CN102559725A - Human stem cell growth factor as well as production method and application of polyethylene glycol (PEG) modified human stem cell growth factor - Google Patents

Abstract

The invention discloses a human stem cell growth factor as well as a production method and application of a polyethylene glycol (PEG) modified human stem cell growth factor. The production method comprises the following steps of: fusing an h-SCF (Stem Cell Factor)-alpha sequence and an SUMO (Small Ubiquitin-Related Modifier) sequence and constructing to obtain an SUMO-rhSCF-alpha fused gene expression vector; transferring the SUMO-rhSCF-alpha fused gene expression vector into a host bacteria to obtain engineering bacteria; culturing the engineering bacteria and inducing to express an SUMO-rhSCF-alpha fused protein; and cutting off an SUMO part to obtain an rhSCF-alpha protein. By using the production method, the high soluble expression and large-scale purification of the rhSCF-alpha protein in cell plasmas are realized, and the activity of the obtained rhSCF-alpha protein is high. The invention also discloses the production method of the PEG modified human stem cell growth factor; the half-life period of the PEG modified human stem cell growth factor obtained by using the method is remarkably prolonged, and the activity of the PEG modified human stem cell growth factor in promoting the proliferation of red blood cells is also remarkably improved; and the PEG modified human stem cell growth factor can be applied to the preparation of medicines for hypohemia therapy, reconstruction and recovery of a hematopoietic function after chemoradiotherapy and a bone marrow transplantation operation, stem cell ex-vivo expansion and gene therapy and cosmetics for promoting the metabolism of epidermal cells, repairing aged and damaged skin cells, delaying the aging of skin and the like.

Description

The working method of human stem cell growth and polyethyleneglycol modifiedization product thereof and purposes

Technical field

The invention belongs to the medical biotechnology field, be specifically related to efficiently expressing and purification process of human stem cell growth, and the preparation method and its usage of polyethyleneglycol modifiedization human stem cell growth.

Background technology

Stem cell factor (SCF) is a kind of acid glycoprotein that is produced by the stroma cell in the bone marrow microenvironment; It is a kind of important hematopoietic cytokine; Mainly act on early stage hemopoietic stem cell and original HPC, promote its propagation and differentiation, keeping the hematopoietic cell survival; Promote hematopoietic cell proliferation and differentiation, regulation and control respectively are to play an important role in the growing of hematopoietic cell.The people SCF assignment of genes gene mapping is last at human chromosome 12q22～12q24, and long 1.4kb contains 7 introns and 8 exons.The natural human STEMCELLFACTOR has two kinds of existence forms: full-length molecule hSCF-α (the expressing protein size is about 45KDa for the complete opening code-reading frame 972bp of ripe mRNA, 323 amino acid of encoding) and the Ca that guards at the C end ²⁺Rely on sugared recognition structure territory (calcium dependent carbohydrate recognition domain; CRD) disappearance 78 amino acid whose truncation type molecule hSCF-β (the long 738bp of ripe mRNA in; 245 amino acid of encoding, the size of expressing protein is about 29KDa).They are the different products of same coding mRNA due to the different loci shearing.

Along with going deep into of research, it is found that human stem cell growth has the various biological function, can be widely used in numerous areas such as biological medicine, fine chemistry industry.Particularly stem cell fast development in recent years utilizes stem cells technology to carry out disease treatment and has very big development prospect, especially has great meaning for hemopathic treatment.Bone marrow transplantation and HSCT have become the hemopathic a kind of main means of treatment in the modern medicine, and blood is joined the type difficulty, stem cell population is few but also exist, problems such as amplification difficulty.Isolate adult stem cell if can join the identical people's of type its hetero-organization and the organ from the patient or with it, in the directed differentiation of external realization hematopoietic cell, rapid amplifying, this will be to being significant with HSCT.But natural solvable type SCF is very little at human body and animal body intensive amount, and the concentration in blood is 3.3ng/ml, thus can not be from natural tissues separation and purification q.s sample, chamber and clinical application research experimentize.At the reorganization stem cell factor of expression in escherichia coli, often form soluble, the active inclusion body of lifeless matter, even renaturation does not have activity or active the reduction in vivo yet, and shortcomings such as aminoacid deletion, purification process complicacy often appear in amalgamation and expression.

SUMO expressing fusion protein system is a kind of novel protein expression system of developed recently; Compare with other protein expression system; Has following some advantage: 1) SUMO (small ubiquitin-related modifier; The little ubiquitin relevant modifications factor) not only can improve the expression amount of protein in the host bacterium, and can increase proteic solubility greatly; 2) excise fusion tag like clockwork.Because fusion tag can influence the 26S Proteasome Structure and Function of recombinant protein, so fusion rotein must excise fusion tag, adopts chemistry or Enzymology method usually, like Xa factor, zymoplasm etc.Yet enzyme is cut fusion rotein a lot of problems are arranged, productive rate is low, albumen precipitation, enzyme tangent condition complicacy, proteolytic enzyme costliness etc., often produces unexpected N-end in addition.Different with other proteolytic enzyme, the tertiary structure of SUMO enzyme identification SUMO label, thereby can excise exactly, can not produce the N-end of extension, and enzyme tangent condition (pH, temperature and ionic strength) is very loose.But whether the SUMO expression system can be used for the expression of SCF do not appear in the newspapers as yet.

