CN100494213C - Process for preparing synergistic factor in artificial blood, nucleotide sequence used thereby - Google Patents

Abstract

The invention relates to biological pharmaceutical field, and specifically to a preparation method for producing artificial oxygen carrier synergic factors. The invention also relates to specific nucleotide sequence used in the method.

Description

A kind of preparation method of artificial blood synergistic factor and used nucleotide sequence produced

Technical field

The present invention relates to biomedicine field.Particularly, the present invention relates to a kind of preparation method who produces the artificial blood synergistic factor, the invention still further relates to the special nucleotide sequence that uses in the described method.

Background technology

Hematopoietic stem cell transplantation can be divided into by the hemopoietic stem cell source: bone marrow transplantation (BMT), peripheral blood hematopoietic stem cells transplant (PBSCT) and umbilical hemopoietic stem cell is transplanted (CBSCT).PBSCT can be divided into from body (Auto-PBSCT) and recessive allele PBSCT (Allo-PBSCT).Over past ten years, PBSCT is owing to than BMT following advantage being arranged and being subject to people's attention day by day: it is rapid that (1) transplants the back hematopoietic function recovery, reduced complication and hospital stayss such as infection: (2) radiosensitivity is low, implantation rate height in vivo: (3) gather convenient, avoid anesthesia and operation, reduce the spending of patient's funds: (4) increase T cell and NK cell quantity in the graft, and immunologic function is recovered rapidly: (5) are accepted can gather PBSC when reason such as radiotherapy can't be gathered marrow to tumor cell invasion, fibrosis or pelvis part are arranged in the marrow.Based on these advantages, PBSC has replacement marrow just to a great extent as the trend from body and the main source of human stem cell of heteroplastic transplantation in recent years.But the speed dependent of hematopoietic function recovery is in the quantity of the hematopoietic stem of implanting (HSC/HPC) after the hematopoietic stem cell transplantation.Normal people's peripheral blood CD34 ⁺Cell only accounts for 0.01-0.1% of mononuclearcell, and guarantees the CD34 of implantation ⁺The cell minimum is 1 X 10 ⁶/ Kg.Collection enough is used for the hemopoietic stem cell number of Auto-PBSCT or Allo-PBSCT from normal people or patient's peripheral blood, need rise the blood volume can obtain through repeatedly concentrating surplus several liters even 10.Repeatedly blood sampling is not only all very unfavorable to medical worker and patient, and spending is expensive.The scheme of mainly uniting use at present clinically with lists such as hematopoietic cytokine such as G-CSF, SCF, GM-CSF usefulness or several cytokine, or chemotherapy drugs in combination cytokine scheme is mobilized peripheral hematopoietic stem cells.But G-CSF, SCF and GM-CSF need administration in many days (4-10 days), still need repeatedly to gather, and the expense height is difficult to predict the optimal acquisition time, and individual difference is bigger clinically, and can not effectively mobilize some individuality.Therefore, seek a kind of a large amount of hemopoietic stem cells that exist in the marrow that make and effectively mobilize preparation or scheme in the peripheral blood fast, carry effectively that the content of hemopoietic stem cell reduces times of collection in the peripheral blood, improve collecting efficiency, have important clinical significance.

Chemokine draws attention day by day to the mobilized effects of hematopoietic stem in recent years.Chemokine (Chemokines) is one group white corpuscle had the secretor type single chain protein matter of chemotaxis, and molecular weight is 8-12KD, contains two pairs of disulfide linkage, and alkalescence can heparin-binding.GRO (growth related oncogene) family is that a class belongs to ELR ⁺The chemokine of CXC family comprises GRO α, GRO β, GRO γ.The GRO assignment of genes gene mapping is made up of 4 exons and 3 introns at 4q12-13.Total length GRO β (1-73) contains 73 amino acid, and main biological characteristics is chemotactic, activation neutrophil leucocyte.Nineteen ninety-five King etc. find that the varient GRO β-T or the GRO β (5-73) of GRO β N-end 4 amino acid of disappearance (APLA) are a kind of novel chemokine, and it is made up of 69aa, and molecular weight is 7550, and theoretical iso-electric point is 9.75.

