 μ L LB liquid nutrient mediums, coat and be added with in corresponding antibiotic LB solid culture ware, cultivate 12～16h in 37 ℃.

(8) the connect enzyme of triploid prokaryotic expression carrier of screening recombinant plasmid: recombination human thymus peptide beta 4 β 4 is cut detection, picking mono-clonal the LB solid medium obtaining from above-mentioned steps 7, be seeded in the antibiotic LB liquid nutrient medium of 50～100 μ g/mL that 10mL is added with, prepare plasmid according to above-mentioned steps 2 methods, through restriction enzyme NcoI and XhoI digestion and the detection of 1% agarose gel electrophoresis, enzyme cut in product, there is carrier strap and 474bp goal gene band carry the connect expression vector of triploid encoding gene of recombination human thymus peptide beta 4 β 4, its source clone is and carries the connect engineering bacteria of triploid gene of recombination human thymus peptide beta 4 β 4.Shown in accompanying drawing 1, be and comprise the connect enzyme of expression vector of triploid encoding gene of recombination human thymus peptide beta 4 β 4 and cut detected result.NcoI and the XhoI double digestion detected result of the plasmid extracting in step 7 transformed clone that wherein front 4 swimming lanes are from left to right 1,2,3, No. 4 random pickings, 5-8 swimming lane is respectively to cut the corresponding plasmid DNA sample of product with 1,2,3, No. 4 enzyme.The 9th swimming lane is the empty carrier pET22b plasmid DNA sample that enzyme is not cut.Restriction enzyme mapping demonstration, the plasmid DNA of 4 Clone Origins of random picking is all the recombinant plasmid being made up of goal gene and carrier, after NcoI and XhoI double digestion, has carrier strap and the 474bp goal gene band that approaches 500bpMarker band to produce.

(9) determined dna sequence: entrust Dalian precious biotinylated biomolecule Engineering Co., Ltd to analyze with full-automatic determined dna sequence instrument.

The recombinant plasmid that step 8 enzyme is cut after detection carries out determined dna sequence detection, and sequencing result is in full accord with the synthetic sequence of design.If need to list sequencing result, it is exactly supplementary nucleotide sequence before document.

Sequencing result:

Acgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggatatgagcgacaaaccggatatggcggaaatcgaaaaattcgataaaagcaaactgaaaaaaaccgaaacccaggaaaaaaatccgctgccgagcaaagaaaccattgaacaggaaaaacaggcgggcgaaagcggcggcggcagcggcggcggcagcatgagcgacaaaccggatatggcggaaatcgaaaaattcgataaaagcaaactgaaaaaaaccgaaacccaggaaaaaaatccgctgccgagcaaagaaaccattgaacaggaaaaacaggcgggcgaaagcggcggcggcagcggcggcggcagcatgagcgacaaaccggatatggcggaaatcgaaaaattcgataaaagcaaactgaaaaaaaccgaaacccaggaaaaaaatccgctgccgagcaaagaaaccattgaacaggaaaaacaggcgggcgaaagcctcgagcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttg

The comparison result (DNAssist2.0) of order-checking institute's calling sequence and implementation sequence:

The explanation of above-mentioned comparison result, the external source fragment that restructuring enters expression vector pET22b is exactly to design in advance the synthetic recombination human thymus peptide beta 4 β 4 triploid encoding sequence of connecting.In the above results, shown in " sequencing result " not with implementation sequence one to one nucleotide sequence be the sequence of expression vector.

embodiment 2

This embodiment is that the engineering bacteria that utilizes embodiment 1 to build is produced human thymosin β 4 triploid albumen of the present invention.The affinity tag that this embodiment uses is 6 histidine-tagged (being His-tag).This label is introduced in the time of construction of expression vector, directly utilized carrier with His-tag.Column material is Ni chelating dextran.