Polyoxyethylene glycol (PEG) is modified; Or title PEGization (PEGylation); It is a hot technology that grows up gradually the twentieth century later stage seventies; Being at present protein to be carried out one of most important technology of chemically modified, is with the effective way that solves or alleviate the problems that albumen and polypeptide exist in medicinal process.Can increase the Degradation of the anti-proteolytic ferment of protein and polypeptide drugs greatly with PEG modifying protein and polypeptide drugs.To protein drug, because after the PEG modification, its circulating half-life phenomenal growth in vivo, after PEG modified, pharmaceutical grade protein drug effect in vivo increased.But; With protein and the polypeptide after the PEG coupling, its space conformation, sterically hindered; The static associativity; Physico-chemical properties such as hydrophobicity all can change, and the avidity that is connected with acceptor often reduces because of the variation of these physico-chemical properties, thereby cause the external drug effect of polyethyleneglycol modified back protein and polypeptide drugs to reduce.

This shows that pharmaceutical grade protein is prone to by proteasome degradation in vivo, and can prolong its transformation period, but its external drug effect generally can reduce through the pharmaceutical grade protein of PEG modificationization.Therefore, how to improve proteic promoting erythrocyte proliferation activity of SCF and prolong half-life simultaneously, need systematic research.

Summary of the invention

An object of the present invention is to provide a kind of working method of human stem cell growth.

Another object of the present invention provides a kind of preparation method of polyethyleneglycol modifiedization human stem cell growth.

The technical scheme that the present invention adopted is:

1, a kind of fusion gene, its coding SUMO-rhSCF-alpha fusion protein.

Preferably, described fusion gene has the nucleotide sequence shown in the SEQ ID NO.1.

2, the expression vector that contains above-mentioned fusion gene.

3, the working method of human stem cell growth comprises the steps:

1) changes in the host bacterium 2 expression vector over to abduction delivering SUMO-rhSCF-a fusion rotein;

2) separation and purification SUMO-rhSCF-a fusion rotein, excision SUMO part, purifying rhSCF-α albumen.

4, according to the resulting reorganization of 3 method rhSCF-α albumen.

5, a kind of preparation method of polyethyleneglycol modifiedization human stem cell growth comprises the steps:

1) 4 described rhSCF-α albumen and mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD) were reacted 10～14 hours at 24～26 ℃ or 3～5 ℃, add the glycocoll termination reaction;

2) separation and purification obtains polyethyleneglycol modified human stem cell growth.

Preferably, step 1) is at pH6.0, carries out in the phosphate buffered saline buffer of 50mmol/L, and the proteic concentration of said rhSCF-α is 4.5～5.5mg/mL.

Preferably, the mass ratio of said rhSCF-α albumen and mono methoxy polyethylene glycol propionic aldehyde is 2～3: 1.

Preferably, said mono methoxy polyethylene glycol propionic aldehyde is a 20kDa mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD-20kDa).

6, the polyethyleneglycol modifiedization human stem cell growth for preparing of above method is at the reconstruction of preparation treatment anaemia, chemicotherapy and bone marrow transplantation postoperative hemopoietic function and recovery, ex vivo expansion of stem cell, gene therapy medicament and promoting epidermic cell metabolism, repairing the application aspect aging and the makeup such as injured skin cell, delaying skin aging.

Beneficial effect of the present invention is:

1) the present invention carries out amalgamation and expression with rhSCF-α gene and small molecules ubiquitin appearance modified protein (SUMO) gene, and the abduction delivering condition is optimized, and has realized that rhSCF-α albumen is at intracytoplasmic solubility high expression level.

2) the present invention designs synthetic SUMO-rhSCF-alpha fusion protein and has the His label; Behind nickel ion affinity chromatograph and SUMO proteolysis removal SUMO albumen and His label; Can realize the proteic large scale purification of rhSCF-α, the rhSCF-α protein-active of gained is high.

3) human stem cell factor carry out polyethyleneglycol modifiedization, adopts the mPEG-ALD modifier, modify mild condition, do not introduce other reactive group when crosslinked, less to the protein active influence, be convenient to the purifying and the evaluation of product simultaneously.Transformation period through the resulting polyethyleneglycol modifiedization human stem cell growth of this modifying method obviously prolongs, and its promoting erythrocyte proliferation activity also is significantly improved.

Description of drawings

Fig. 1 is rhSCF-α gene PCR product 1% agarose gel electrophoresis figure, and length is 935bp;

Fig. 2 is SUMO-rhSCF-alpha fusion gene PCR product 1% agarose gel electrophoresis figure, and length is 1300bp;

Fig. 3 is plasmid plasmid pET-3c (+) electrophorogram;

Fig. 4 be SUMO-rhSCF-α recombinant plasmid enzyme cut the detected result electrophorogram (M:maker, 1 is BamH I single endonuclease digestion, 2 is plasmid pET-3c-SUMO-rhSCF-α, 3 is Nde I and BamH I double digestion, 4 is Nde I single endonuclease digestion;

Fig. 5 is that the different IPTG concentration of SDS-PAGE analysis is induced Recombinant Protein Expression (M:maker, 1: do not induce; 2～9:IPTG adds concentration and is respectively 0.1,0.3,0.5,0.7,0.9,1.0,1.5,2.0mM);