The main biological function of recent findings GRO β-T is except that having than total length GRO β stronger chemotactic, mobilization and the activation neutrophil leucocyte usefulness, also have quick Effects of Bone Marrow Stem Cells Mobilization and enter usefulness and very strong hematopoiesis synergistic activity (promptly stimulating CFU-GM propagation) in the peripheral blood, so with its called after hematopoietic synergistic factor (hematopoietic synergistic factor, be called for short HSF), and this activity of total length GRO β is very low.U.S. Smith Ke Laisi Beecham (Smithkline Beecham Corporation) scientific research personnel AG.King etc. regulates peptide SK﹠amp in research hematopoiesis; Find HSF in the F107647 compound function course, they pass through SK﹠amp; F107647 stimulates people's TF274 marrow stromal cell and mouse C6 marrow stromal cell, utilizes heparin column and anti-GRO β affinity column to isolate people HSF and mouse HSF respectively.Clone coding people HSF and GRO β gene with the RT-PCR method simultaneously, subclone advances on the prokaryotic expression plasmid pEAKn, and obtains recombinant human HSF expression through chemical induction in E.coli.They are for the recombinant human HSF that obtains terminal the no initial sub-methionine(Met) of N-(Met) and be convenient to purifying, contain 12 aa sequence labels (DET-1) and enteropeptidase cleavage site, i.e. DET-1-DDDDK at people HSF and one of GRO β N end introducing.Fusion rotein DET-1-DDDDK-HSF (or DET-1-DDDDK-GRO β) in E.coli almost equivalent express with inclusion body and soluble form.Through renaturation → anti-DET-1 monoclonal antibody affinity chromatography post separation → enteropeptidase cutting → affinity post separation → RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC) is separated once more, obtains the pure product of recombinant human HSF at last.This technology is complicated, the cost height.Therefore exist the needs of simplifying people HSF preparation technology method.

Summary of the invention

Therefore, based on above-mentioned needs, one aspect of the present invention relates to the polynucleotide sequence shown in SEQ ID NO.:1.Described polynucleotide sequence is in 69 codons of above-mentioned coding people HSF gene, have 34 and replace with that people intestinal bacteria preference codon obtains, they are the 4th, 5,7,8,13,14,16,17,20,21,22,23,24,25,27,28,29,32,33,36,37,38,40,41,42,43,48,50,51,52,53,56,61,65.

The invention still further relates to the carrier that contains described polynucleotide sequence.

The invention still further relates to the positive expression plasmid that contains described carrier.

The invention further relates to hematopoietic synergistic factor by described positive expression plasmid expression.The described factor is mainly with the formal representation of inclusion body.

The invention further relates to a kind of method of producing the artificial blood synergistic factor, it may further comprise the steps:

1) the polynucleotide sequence subclone that contains the hematopoietic synergistic factor of encoding is to expression vector, and screening contains the clone of positive expression plasmid;

2) described positive expression plasmid is changed in the intestinal bacteria, abduction delivering is with the hematopoietic synergistic factor of inclusion body formal representation;

3) with described inclusion body sex change cracking, separate through Sephacryl S-100, reclaim target protein;

4) after the target protein renaturation, separate with SP-SepharoseFF;

The polynucleotide sequence that it is characterized in that described coding hematopoietic synergistic factor is the polynucleotide sequence shown in the SEQ IDNO.:1.

The invention further relates to described hematopoietic synergistic factor and be used for preparing the purposes that mobilizing hematopoietic stem cells enters the medicine of peripheral blood.

Description of drawings

Fig. 1 shows the PCR product agarose gel electrophoresis result that primer amplification is later.

Fig. 2 shows the structure of expression plasmid pET30a-hHSF.

Fig. 3 shows that the enzyme of positive colony pET30a-hHSF cuts the evaluation collection of illustrative plates.

Fig. 4 shows the SDS-PAGE protein electrophoresis collection of illustrative plates of pET30a-hHSF/BL21 (DE3) abduction delivering different time.

Fig. 5 shows the separation graphic representation of Sephacryl S-100 purifying rhHSF.

Fig. 6 shows the separation graphic representation of SP Sepharose purifying rhHSF.

Fig. 7 shows the SDS-PAGE collection of illustrative plates of the rhHSF of purifying.

Fig. 8 shows the SDS-PAGE UV scanning figure of the rhHSF of purifying.

Fig. 9 shows the molecular weight of the rhHSF that SDS-PAGE measures.

Figure 10 shows the rhHSF molecular weight that MALDI-TOF-MS measures.

Figure 11 shows rhHSF amino acid composition analysis collection of illustrative plates.

Figure 12 shows the absorption spectrum curve of rhHSF.

Preferred forms of the present invention

The method that following reference example and experimental example are described polynucleotide of the present invention and the described HSF of preparation of the present invention in detail its objective is further illustration the present invention, rather than limits its scope.