(1) recombination human thymus peptide beta 4 β 4 abduction delivering of triploid in engineering bacteria of connecting: the recombination human thymus peptide beta 4 β 4 of the activation triploid engineering bacteria (or engineering bacteria mono-clonal of fresh conversion) of connecting is inoculated in and is added with in the antibiotic fermention medium of 50～100 μ g/mL, be cultured to OD in 37 ℃, 250rpm/min shaking table ₆₀₀reach more than 0.4, add inductor IPTG, in 30 ℃, 250rpm/min shaking table continuation cultivation 2-4h, in 8000rpm/min, the centrifugal collection thalline of room temperature, thalline is resuspended in proper volume protein sample-loading buffer, and boiling water bath effect 5～10min, is chilled to room temperature, in the centrifugal 10min of 12000rpm/min, supernatant liquor comprises the cell pyrolysis liquid of target protein.

Fermention medium: glucose 10-20g/L, yeast powder 15-25g/L, proteolysis mixture 10-20g/L, mineral salt solution 10-25g/L(sodium-chlor: Repone K: Sodium phosphate dibasic: potassium primary phosphate is dissolved in respective volume distilled water according to the consumption order of every liter of 10-25g/L according to the ratio of mass ratio 8:0.2:1.5:0.3.）。

Fermentation culture conditions: 37 ℃ of culture temperature, pH7.0～7.2, dissolved oxygen DO20～50%, air flow quantity 1VVM～4.5VVM, mixing speed 300rpm/min～1100rpm/min, inoculum size 5%～10%, seed culture medium is conventional LB substratum, microbiotic 50～100 μ g/mL, stream adds glucose controlled concentration and is no more than 1.5%, dissolved oxygen DO controls 40% left and right, and the time point of results engineering bacteria is OD ₆₀₀reach more than 80, target protein recombination human thymus peptide beta 4 β 4 expression effect of triploid in engineering bacteria of connecting is shown in accompanying drawing 2.In Fig. 2, sample is respectively from left to right: the 1st swimming lane is protein molecule quality standard product, be Marker, the 2nd swimming lane be not with IPTG induction, but transforming restructuring has the connect engineering bacteria cell pyrolysis liquid sample of triploid gene of recombination human thymus peptide beta 4 β 4, the 3rd swimming lane to the 8 swimming lanes are to transform restructuring to have the recombination human thymus peptide beta 4 β 4 engineering bacteria cell of the triploid gene cell pyrolysis liquid sample after IPTG induces respectively 1h, 2h, 3h, 4h, 5h and 6h of connecting.This result clearly demonstrates goal gene and can under IPTG induction, in engineering bacteria, cross and express, and its expression amount extends and increases gradually with induction time.

The fermention medium of engineering bacteria is: glucose 1-2g/L, yeast powder 20-30g/L, proteolysis mixture (Tryptone that oxfoid company produces) 5-10g/L, mineral salt 1-2.5g/L; Wherein mineral salt consist of sodium-chlor: Repone K: Sodium phosphate dibasic: potassium primary phosphate is according to the mass ratio composition of 8:0.2:1.5:0.3.The fermentation culture conditions of engineering bacteria is: 37 ℃ of culture temperature, pH7.0～7.2, dissolved oxygen DO20～40%, air flow quantity 2VVM～5.5VVM, mixing speed 500rpm/min～700rpm/min, inoculum size 5%～10%, seed culture medium is conventional LB substratum, microbiotic 50～100 μ g/mL, stream adds glucose controlled concentration and is no more than 1.5%, dissolved oxygen DO controls 35% left and right, and the time point of results engineering bacteria is OD ₆₀₀reach more than 80.