Fig. 6 is that Recombinant Protein Expression (M:maker, 1: do not induce are induced in the SDS-PAGE analysis when different OD600; 2～6:OD600 is respectively 0.4,0.6, and 0.8,1.0,1.2);

Fig. 7 is that SDS-PAGE analyzes in differing temps, induces different time to induce Recombinant Protein Expression (M:maker; 1,4,7: induce 4h, 12h, 24h respectively at 37 ℃; 2,5,8: induce 4h, 12h, 24h respectively at 20 ℃; 3,6,, 9: induce 4h respectively at 15 ℃, 12h, 24h);

Fig. 8 is the electrophoresis detection figure (M:maker of target protein SUMO-rhSCGF-α; 1: e. coli bl21; 2:0.1M IPTG induces e. coli bl21; 3: the e. coli bl21 that contains plasmid pET-3c-SUMO-rhSCF-α is not induced; 4:0.1M IPTG induces the e. coli bl21 that contains plasmid pET-3c-SUMO-rhSCF-α; 5: induce back albumen supernatant; 6: induce the back albumen precipitation);

Fig. 9 is the 12%SDS-PAGE electrophorogram that the enzyme of SUMO-rhSCF-α is cut product;

Figure 10 is the proteic western blot of rhSCF-α detection figure;

Figure 11 is rhSCF-α, mPEG-rhSCF-α form GM-Colonies respectively with the collaborative BALA/c of stimulation of rmGM-CSF mouse monokaryon cell a experimental result picture;

Figure 12 is hSCF-α or PEG-hSCF-α albumen at 37 ℃ Detection of Stability figure;

Figure 13 is hSCF-α or PEG-hSCF-α albumen transformation period detection figure in the SD rat.

Embodiment

Below in conjunction with embodiment the present invention is further described, but is not limited thereto.

Embodiment 1: human stem cell growth recombinant expressed

One, makes up the SUMO-rhSCF-alpha fusion gene

1, design of primers:

Design primer (seeing table 1) respectively according to SUMO gene order (Genebank no:NC_011677.1) and rhSCF-α gene order (GenBank:AB009244.1).

Table 1 is used for the primer of synthetic SUMO-rhSCF-full length sequence

Annotate: overstriking is the SUMO gene order; The underscore overstriking is the His sequence label; Under to draw solid line be 15 peptide linker gene orders; The restriction enzyme site of italic for adding; Under to draw dotted line be rhSCF-α gene order.

2, PCR reaction:

(1) from human umbilical cord mesenchymal stem cells (hUCMSCs), extracts mRNA, adopt the amplification of RT-PCR method to obtain the cDNA of rhSCF-α with ordinary method.

(reaction system of setting up 20 μ l can be used for rt 1ng～total RNA of 5 μ g or 1～500ngmRNA) to use the M-MLV reversed transcriptive enzyme to carry out the first chain cDNA synthetic

1) will descend component to add in the Eppendorf tube of no RNase: oligo (dT) 1 μ l, extractive hUCMSCs RNA 2 μ l, 10mM dNTP mixture 1 μ l adds 3d water 9 μ l to TV 13 μ l.

2) mixture is at 65 ℃ of heating 5min, rapid cooled on ice, of short duration centrifugal after, add following component: 5 * the first chains synthesize damping fluid 4 μ l, 0.1M DTT 2 μ l, TV 19 μ l.

3) in centrifuge tube, gently various components are mixed, of short duration centrifugal after, under 37 ℃, hatch 2min.

4) at room temperature add 1 μ l M-MLV reversed transcriptive enzyme, blow and beat mixing gently.

5) hatch 50min under 37 ℃.

6) 70 ℃ of heating 15min termination reactions.

(2) with primer S1, S2 amplification SUMO gene and middle 15 peptide linker genes, reaction system and program are following:

The PCR reaction system:

The PCR response procedures:

After reaction finished, reaction product was identified with 1% agarose gel electrophoresis.Glue reclaims and purified pcr product.

(3) with P1, P2 primer amplification rhSCF-α gene and middle 15 peptide linker genes, reaction system and program are following:

The PCR reaction system:

The PCR response procedures:

After reaction finished, reaction product was identified with 1% agarose gel electrophoresis.RhSCF-α PCR product electrophoresis result shows that rhSCF-α PCR product (comprising whole rhSCF-α mature peptide encoding soxs) length is 935bp, conforms to expected results

(see figure 1).Glue reclaims and purified pcr product.

(4) carry out recombinant PCR with the product after step (2) and the recovery of (3) glue as common template and amplify the SUMO-rhSCF-alpha gene fragment, and introduce Nde I and BamH I restriction enzyme site respectively in upstream and downstream.With S1 is upstream primer, and P2 is a downstream primer, and reaction system and program are:

The PCR reaction system:

The PCR response procedures:

Reaction product is identified with 1% agarose gel electrophoresis.Glue reclaims and purified pcr product SUMO-rhSCF-α, and sequence is shown in SEQ ID NO.1.Plasmid extraction and glue recovering step reclaim the test kit specification sheets by OMEGA glue to carry out.SUMO-rhSCF-α PCR product electrophoresis result shows that SUMO-rhSCF-α PCR product (comprising SUMO and rhSCF-α gene (915bp) and middle 15 peptide linker genes) length is 1300bp, and (see figure 2) conforms to expected results.