Embodiment 1

The acquisition of hHSF gene

The present invention will encode, and ' end is introduced the Ndel restriction enzyme site for the dna fragmentation 5 of hHSF, 3 ' end is introduced the BamHI restriction enzyme site, be connected to expression plasmid pET30a (available from Invitrogene, the U.S.) between NdeI and the BamHI restriction enzyme site, structure is the expression plasmid of initiator codon with ATG, obtain the polynucleotide sequence shown in SEQ ID NO.:1, it is in 69 codons of coding people HSF gene, have 34 and replace with that the intestinal bacteria preference codon obtains, they are the 4th, 5,7,8,13,14,16,17,20,21,22,23,24,25,27,28,29,32,33,36,37,38,40,41,42,43,48,50,51,52,53,56,61 and 65.Its concrete replacement sees Table 1.

Table 1

No	aa	Codo n	optimal	No	aa	Codon	optimal	No	aa	Codon	optimal
												1	T	ACT		25	K	AAG	AAA	49	N	AAC
2	E	GAA		26	S	TCC		50	P	CCC	CCG
												3	L	CTG		27	P	CCC	CCG	51	A	GCA	GCT
4	R	CGC	CGT	28	G	GGA	GGT	52	S	TCG	TCC
												5	C	TGC	TGT	29	P	CCC	CCG	53	P	CCC	CCG
6	Q	CAG		30	H	CAC		54	M	ATG
												7	C	TGC	TGT	31	C	TGC		55	V	GTT
8	L	TTG	CTG	32	A	GCC	GCT	56	K	AAG	AAA
												9	Q	CAG		33	Q	CAA	CAG	57	K	AAA
10	T	ACC		34	T	ACC		58	I	ATC
												11	L	CTG		35	E	GAA		59	I	ATC
12	Q	CAG		36	V	GTC	GTT	60	E	GAA
												13	G	GGA	GGT	37	I	ATA	ATC	61	K	AAG	AAA
14	I	ATT	ATC	38	A	GCC	GCT	62	M	ATG
												15	H	CAC		39	T	ACA		63	L	CTG
16	L	CTC	CTG	40	L	CTC	CTG	64	K	AAA
												17	K	AAG	AAA	41	K	AAG	AAA	65	N	AAT	AAC
18	N	AAC		42	N	AAT	AAC	66	G	GGC
												19	I	ATC		43	G	GGG	GGT	67	K	AAA
20	Q	CAA	CAG	44	Q	CAG		68	S	TCC
												21	S	AGT	TCC	45	K	AAA		69	N	AAC
22	V	GTG	GTT	46	A	GCT		70		TGA	TAA
												23	K	AAG	AAA	47	C	TGT
24	V	GTG	GTT	48	L	CTC	CTG

One. the extraction of plasmid DNA

1. a small amount of of plasmid DNA is extracted (alkaline lysis)

From the bacterium liquid of 3ml incubated overnight, get 1.5ml and pour in the Eppendoff pipe, the centrifugal 1min of 7000rpm, supernatant discarded.Precipitation adds the Solution I (25mMTrisHCl, pH8.0,10mMEDTA, the glucose of 50mM) of 100 μ L, and vibration suspends, mixing.(0.2M NaOH, 1%SDS), mixing is placed 5min on ice to add the Solution II of 200 μ L.(the 3M Potassium ethanoate, pH4.8), mixing is placed 10min on ice to add the Solution III of 150 μ L.The centrifugal 10min of 15000rpm, supernatant change among another Eppendoff, add the 1ml dehydrated alcohol, and room temperature is placed 30min.15000rpm is centrifugal, and 10min abandons supernatant.Precipitation adds 0.5ml75% ethanol washes once, the centrifugal 5min of 15000rpm, and the precipitation room temperature is placed dry.It is standby to add among the 30 μ L TE contain 20 μ g/ml RNase A (available from magnificent biotech company) dissolving.