The immunoblotting assay of target protein: transferring film is to pvdf membrane or nitrocellulose filter after SDS-PAGE electrophoresis for the cell pyrolysis liquid that comprises target protein, and conventional western blot operation is carried out qualitative and quantitative analysis to expression product.The recombination human thymus peptide beta 4 β 4 western blotting qualification result of triploid in engineering bacteria of connecting is shown in accompanying drawing 3.It is not dyed that result shown in Fig. 3 is that the 1st swimming lane protein dyes Marker(in advance from right to left successively, in electrophoresis process, just can observe), the 2nd swimming lane is that cell pyrolysis liquid sample, the 3-8 swimming lane of empty expression vector transformed bacteria is all the recombination human thymus peptide beta 4 β 4 triploid genetic engineering bacterium cell pyrolysis liquid sample of connecting.Diagram result is clear to be shown, the albumen that expression is crossed at 21kDa place is exactly the recombination human thymus peptide beta 4 β 4 of the experimental design triploid albumen of connecting.

The micro-target protein reclaiming through Western qualitative detection, from PAGE glue the inside is carried out to determined amino acid sequence, and detected result is consistent with implementation sequence of the present invention.

(2) the recombination human thymus peptide beta 4 β 4 triploid separation and purification of connecting: cultivate according to above-mentioned steps 9 working method, induction, collecting cell, cell is resuspended in the PBS damping fluid of pH7.2～7.4, 4 ℃ of high-speed homogenizations are with smudge cells, in 12000rpm/min high speed centrifugation to remove cell relic, supernatant liquor upper prop through boiling the thick pure protein sample liquid that removes most of foreign protein is to corresponding affinity column, post in producer's suggestion is pressed, column flow rate, under column temperature, carry out chromatography operation, phosphate buffered saline buffer washes away after impurity albumen, target protein by the phosphoric acid buffer elution of bound containing 1.7% imidazoles on affinity chromatography column material, elutriant is after dialysis desalting, the lyophilize of cryoconcentration final vacuum, obtain target protein lyophilized powder.It is the recombination human thymus peptide beta 4 β 4 triploid sterling of connecting.See accompanying drawing 4 and Fig. 5.Figure 4 shows that the connect SDS-PAGE glue detected result of triploid purification result of recombination human thymus peptide beta 4 β 4, is to utilize the color atlas of protein separation system through Ni post affinitive layer purification target protein shown in Fig. 5.Wherein, in Fig. 4, A swimming lane is the cell pyrolysis liquid sample after abduction delivering finishes, B swimming lane is that the cell pyrolysis liquid after abduction delivering finishes is removed the thick purifying protein quality sample after most foreign proteins through boiling 10min, and C, D, tri-swimming lanes of E are respectively after concentrated 1 times of Ni post affinity chromatography elutriant, 2 times, 4 times rear reserved protein samples.Fig. 5 clear demonstration utilizes Ni post affinitive layer purification target protein, can effectively remove foreign protein (stream is worn shown in peak), and target protein can reach higher purity (shown in elution peak).This result clearly demonstrates, and the separation purification method that this patent is set up is relatively applicable to the recombination human thymus peptide beta 4 β 4 triploid separation and purification of connecting.

Connect triploid immunocompetence of recombination human thymus peptide beta 4 β 4 is measured: rosettes measuring sees attached list 1, and specific experiment is as follows: (1) aseptic venous blood samples 5mL, and add and have in advance heparin liquid in vitro, mix anti-freezing; (2) get 2mL blood sample, slowly add in the test tube that fills 2mL lymphocyte separation medium, note not upsetting boundary liquid level; (3) 2000 revs/min of horizontal centrifugals 20 minutes; (4) carefully draw plasma layer and separate the membranaceous buffy coat of white between liquid layer, PBS liquid washed twice.Counting, by cell furnishing 2X106/ml, then 45 ℃ of 30min desensitizations; (5) by the fresh sheep red blood cell (SRBC) PBS liquid washing of preserving three times, centrifugal, abandon supernatant, packed red cells is made into 1% cell suspension (8X10 7/mL) with PBS liquid; (6) 0.2mL lymphocyte suspension and 0.2mL red cell suspension are mixed, then add 0.2mL calf serum to mix, then add respectively 8ul, 10ul, 12ul Zadaxin; (7) 37 ℃ of water-baths 30 minutes, low-speed centrifugal 1000rpm/min, 5min, then put 4 ℃ and spend the night; (8) discard most of supernatant after taking out, shake up gently, add smear after 2 fixed numbers of 0.8% glutaraldehyde minute, add 1 Ji's nurse Sa dye liquor after seasoning, coated with cover glass, high power lens is observed.All lymphocytes adsorb 3 or the positive bow structure cell of above sheep red blood cell (SRBC) person (seeing accompanying drawing 6) around; (9) 200 lymphocytes of count random counting.Experimental result sees attached list 1.