SUMO-rhSCF-α full-length gene order (SEQ ID NO.1):

ATGCATCATCATCATCATCAC GGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATCATTGAGGCTCACAGAGAACAGATTGGTGGT

Annotate: italic is the His sequence label; Under to draw solid line be the SUMO gene order; Overstriking is 15 peptide linker gene orders; The underscore overstriking is a rhSCF-α gene order.

Two, make up the pET-SUMO-rhSCF-a recombinant expression plasmid:

1, enzyme is cut and the glue recovery: use Nde I and BamH I double digestion SUMO-rhSCF-alpha gene fragment and expression vector pET-3c respectively, glue reclaimed after enzyme was cut the product agarose gel electrophoresis.The plasmid of plasmid extraction and glue recovering step employing OMEGA company is taken out for a short time with glue recovery test kit specification sheets and is carried out.The pET-3c electrophoresis result is seen Fig. 3.

2, connection and conversion: the enzyme switchback receipts product of the enzyme switchback of SUMO-rhSCF-alpha gene fragment being received product and expression vector pET-3c is connected 16h in 16 ℃ with the T4 ligase enzyme; Be converted in the DH5 α competent cell, utilize the amicillin resistance screening positive clone then.

3, identify: picking positive colony list colony inoculation is in the LB substratum that contains penbritin; Behind 37 ℃ of shaking culture 16h; Carrying out bacterium colony PCR and enzyme behind the extracting plasmid cuts and identifies and feeding sample carries out sequencing analysis to biological order-checking company that the result shows the success of pET-SUMO-rhSCF-α construction of recombinant expression plasmid.SUMO-rhSCF-α recombinant plasmid enzyme is cut detected result and is seen Fig. 4.

Select several the evaluation with PCR and identify the recon that is positive, be inoculated into 5ml and contain in the LB substratum of penbritin of 100 μ g/ml through double digestion, 37 ℃, 180rpm incubated overnight, 15% glycerine is protected kind.

Three, the expression of SUMO-rhSCF-α in intestinal bacteria:

1, the conversion of recombinant plasmid pET-3c-SUMO-rhSCF-α: get and identify that correct recombinant plasmid pET-3c-SUMO-rhSCF-α is converted in the competent cell of e. coli bl21 (DE3).Screening positive monoclonal bacterial strain on resistance (penbritins of 100 μ g/ml) flat board.

2, the abduction delivering of SUMO-rhSCF-α: a plurality of mono-clonal bacterium colonies of random choose are inoculated in respectively in the 4ml LB substratum (containing 100 μ g/ml penbritins), and 37 ℃, 250rpm is cultured to OD600=0.6～1.0.Get 1ml and do not induce contrast, all the other add IPTG to final concentration 1mmol/L, under 37 ℃, induce 4h, and centrifugal collection thalline carries out 12% SDS-PAGE analysis after bacterial sediment is handled.Select after the higher mono-clonal enlarged culturing of expression amount respectively under differing temps, carry out abduction delivering under different inductor concentration and the different OD600, induce result (Fig. 5～7) through 12% SDS-PAGE electrophoretic analysis.The optimal expression condition of confirming SUMO-rhSCF-α is: 37 ℃ of cultivations contain e. coli bl21 (DE3) 2～3h to OD of recombinant plasmid pET-3c-SUMO-rhSCF-α ₆₀₀During=0.8～1.2 left and right sides, adding inductor IPTG is 0.1mM to ultimate density, and low temperature is induced 20h for 20 ℃ then, can make the target protein expression amount the highest, and is mainly solubility expression (Fig. 8).The bacterial strain of choosing the solubility high expression level carries out bacterial classification and preserves and next-step operation.

Four, rhSCF-α protein purification

According to the optimal conditions of the expressing fusion protein of above-mentioned establishment, after the enlarged culturing, collect thalline; UW (100w; Ultrasonic 5s, 5s at interval) broken bacterium, centrifugal collection supernatant; Slough imidazoles through twice Ni-NTA affinity chromatography (the concrete grammar step is undertaken by the Ni-NTA of QIAGEN company resin affinity chromatography specification sheets) and Sephadex G-25 molecular sieve then, once specific SUMO proteolytic enzyme enzyme is cut and can be removed fusion tag.Concrete steps are following:

1, the purifying of SUMO-rhSCF-alpha fusion protein

(1) nickel sepharose FF dress post, 1.6 ' 20m, column volume are 20ml.

(2) with 2-5 bed volume of damping fluid balance, column volume is 20ml;

(3) with appearance on the SUMO-rhSCF-α albumen supernatant of aforementioned preparation, flow velocity is 1ml/min;

(4) with 2-5 bed volume of damping fluid flushing, flow velocity is 2ml/min;

(5) with the buffer solution elution foreign protein that contains the 20mM imidazoles, flow velocity is 2ml/min, and the detection wavelength is 280nm, and electrophoresis keeps sample;

(6) with the buffer solution elution SUMO-rhSCF-alpha fusion protein that contains the 200mM imidazoles, flow velocity is 2ml/min, and the detection wavelength is 280nm, collects elution peak;

(7) wash 5 column volumes with 0.5molNaOH and each stream of pure water, wash 3 column volumes with 20% ethanol stream again, flow velocity is 2ml/min, and pillar places 4-8 ℃ of environment to preserve;

(8) 12%SDS-PAGE detects the molecular weight size and the purity of SUMO-rhSCF-alpha fusion protein.The aminoacid sequence of SUMO-rhSCF-alpha fusion protein is shown in SEQ ID NO.2.