2. a large amount of preparations (alkaline lysis) of plasmid DNA

The bacterium liquid of 50ml incubated overnight is put into the 50ml centrifuge tube, and 4 ℃, the centrifugal 5min of 7000rpm abandons supernatant, and thalline adds 6ml Solution I, the vibration mixing.Add 9ml SolutionII, turn upside down for several times mixing, ice bath 5min gently.Add 9ml Solution III, the mixing that turns upside down, ice bath 10min.4 ℃, the centrifugal 15min of 15000rpm, supernatant change another 50ml centrifuge tube over to.Add isopyknic Virahol (Beijing Chemical Plant), mixing, room temperature is placed 10min, the centrifugal 15min of 14000rpm room temperature.Precipitation adds 75% ethanol 1ml, places a moment, and precipitation changes in the 1.5ml Eppendoff pipe after suspending.The centrifugal 2min of 15000rpm, supernatant discarded.After the precipitation vacuum is drained, add and contain among the 500 μ L TE of 20 μ g/ml Rnase A 37 ℃ of enzymolysis RNA 1hr.With phenol/chloroform (Beijing Chemical Plant) extracting twice of isopyknic 1:1, twice of chloroform extracting.The 3mol/L NaAc (pH4.6) that adds 50 μ L, the 1ml dehydrated alcohol, mixing is placed 10min for-20 ℃, the centrifugal 10min of 15000rpm, precipitation 75% washing with alcohol, the centrifugal 2min of 15000rpm, abandon supernatant, twice, precipitation vacuum-drying, with the dissolving of 480 μ L sterilized waters, add 4mol/L NaCl 120 μ L and 13%PEG 600 μ L, mixing, ice bath 40min, 4 ℃, the centrifugal 10min of 15000rpm, abandon supernatant, precipitation washes twice with 75% ethanol, vacuum-drying.It is standby to add 400 μ L TE dissolving.

Two .DNA reorganization

1.PCR amplification

Below be described PCR primer:

P1 (38mers): 5 ' introduces the NdeI restriction enzyme site

5’CC CAT?ATG?ACT?GAA?CTG?CGT?TGT?CAG?TGT?CTG?CAG?ACC3’

P2(58mers)：

5’T?AAC?TTT?AAC?GGA?CTG?GAT?GTT?TTT?CAG?GTG?GAT?ACC?CTG?CAG?GGT

CTG?CAG?ACA?CTG3’

P3(58mers)：

5’AG?TCC?GTT?AAA?GTT?AAA?TCC?CCG?GGT?CCG?CAC?TGC?GCT?CAG?ACC

GAA?GTT?ATC?GCT?AC3’

P4(56mers)：

5’GA?AGC?CGG?GTT?CAG?ACA?AGC?TTT?CTG?ACC?GTT?TTT?CAG?GGT?AGC?GAT

AAC?TTC?GGT3’

P5(58mers)：

5’TCTG?AAC?CCG?GCT?TCC?CCG?ATG?GTT?AAA?AAA?ATC?ATC?GAA?AAA?ATG

CTG?AAA?AAC?GGC3’

P6 (38mers): introduce the BamHI restriction enzyme site

5’CG GGA?TCC?TTA?TTA?GTT?GGA?TTT?GCC?GTT?TTT?CAG?CAT3’

P1, P2, P3, P4, P5, P6 are made into 50 μ mol/L concentration.The first round is carried out two fragment assemblies (P1 and P2, P3 and P4, P5 and P6 successively).(P1, P2 are template with P1 and P2, be again primer) respectively get 1 μ L, add in the reaction system of 50 μ L and (include 2.5mmol/L dNTP (Promega company product) 8 μ L, 10x buffer 5 μ L, Taq enzyme 1-2U can add an amount of magnesium ion in case of necessity, supplies volume with the disinfectant redistilled water), the surface adds one deck mineral oil, puts into the PCR instrument.P3 and P4, P5 and P6 also so handle, and set response procedures according to the situation of template and primer, and described response procedures is the known technology of those of ordinary skills, amplifies P1-2, P3-4, P5-6 respectively.Again P1-2 and P3-4 respectively being got 1 μ L is template, adds each 0.5 μ mol/L of P1, P4 respectively and carries out second and take turns PCR, and 4 fragment assembly product P 1-4 increase.Then P1-4, P5-6PCR product respectively being got 1uL is template, adds each 0.5 μ mol/L of P1, P6 respectively and carries out third round PCR, and 6 fragment assembly product P 1-6 increase.The PCR product is seen Fig. 1.

2.DNA agarose electrophoresis

Take by weighing an amount of agarose, add the 0.5xTBE of respective amount, be made into corresponding concentration, melt postcooling to 40-50 ℃, add a small amount of EB, pour in the electrophoresis chamber that is inserted with comb, treat thoroughly to solidify, transfer to comb, sample mixes with sample loading buffer, adds well, connects power supply, 50-80V electrophoresis.