The table 1 rosettes measuring recombination human thymus peptide beta 4 β 4 triploid immunologic competence of connecting

Experimentation on animals detects recombination human thymus peptide beta 4 β 4 triploid organism of connecting and learns active: 30 of Kunming mouses, take off mouse back hair with trichogen, then be divided into two groups, one group is contrast, smears one group of every day three recombination human thymus peptide beta 4 β 4 triploid purifying protein of connecting.Every sampling in 7 days, contrast and smear each 3,4% paraformaldehyde is fixed, dehydration, embedding, section, HE dyeing, microscopy observations.See accompanying drawing 7 and accompanying drawing 8.Fig. 7 result shows, in treatment time section, the recombination human thymus peptide beta 4 β 4 of the purifying triploid treatment group experimental mouse hair regeneration speed of connecting is greater than undressed control group experimental mouse, and the recombination human thymus peptide beta 4 β 4 that purifying the is described triploid of connecting can promote experimental mouse hair regeneration.Promote that with natural human Thymosin β4 the activity of hair regeneration is similar.The histological stain result of Fig. 8 further illustrates, the recombination human thymus peptide beta 4 β 4 of the purifying triploid treatment group of connecting, and it promotes experimental mouse hair regeneration to realize by hair follicle stimulating cell activation.

Claims (4)

1. human thymosin β 4 triploid albumen, is characterized in that, the aminoacid sequence of this albumen is as shown in SED ID NO:1.

MDMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESGGGSGGGSMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESGGGSGGGSMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESLEHHHHHH。

2. the triploid encoding gene of human thymosin β 4, is characterized in that, the sequence of this encoding gene is as shown in SED ID NO:2.

CCATGGATatgagcgacaaaccggatatggcggaaatcgaaaaattcgataaaagcaaactgaaaaaaaccgaaacccaggaaaaaaatccgctgccgagcaaagaaaccattgaacaggaaaaacaggcgggcgaaagcGGCGGCGGCAGCGGCGGCGGCAGCatgagcgacaaaccggatatggcggaaatcgaaaaattcgataaaagcaaactgaaaaaaaccgaaacccaggaaaaaaatccgctgccgagcaaagaaaccattgaacaggaaaaacaggcgggcgaaagcGGCGGCGGCAGCGGCGGCGGCAGCatgagcgacaaaccggatatggcggaaatcgaaaaattcgataaaagcaaactgaaaaaaaccgaaacccaggaaaaaaatccgctgccgagcaaagaaaccattgaacaggaaaaacaggcgggcgaaagcCTCGAG。

3. the separation purification method of human thymosin β 4 triploid albumen, is characterized in that, the method comprises: adopt affinity column to carry out separation and purification.

4. the separation purification method of human thymosin β 4 triploid albumen as claimed in claim 3, is characterized in that, described separation purification method comprises:

The first step, thick purifying: by resuspended to abduction delivering human thymosin β 4 triploid albumen thalline waters or PBS, boil rear centrifugal collection supernatant liquor;

Second step, utilizes affinity column to carry out purifies and separates to supernatant liquor, and column material used is the column material of specific binding affinity tag.

Images