The aminoacid sequence of SUMO-rhSCF-alpha fusion protein (SEQ ID NO.2):

MHHHHHHGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGGGGSGGGGSGGGGSHMGARGAEREWEGGWGGAQEEEREREALMLKHLQEALGLPAGRGDENPAGTVEGKEDWEMEEDQGEEEEEEATPTPSSGPSPSPTPEDIVTYILGRLAGLDAGLHQLHVRLHALDTRVVELTQGLRQLRNAAGDTRDAVQALQEAQGRAEREHGRLEGCLKGLRLGHKCFLLSRDFEAQAAAQARCTARGGSLAQPADRQQMEALTRYLRAALAPYNWPVWLGVHDRRAEGLYLFENGQRVSFFAWHRSPRPELGAQPSASPHPLSPDQPNGGTLENCVAQASDDGSWWDHDCQRRLYYVCEFPF

2, SUMO-rhSCF-α N fusion rotein takes off imidazoles

(1) Sephadex G-25 dress post, 1.6 * 20cm, column volume are 20ml.

(2) with 2-5 bed volume of damping fluid balance, column volume is 20ml;

(3) with appearance on the SUMO-rhSCF-alpha fusion protein of 4.1 preparations, flow velocity is 3ml/min;

(4) use buffer solution elution, flow velocity is 3ml/min; The detection wavelength is 280nm, collects elution peak;

(5) wash 5 column volumes with 0.5molNaOH and each stream of pure water, wash 3 column volumes with 20% ethanol stream again, flow velocity is 2ml/min, and pillar places 4-8 ℃ of environment to preserve.

3, the enzyme of SUMO-rhSCF-α is cut

The SUMO-rhSCF-alpha fusion protein sample adjustment concentration that to take off imidazoles adds 1U SUMO proteolytic enzyme fusion rotein to 1mg/ml, and 30 ℃ of enzymes are cut 2h.Get enzyme and cut product and carry out Tricine-SDS-PAGE, whether check SUMO-rhSCF-α cuts (see figure 9).

4, enzyme is cut the repurity of the Ni-NTA post of product

All there is 6 * His label in SUMO-rhSCF-alpha fusion protein and SUMO enzyme front, and enzyme is cut product and removed SUMO-rhSCF-α, SUMO, SUMO enzyme through the Ni-NTA column purification once more, the rhSCF-α albumen that obtains recombinating, and concrete grammar is with 1.

5, RP-HPLC analyzes rhSCF-α purity of protein

(reverse-phase high-performance liquid chromatography RP-HPLC) detects the rhSCF-α purity of protein for preparing with RPLC.RhSCF-α albumen is after the desalination of Sephadex G-25 molecular sieve, and adjustment concentration is about 0.5mg/ml, gets 10 μ l on appearance extremely with the good C18 chromatographic column of 0.1% trifluoroacetic acid balance (4.6 * 250mm Zorbax, 300 SB-C18 column).Use the acetonitrile gradient wash-out of flow velocity as the 30-70% of 1ml/min.The purity of measuring recombinant protein is 98.3%.

Five, the proteic western blot of reorganization rhSCF-α test experience

This experiment adopts the DAB explicit representation to obtain western blot result; Shown in figure 10, there is a single band Western blot colour developing back about 45 KD, conform to SDS-PAGE result; Show that the albumen that this experiment is purified into is needed target protein rhSCF-α; Sequence shown in SEQ ID NO.3 (totally 305 aa, pI:5.06, Mw:33859.41Da).Concrete steps are following:

1. with the sampling carrying out of the rhSCF-α albumen behind purifying 12%SDS-PAGE.

2. commentaries on classics film: after electrophoresis is accomplished, downcut the target protein fragment, change film, electrophoresis 200mA 1h according to dying the Marker indication in advance;

3. sealing: film is taken out, after putting into pure water and washing, add confining liquid (skim-milk), shaking table 60r/min, 1h;

4. incubate one anti-: clean with pure water after outwelling confining liquid, add one anti-(rhSCF-α rabbit one anti-monoclonal antibody, dilution in 1: 3000) back in hybridization bag 4 ℃ spend the night;

5. incubating two resists: film is taken out from one resists, wash 3 times with PBST, (60r/min) shakes 10min on each shaking table, incubates for two anti-(mouse-anti people two resists, and dilutes at 1: 1000) then, in hybridization bag, shakes 1h (60r/min) then;

6. wash film: take out film, wash 2 times with PBST, (60r/min) shakes 5min on each shaking table, again with PBS wash 1 time (60r/min, 5min);

7. develop:

(1) with luminescent solution A liquid and each 400 μ l mixing of B liquid; With film submergence 2min wherein, to take out and load onto compressing tablet after wrap with preservative film the back and begin to develop, 20s makes public under the lucifuge; Take out film and put into developing solution 2min; Put into stop bath 2min again after film cleaned in water, take out and dry after in clear water, cleaning, take pictures;

(2) DAB development process (enhancement type HRP-DAB substrate colouring reagents box (Tiangen)): get 1 * HRP damping fluid 1ml, add reagent A, B, each 50 μ l mixing of C, under the lucifuge, film is submerged into 5min, take pictures.