1) recovery of DNA

Downcut separated DNA fragment band in the agarose electrophoresis under ultraviolet lamp, the dna fragmentation gel recovery test kit of producing with the precious biotech firm in Dalian reclaims.

2) alcohol precipitation of DNA

The solution that contains target DNA adds the 3mol/LNaAc (pH4.6) of 10% volume, and the dehydrated alcohol deposit D NA that diploid is long-pending is centrifugal, will precipitate and use 75% washing with alcohol more once, and low temperature is drained, and it is standby to be dissolved in TE.

3) enzyme of DNA is cut

The reaction system of 50 μ L contains plasmid 3～5 μ g, 10 x buffer, 5 μ L, NdeI and BamHI restriction enzyme 5-10U (Dalian precious biotech firm), add water to volume required, 37 ℃ of insulation 3～4hr.

4) connection of DNA

The reaction system of 20 μ L contains 10x buffer 2 μ L, dna ligase 1U, and linear carrier and insertion target DNA fragment mole ratio are generally 1:4, mixing, 4 ℃ spend the night (16hr).

3. the preparation of competence bacteria

Intestinal bacteria are inoculated on the LB culture medium flat plate rule 37 ℃ of overnight incubation.Choose single colony inoculation in 3mlLB, cultivate 16hr for 37 ℃.Get the 2.5ml culture and be connected among the 200ml LB, cultivate 2～3hr for 37 ℃, to OD ₅₅₀=0.55～0.65, with the centrifugal 10min of 4000rpm, collect thalline, be resuspended in the 0.1mol/L MgCl of 1/2 volume precooling ₂In, the centrifugal 5min of 4000rpm collects the 0.1mol/L CaCl that thalline is suspended in 1/2 volume precooling ₂In, ice bath 30min, the centrifugal 5min of 4000rpm, thalline are resuspended in 15-20% glycerine-0.1mol/LCaCl of 5-6ml ₂In, 200 μ L/ manage packing ,-50 ℃ of preservations.

4. plasmid transforms

Get 100 μ L competence bacterias, add an amount of plasmid DNA or connect product, mixing gently, ice bath 40min, 42 ℃ of heat-shockeds 90 seconds, ice bath 2min adds 0.6ml LB substratum, 37 ℃ of shaking culture 1.5hr, get an amount of thalline, be applied to and contain on the antibiotic flat board, 37 ℃ of overnight incubation.

5. colony screening and evaluation

1) blue hickie screening

The T carrier is connected product transform DH5 α competence bacteria.The LB substratum that will contain 1.5% agarose dissolves with microwave oven, be cooled to and add penbritin (final concentration is 100 μ g/ml) about 60 ℃, pour flat board into, wait to solidify the back and coat 20mg/mlX-gal 16 μ L and 100mmol/L IPTG 4 μ L on its surface, place 1～2hr for 37 ℃, get converted product 200 μ L coated plates, 37 ℃ are spent the night.Blue hickie appears in the bacterium colony that grows, and can place 1-2hr so that it is clearer at 4 ℃ in case of necessity.Choose hickie and identify positive colony.Positive colony pET30a-hHSF restriction enzyme mapping is seen Fig. 3.

The structure of concrete pET30a-hHSF is seen Fig. 2.

2) restriction enzyme is identified

With the plasmid DNA of a small amount of alkaline lysis method of extracting, select suitable endonuclease digestion for use, the agarose electrophoresis of proper concn is identified, determines whether restriction enzyme site coincide, and whether clip size is correct.

3) PCR method is identified

Plasmid DNA with a small amount of alkaline lysis method of extracting is a template, carries out PCR with primer P1 and P6, and the agarose electrophoresis of proper concn identifies determined whether special district band, whether clip size is correct.

6. bacterial classification is preserved

Bacterial classification is rule on the LB flat board, grows single bacterium colony next day, choose a single bacterium colony to 3ml LB substratum,

37 ℃ of shaking culture 12-16hr, 1:100 is seeded in another fresh LB liquid nutrient medium in proportion, 37 ℃ of shaking culture 12-16hr, 1:100 is seeded in another fresh LB liquid nutrient medium in proportion, and 37 ℃ of shaking culture are to OD ₆₀₀=0.4-0.6 adds glycerine to final concentration 15%～20% ,-50 ℃ of preservations.

Three. expression and the purifying of recombinant protein in E.coli

1. the abduction delivering of recombinant protein

1) a small amount of abduction delivering of recombinant protein

Choose the mono-clonal bacterium colony in the 3ml substratum that contains kantlex (final concentration 50 μ g/ml), incubated overnight is inoculated into another by 1% and manages in the 3ml substratum, is cultured to OD ₆₀₀Be about 0.4-0.6, (IPTG Promega), continues to cultivate about 4hr, collects thalline, and SDS-PAGE identifies Recombinant Protein Expression to add the inductor isopropylthio-.Its expression of results is seen Fig. 4.

2) a large amount of abduction deliverings of recombinant protein

Choose single colony inoculation in 3ml LB substratum, cultivate 12hr for 37 ℃, be inoculated in the 100ml LB substratum by 0.5%, 37 ℃ of 250rpm incubated overnight, be inoculated in 250ml high density broth culture by 4%, 37 ℃ are cultured to logarithmic phase, add inductor 0.5mmol/L IPTG abduction delivering 4～5hr.Collect thalline, ultrasonication, separation and purification recombinant protein.

2. the purifying of recombinant protein

1) ultrasonication of thalline

Thalline is with solution A (50mM Tris-HCl, pH8.0, the 1%Tritonx-100 of 10 times of volumes, 10mM EDTA) suspends, 4 ℃ of ultrasonication thalline, 360W, each 20 seconds working hours, 40 seconds intermittent times, 12 times, 12000rpm is centrifugal, collects respectively and goes up cleer and peaceful precipitation, will precipitate part and repeat above-mentioned steps twice, the SDS-PAGE electrophoresis is identified the existence form (be present in supernatant be soluble form, be present in precipitation be inclusion body form) of recombinant protein in thalline.

2) washing of inclusion body

To precipitate the part add isopyknic inclusion body washings B (50mM Tris-HCl, pH8.0), mixing, 4 ℃ of centrifugal 10min of 12000rpm must precipitate, and repeat once.

3) dissolving of inclusion body

Inclusion body after the washing adds inclusion body lysate (50mM Tris-HCl, 4M Guanidinium hydrochloride, 2M urea, 150mM NaCl, 200mM beta-mercaptoethanol) 40ml/L inoculum, and dissolving is spent the night, 14000rpm, and 4 ℃ of centrifugal 30min collect supernatant.SDS-PAGE identifies inclusion body purity.

4) the solubilization of inclusion bodies thing is crossed 2 times of column volume balance liquid equilibrated Sephacryl S-100 (Pharmacia Corp, the U.S.).The separation curve that obtains is seen Fig. 5.

5) renaturation of inclusion body

Under ice bath and stirring state, will dropwise add the renaturation solution from the recombinant protein of Sephacryl S-100 separated and collected, behind 4 ℃ of renaturation 48hr, A fully dialyses with dialyzate, changes liquid 7-8 time, each dialysis liquid measure is at least 10 times of dialysis sample volume, dialysis 48hr.

6) cross SP Sepharose post: SP Sepharose post on the dialyzed sample is separated, earlier with elutriant A wash-out, again with elutriant B wash-out.

7) the sample isolating target protein of SP Sepharose post of dialysing with dialyzate B.It separates curve and sees Fig. 6.

8) leave and take small amount of sample and be used for physico-chemical property and identify, add 1% human albumin in all the other samples, Sterile Filtration behind the mixing, it is standby to put-20 ℃ of preservations.

9) processing of resin

(1) processing of CM-Sepharose FF

With the 2mol/L NaCl washing of 2 times of column volumes, flow velocity is fast earlier for the exhausted resin.0.5mol/L NaOH-1.5mol/L NaCl with 2 times of volumes handles 20-40min then.Be washed with water to about pH7.0, standby with 3-5 column volume of sample-loading buffer balance.Short-term is preserved and is added small amount of N aN ₃, prolonged preservation is in 20% ethanol.

(2) processing of SP-Sepharose High Performance

The exhausted resin with the 2.0mol/L NaCl washing of 1 times of column volume, is handled 10-15min earlier, handles 0.5-1h with 1.0mol/L NaOH then, is washed with water to about pH7.0, and is standby with at least 3 column volumes of sample-loading buffer balance.Prolonged preservation is in 4 ℃ of 20% ethanol-0.2mol/L sodium acetate.