RhSCF-α protein sequence (SEQ ID NO.3):

HMGARGAEREWEGGWGGAQEEEREREALMLKHLQEALGLPAGRGDENPAGTVEGKEDWEMEEDQGEEEEEEATPTPSSGPSPSPTPEDIVTYILGRLAGLDAGLHQLHVRLHALDTRVVELTQGLRQLRNAAGDTRDAVQALQEAQGRAEREHGRLEGCLKGLRLGHKCFLLSRDFEAQAAAQARCTARGGSLAQPADRQQMEALTRYLRAALAPYNWPVWLGVHDRRAEGLYLFENGQRVSFFAWHRSPRPELGAQPSASPHPLSPDQPNGGTLENCVAQASDDGSWWDHDCQRRLYYVCEFPF

Embodiment 2

Polyethyleneglycol modifiedization of human stem cell factor 1

1) PEG modifies: at pH=6.0; In the phosphoric acid buffer of 50mmol/L, rhSCF-α protein concentration was 5mg/mL, added the mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD-20kDa) of 20kDa by 2: 1 mass ratioes; 25 ℃ were reacted 12 hours, added the glycocoll termination reaction.

2) separation and purification: adopt Superdex 200 molecular sieve column chromatographies, with pH7.0, the 50mmol/L PB that contains 150mmol/L NaCl is a moving phase, collects second and washes the peak, obtains purity and be 92.4% mPEG-ALD-20kDa-rhSCF-a.

Embodiment 3

Polyethyleneglycol modifiedization of human stem cell factor 2

1) PEG modifies: at pH=6.0; In the phosphoric acid buffer of 50mmol/L, rhSCF-α protein concentration was 5.5mg/mL, added the mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD-20kDa) of 20kDa by 2: 1 mass ratioes; 4 ℃ were reacted 10 hours, added the glycocoll termination reaction.

2) separation and purification: adopt Superdex 200 molecular sieve column chromatographies, with pH7.0, the 50mmol/L PB that contains 150mmol/L NaCl is a moving phase, collects second and washes the peak, obtains purity and be 93.5% mPEG-ALD-20kDa-rhSCF-a.

Effect experiment

RhSCF-α albumen for preparing with embodiment 1 and embodiment 2 modify the mPEG-ALD-20kDa-rhSCF-a that obtains and carry out following effect experiment.

1. colony forms experimental identification rhSCF-α biological activity

Selecting the mouse monokaryon cell is that model carries out colony generation experiment, estimates rhSCGF-α albumen and the proteic promoting erythrocyte formation characteristic of PEG modificationization rhSCF-α.

Experimental procedure is following:

With Ficoll (1.083mg/ml, sigma) density gradient centrifugation separates adult BALA/c mouse bone marrow cells mononuclearcell, counting, the adjustment cell density is 2 * 10 ⁵/ ml, enchylema and isopyknic methylcellulose gum semisolid medium mixing carry out semisolid colonies and cultivate.Experiment is divided into rhSCF-α group, mPEG-rhSCF-α group and forms GM-Colonies with the collaborative BALA/c of stimulation of rmGM-CSF mouse monokaryon cell respectively.Each group is established 3 repetitions, places 37 ℃, 5%CO ₂, cultivate 7d under the saturated humidity condition, counting is counted 1 cell colony greater than 50 cells then, the experiment triplicate.Experimental result is seen Figure 11.

2. subcutaneous injection rhSCF-α and mPEG-rhSCF-α pharmacokinetic parameter

With the solution of the rhSCF-α of purifying preparation and mPEG-rhSCF-α with 5 groups of the amount subcutaneous injection SD rats (280-300g) of every 50ug, 5 every group, every injection volume 1mL.Injection back different time sections is got blood 0.3 mL from the mouse eye socket; Add 0.1mL at once, in the EDTA-2K anti-coagulant of 15mg/mL, centrifugal (5000r/min; 10min); Supernatant is in-70 ℃ of preservations, and with the content of rhSCF-α in the ELISA kit measurement blood plasma, experimental result is seen table 2 and Figure 12,13.

Table 2 subcutaneous injection hSCF-α or PEG-hSCF-α pharmacokinetic parameter

More than experiment shows, the polyethyleneglycol modifiedization human stem cell growth that is prepared by the inventive method can be used for preparing medicines such as the reconstruction of treating anaemia, chemicotherapy and bone marrow transplantation postoperative hemopoietic function and recovery, ex vivo expansion of stem cell, gene therapy; Also can be used for preparation promotes epidermic cell metabolism, repairs aging and makeup such as injured skin cell, delaying skin aging.

Above embodiment is merely and introduces preferred case of the present invention, and to those skilled in the art, any conspicuous variation and the improvement in the scope that does not deviate from spirit of the present invention, carried out all should be regarded as a part of the present invention.