4. the evaluation of recombinant protein

1) SDS-PAGE protein electrophoresis

The according to the form below preparation concentrates glue and separation gel (unit/ml)

Table 2

	H 2O	30％Acr- Bis	1.5mol/L?Tris -HCl?Ph8.8	10％SDS	10％APS	TEMED
							5% concentrates glue	4.6	1	2*	0.08	0.07	0.008
15% separation gel	4.7	10	5.0	0.2	0.09	0.008
							18% separation gel	2.7	12	5.0	0.2	0.09	0.008

The upper strata pours into concentrated glue after the glue polymerization to be separated, extracts comb after the polymerization fully, the now good sample of adding place in well, and deposition condition is constant current 20mA.Add an amount of sample-loading buffer during sample preparation, boil 10min.

2) determining the protein quantity

SDS-PAGE-dyeing-colorimetry: the rhSCF with accurate amount is a standard, to gel-colored, compares the ultraviolet absorption value of a certain amount of rhSCF and rhHSF through the gel UV scanning with Xylene Brilliant Cyanine G R-250, extrapolates rhHSF content.

The purity of the target protein of gained is identified and is seen Fig. 7.

Make inclusion body be dissolved in that purity is 75% after the denaturing agent, through the SP-Sepharose purifying and after filtering, non-reduced type rhHSF purity is 97%, sees Fig. 8.

Four. the recombinant protein physical and chemical property determining

1. molecular weight determination

1) SDS-PAGE method

Preparation 18% SDS-polyacrylamide denaturant gel carries out SDS-PAGE to the rhHSF of purifying and analyzes, and measures migration distance.With the proteinic migration distance of the different molecular weight of small molecular weight protein marker is X-coordinate, with its molecular weight the numerical digit ordinate zou is mapped, draws the typical curve that concerns between a proteinic molecular weight and its migration distance.According to the migration distance of rhHSF, from typical curve, find corresponding molecular weight values, see Fig. 9.

2) measure with the flight time mass spectrum (MALDI-TOF-MS) of ABI company by centralab of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, see Figure 10.

2. amino acid sequence analysis

Measure with the Applied Biosystems Procise of ABI company by centralab of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.The result shows that terminal 10 amino acid of N-are TELRCQCLQT.

3. amino acid composition analysis

Measure with Waters company 2690 pumps, 996 diode-array detectors, hydrolysis amino acid analytical column AccQ.TagrM by centralab of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, see Figure 11.

4. absorption spectrometry

Separate rhHSF with the C18RP-HPLC of centralab of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with Waters HPLC-UVmodel 996

Diode-array detector detects the absorption spectrum curve of rhHSF in wavelength 210nm-300nm scope, see Figure 12.

Five .rhHSF are to the quick mobilized effects of rhesus monkey peripheral blood hematopoietic ancestral cells

This experiment is helped through by Chinese Radiation Medical Science Inst., Chinese Academy of Military Medical Science (CN director Luo Qingliang laboratory, in the rhesus monkey body, observed rhHSF separately or with the influence of rhG-CSF combined utilization to peripheral blood CFU-GM number, peripheral blood CD34+ cell number and peripheral hemogram.

1. laboratory animal grouping

Eight rhesus monkey body weight 4.2 ± 0.33 (3.6-4.5) kg, fed back inspection animal physique stalwartness, no wound deformity and body surface focus of infection, body temperature and blood picture through 10 days normal, animal quality conformity certification number: BDW95002.Animal carries out external wind in buffer compartment and drenches cleaning, solitary the raising in cages in enteron aisle sterilising treatment back layer fluid room in the room.The room temp in laminar flow room is 22 ± 2 ℃, and relative humidity is 70%, fluorescent lamp control diel rhythm.Every door bolt gives calibrated bolck material, apple, corn distiller's dried grain, Semen arachidis hypogaeae etc., and cold water is put the cage side and freely drunk, animal breeding plant conformity certification number: B98020.

Experiment is divided into rhHSF and two groups of rhG-CSF+rhHSF (limited because of size of animal, as not establish not administration control group).

2. medication

The subcutaneous injection 10 μ g/kg rhG-CSF of rhG-CSF+rhHSF treated animal elder generation, once a day, successive administration 4 days, the 5th day two treated animals while single subcutaneous injection 500 μ g/kgrhHSF (stoste 0.3mg/ml).

3. observation index

1) peripheral hemogram (WBC, RBC, HGB, HCT, PLT, SCC, LCC)

Detect with the F-820 blood-counter system

2) the semi-solid peripheral blood CFU-GM of agar cultivates

3) detect peripheral blood CD34+ cell count with flow cytometer.

4) body temperature and general situation

See Table 2 the detection time that each treated animal detects index.

Detect index and testing time after table 2 rhHSF and the rh-CSF+rhHSF administration

4.PBMC separation

3ml anticoagulant heparin peripheral blood is slowly added in the 5ml lymphocyte separation medium with the long-pending RPMI-1640 cell culture fluid dilution back of isoploid, and the centrifugal 20min of 2000rpm draws the intermediate cell layer, carries out cell counting with the F-820 automatic hematology analyzer.

5. the cultivation of peripheral blood CFU-GM

The CFU-GM culture system sees Table 2.Each sample is put 37 ℃ of 5%CO with 3 culture dish ₂Cultivating counting colony number after 14 days in the incubator, is a colony with the cell mass that contains 50 above cells.

6. detect peripheral blood CD34+ cell

Mouse anti human CD34 monoclonal antibody and control antibodies and peripheral blood are hatched behind the 30min with erythrocyte cracked liquid splitting erythrocyte 15min, the centrifugal 5min of 1500rpm, supernatant is removed in suction, with cell precipitation with the PBS washed twice, with the 1mlPBS suspension cell, detect peripheral blood CD34+ cell behind the centrifugal 5min of 1500rpm with the FACSCalibur flow cytometer.

The rhHSF that experimental results show that purifying in the rhesus monkey body can mobilize CD34+ hematopoietic stem, CFU-GM and neutrophil leucocyte to peripheral blood fast.

The rhesus monkey test-results shows that single subcutaneous injection rhHSF 250-1000 μ g/Kg can make rhesus monkey peripheral blood CFU-GM increase by 3.8 to 11.2 times, continue 2-4hr, and are suitable with independent injection G-CSF10 μ g/Kg/d X 4d peripheral blood CFU-GM rising amplitude.45min peripheral blood CFU-GM adds 58 times behind the single injection hHSF250 μ g/Kg.Peripheral blood CFU-GM increased 11.2-17 doubly after rhesus monkey gave G-CSF 10 μ g/Kg/d X 4d, CFU-Meg is elevated to preceding 26 times of administration after giving single dose hHSF 250 μ g/Kg on the 5th day again, CFU-GM raise 2 times when list was with G-CSF, CFU-Meg raises 200 times, illustrates that hHSF and G-CSF do the mobilization of group cell to hematopoiesis obvious synergy is arranged.People HSF can be separately or is united with G-CSF and to be used for the mobilizing hematopoietic cell.Can shorten administration date of G-CSF with the G-CSF combined utilization, reduce the daily dosage portion of G-CSF, reduce the number of times of gathering stem cell, thereby alleviate the side effect of using G-CSF clinically, reduce exploitation disease, and quicken the recovery that peripheral blood hematopoietic stem cells is transplanted the back hemopoietic system.The applicant uses PCR method synthetic coding people HSF gene, and in E.coli, obtain expression up to 30% with non-fusion rotein form, optimized renaturation condition and separation purifying technique, finally obtained purity and reach the pure product of 95% above recombinant human HSF, this product N-end does not contain methionine(Met).Prove quick mobilizing hematopoietic stem cells of product energy of the present invention and neutrophil leucocyte to peripheral blood in the rhesus monkey in vivo test, and obvious synergy is arranged with G-CSF.

Though described the present invention according to above specific embodiment, but should be realized that and to carry out multiple modification and change by those skilled in the art to the present invention, and these modifications or change are also contained in the defined scope of the present invention of appended claim.

Sequence table

＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences

＜120〉a kind of preparation method of artificial blood synergistic factor and used nucleotide sequence produced

<130>40385CGCNC

<140>2003101242118

<141>2003-12-31

<160>1

<170>PatentIn?version?3.1

<210>1

<211>207

<212>DNA

＜213〉people

<400>1

Claims (4)

1. the polynucleotide sequence shown in SEQ ID NO.:1.

2. the carrier that contains the described polynucleotide sequence of claim 1.

3. the positive expression plasmid that contains the described carrier of claim 2.

4. method of producing the artificial blood synergistic factor, it may further comprise the steps:

1) the polynucleotide sequence subclone that will contain the hematopoietic synergistic factor of encoding is to expression vector, and screening contains the clone of positive expression plasmid;

2) described positive expression plasmid is changed in the intestinal bacteria, abduction delivering is with the hematopoietic synergistic factor of inclusion body formal representation;

3), separate and the recovery target protein with described inclusion body sex change cracking;

4) after the target protein renaturation, carry out the separation and purification target protein;

The polynucleotide sequence that it is characterized in that described coding hematopoietic synergistic factor is the polynucleotide sequence shown in the SEQ IDNO.:1.

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