< 110>Guangzhou Jinan Biological Medicine Research and Development Base Co., Ltd

< 120>working method of human stem cell growth and polyethyleneglycol modifiedization thereof product and purposes

<130>

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1275

<212> DNA

<213> SUMO- rhSCF-α

<400> 1

atgcatcatc atcatcatca cggcatgtcg gactcagaag tcaatcaaga agctaagcca 60

gaggtcaagc cagaagtcaa gcctgagact cacatcaatt taaaggtgtc cgatggatct 120

tcagagatct tcttcaagat caaaaagacc actcctttaa gaaggctgat ggaagcgttc 180

gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgg tattagaatt 240

caagctgatc agacccctga agatttggac atggaggata acgatatcat tgaggctcac 300

agagaacaga ttggtggtgg tggcggaggg agcggtggag ggggcagtgg cggaggaggt 360

agcggtgctc ggggagcaga gagggagtgg gagggaggct ggggaggtgc ccaggaggag 420

gagcgggaga gggaggccct gatgctgaag catctgcagg aagccctagg actgcctgct 480

gggagggggg atgagaatcc tgccggaact gttgagggaa aagaggactg ggagatggag 540

gaggaccagg gggaggaaga ggaggaggaa gcaacgccaa ccccatcctc cggccccagc 600

ccctctccca cccctgagga catcgtcact tacatcctgg gccgcctggc cggcctggac 660

gcaggcctgc accagctgca cgtccgtctg cacgcgttgg acacccgcgt ggtcgagctg 720

acccaggggc tgcggcagct gcggaacgcg gcaggcgaca cccgcgatgc cgtgcaagcc 780

ctgcaggagg cgcagggtcg cgccgagcgc gagcacggcc gcttggaggg ctgcctgaag 840

gggctgcgcc tgggccacaa gtgcttcctg ctctcgcgcg acttcgaagc tcaggcggcg 900

gcgcaggcgc ggtgcacggc gcggggcggg agcctggcgc agccggcaga ccgccagcag 960

atggaggcgc tcactcggta cctgcgcgcg gcgctcgctc cctacaactg gcccgtgtgg 1020

ctgggcgtgc acgatcggcg cgccgagggc ctctacctct tcgaaaacgg ccagcgcgtg 1080

tccttcttcg cctggcatcg ctcaccccgc cccgagctcg gcgcccagcc cagcgcctcg 1140

ccgcatccgc tcagcccgga ccagcccaac ggtggcacgc tcgagaactg cgtggcgcag 1200

gcctctgacg acggctcctg gtgggaccac gactgccagc ggcgtctcta ctacgtctgc 1260

gagttcccct tctag 1275

<210> 2

<211> 426

<212> PRT

<213> SUMO-rhSCF-α

<400> 2

Met His His His His His His Gly Met Ser Asp Ser Glu Val Asn Gln

1 5 10 15

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

20 25 30

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

35 40 45

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

50 55 60

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

65 70 75 80

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

85 90 95

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser His Met Gly Ala Arg Gly Ala

115 120 125

Glu Arg Glu Trp Glu Gly Gly Trp Gly Gly Ala Gln Glu Glu Glu Arg

130 135 140

Glu Arg Glu Ala Leu Met Leu Lys His Leu Gln Glu Ala Leu Gly Leu

145 150 155 160

Pro Ala Gly Arg Gly Asp Glu Asn Pro Ala Gly Thr Val Glu Gly Lys

165 170 175

Glu Asp Trp Glu Met Glu Glu Asp Gln Gly Glu Glu Glu Glu Glu Glu

180 185 190

Ala Thr Pro Thr Pro Ser Ser Gly Pro Ser Pro Ser Pro Thr Pro Glu

195 200 205

Asp Ile Val Thr Tyr Ile Leu Gly Arg Leu Ala Gly Leu Asp Ala Gly

210 215 220

Leu His Gln Leu His Val Arg Leu His Ala Leu Asp Thr Arg Val Val

225 230 235 240

Glu Leu Thr Gln Gly Leu Arg Gln Leu Arg Asn Ala Ala Gly Asp Thr

245 250 255

Arg Asp Ala Val Gln Ala Leu Gln Glu Ala Gln Gly Arg Ala Glu Arg

260 265 270

Glu His Gly Arg Leu Glu Gly Cys Leu Lys Gly Leu Arg Leu Gly His

275 280 285

Lys Cys Phe Leu Leu Ser Arg Asp Phe Glu Ala Gln Ala Ala Ala Gln

290 295 300

Ala Arg Cys Thr Ala Arg Gly Gly Ser Leu Ala Gln Pro Ala Asp Arg

305 310 315 320

Gln Gln Met Glu Ala Leu Thr Arg Tyr Leu Arg Ala Ala Leu Ala Pro

325 330 335

Tyr Asn Trp Pro Val Trp Leu Gly Val His Asp Arg Arg Ala Glu Gly

340 345 350

Leu Tyr Leu Phe Glu Asn Gly Gln Arg Val Ser Phe Phe Ala Trp His

355 360 365

Arg Ser Pro Arg Pro Glu Leu Gly Ala Gln Pro Ser Ala Ser Pro His

370 375 380

Pro Leu Ser Pro Asp Gln Pro Asn Gly Gly Thr Leu Glu Asn Cys Val

385 390 395 400

Ala Gln Ala Ser Asp Asp Gly Ser Trp Trp Asp His Asp Cys Gln Arg

405 410 415

Arg Leu Tyr Tyr Val Cys Glu Phe Pro Phe

420 425

<210> 3

<211> 305

<212> PRT

<213> rhSCF-α

<400> 3

His Met Gly Ala Arg Gly Ala Glu Arg Glu Trp Glu Gly Gly Trp Gly

1 5 10 15

Gly Ala Gln Glu Glu Glu Arg Glu Arg Glu Ala Leu Met Leu Lys His

20 25 30

Leu Gln Glu Ala Leu Gly Leu Pro Ala Gly Arg Gly Asp Glu Asn Pro

35 40 45

Ala Gly Thr Val Glu Gly Lys Glu Asp Trp Glu Met Glu Glu Asp Gln

50 55 60

Gly Glu Glu Glu Glu Glu Glu Ala Thr Pro Thr Pro Ser Ser Gly Pro

65 70 75 80

Ser Pro Ser Pro Thr Pro Glu Asp Ile Val Thr Tyr Ile Leu Gly Arg

85 90 95

Leu Ala Gly Leu Asp Ala Gly Leu His Gln Leu His Val Arg Leu His

100 105 110

Ala Leu Asp Thr Arg Val Val Glu Leu Thr Gln Gly Leu Arg Gln Leu

115 120 125

Arg Asn Ala Ala Gly Asp Thr Arg Asp Ala Val Gln Ala Leu Gln Glu

130 135 140

Ala Gln Gly Arg Ala Glu Arg Glu His Gly Arg Leu Glu Gly Cys Leu

145 150 155 160

Lys Gly Leu Arg Leu Gly His Lys Cys Phe Leu Leu Ser Arg Asp Phe

165 170 175

Glu Ala Gln Ala Ala Ala Gln Ala Arg Cys Thr Ala Arg Gly Gly Ser

180 185 190

Leu Ala Gln Pro Ala Asp Arg Gln Gln Met Glu Ala Leu Thr Arg Tyr

195 200 205

Leu Arg Ala Ala Leu Ala Pro Tyr Asn Trp Pro Val Trp Leu Gly Val

210 215 220

His Asp Arg Arg Ala Glu Gly Leu Tyr Leu Phe Glu Asn Gly Gln Arg

225 230 235 240

Val Ser Phe Phe Ala Trp His Arg Ser Pro Arg Pro Glu Leu Gly Ala

245 250 255

Gln Pro Ser Ala Ser Pro His Pro Leu Ser Pro Asp Gln Pro Asn Gly

260 265 270

Gly Thr Leu Glu Asn Cys Val Ala Gln Ala Ser Asp Asp Gly Ser Trp

275 280 285

Trp Asp His Asp Cys Gln Arg Arg Leu Tyr Tyr Val Cys Glu Phe Pro

290 295 300

Phe

305

<210> 4

<211> 41

<212> DNA

< 213>artificial primer

<400> 4

ggcagtggcg gaggaggtag cggtgctcgg ggagcagaga g 41

<210> 5

<211> 33

<212> DNA

< 213>artificial primer

<400> 5

agaggatccc tagaagggga actcgcagac gta 33

<210> 6

<211> 22

<212> DNA

< 213>artificial primer

<400> 6

cagcatatgc atcatcatca tc 22

<210> 7

<211> 40

<212> DNA

< 213>artificial primer

<400> 7

cctccaccgc tccctccgcc accaccacca atctgttctc 40

Claims (10)

1. fusion gene, its coding SUMO-rhSCF-alpha fusion protein.

2. fusion gene according to claim 1, it has the nucleotide sequence shown in the SEQ ID NO.1.

3. the expression vector that contains claim 1 or 2 said fusion genes.

4. the working method of human stem cell growth comprises the steps:

1) expression vector with claim 3 changes in the host bacterium, abduction delivering SUMO-rhSCF-a fusion rotein;

2) separation and purification SUMO-rhSCF-a fusion rotein, excision SUMO part, purifying rhSCF-α albumen.

5. according to the resulting reorganization of the method for claim 4 rhSCF-α albumen.

6. the preparation method of a polyethyleneglycol modifiedization human stem cell growth comprises the steps:

1) claim 5 described rhSCF-α albumen and mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD) were reacted 10 ~ 14 hours at 24 ~ 26 ℃ or 3 ~ 5 ℃, add the glycocoll termination reaction;

2) separation and purification obtains polyethyleneglycol modified human stem cell growth.

7. preparation method according to claim 6 is characterized in that, step 1) is in the phosphate buffered saline buffer of pH 6.0,50 mmol/ L, to carry out, and the proteic concentration of said rhSCF-α is 4.5 ~ 5.5mg/mL.

8. method according to claim 6 is characterized in that, the mass ratio of said rhSCF-α albumen and mono methoxy polyethylene glycol propionic aldehyde is 2 ~ 3:1.

9. according to claim 6 or 8 described methods, it is characterized in that said mono methoxy polyethylene glycol propionic aldehyde is a 20kDa mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD-20kDa).

10. the polyethyleneglycol modifiedization human stem cell growth for preparing of each method of claim 6 ~ 9 is at the reconstruction of preparation treatment anaemia, chemicotherapy and bone marrow transplantation postoperative hemopoietic function and recovery, ex vivo expansion of stem cell, gene therapy medicament and promoting epidermic cell metabolism, repairing the application aspect aging and the makeup such as injured skin cell, delaying skin aging.

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