CN1445239A - Mutain of human stem cell growth fcator as well as its preparing method and medicine combination - Google Patents

Abstract

A human stem cell growth factor mutein, the DNA moleculae for coding it, its preparing process and its medicinal composition are disclosed. Said mutein contains Arg23Gly, Tyr110Ser and Pro288Ala mutations and has increasing bioactivity and expression in host cell.

Description

Human stem cell growth mutain and method for making thereof and pharmaceutical composition

Invention field

The present invention relates to biological technical field.Specifically, the present invention relates to the recombinant human stem cell factor mutain that a kind of activity and expression amount increase.In addition, the pharmaceutical composition that the invention still further relates to the preparation method of this mutain and contain this mutain.

Background of invention

Since 3 study group of the nineteen ninety U.S. almost report STEM CELL FACTOR simultaneously, carried out research extensively and profoundly all over the world.STEM CELL FACTOR claims mast cell growth factor (MGF) again, the Kit part (KL) and the Steel factor (SLF).It is a kind of acid glycoprotein that is produced by the stroma cell in the bone marrow microenvironment.Its glycosyl is connected on the N and O group of peptide bond, and relative molecular mass 31000～36000 is made up of two same subunit of non-covalent bonded.Iso-electric point PI=3.8.

SCF is encoded by No. 10 karyomit(e) Steel sites mouse.In the people, be positioned at 12 q22～24.SCF has 2 kinds of existence forms: solubility and film mating type.In the people, 248 amino acid whose mRNA (SCF encode ²⁴⁸), in its 6th exon an albumen cleavage site is arranged.MRNA expresses 165 amino acid whose solubility SCF thus.220 amino acid whose mRNA (SCF encode ²²⁰), no albumen cleavage site in its 6th exon.MRNA expresses film mating type SCF thus.In mouse, solubility SCF can be by SCF ²⁴⁸At cutting of the 6th exon or SCF ²⁴⁸And SCF ²²⁰The 7th exon cutting form.SCF is by SCF for film mating type ²²⁰Express.2 kinds of form SCF all have biologic activity (Huang EJ etc., Mol Biol Cell, 1992,3:349).Mouse and people SCF almost have equal biologic activity to human hematopoietic cell, but to the mouse cell, mouse SCF is than people SCF biological effect strong 800 times of (Martin FH etc., Cell, 1990,63 (1): 203).

Gene recombination SCF has identical biologic activity with natural SCF.2 kinds of form SCF play an important role to hematopoiesis.But (Namre, 1991,352 (6338): 809) find mating type SCF than long several weeks of solubility SCF hematopoiesis support, serve as strong to primordial germ cell survival hormesis with mating type SCF to Dolci etc.It is of short duration that solubility SCF activates the c-kit acceptor, and inducing cell surface c-kit receptor down-regulated is rapider.

SCF and other cytokines are induced together and are done and the progenitor cell hyperplasia, prolong its survival time and cause and do and progenitor cell is mobilized.Though the acceptor of SCF does not have remarkable difference at progenitor cell, SCF induces the CFU-E hyperplasia stronger than grain-single progenitor cell, may be other specific factors influence progenitor cell to the reactivity of SCF (.Blood such as Olweus J, 1996 (5), 88:1594).Use SCF and granulocyte colony-stimulating factor (G-CSF) to mouse, peripheral hematopoietic stem cells and progenitor cell promptly reached the peak on the 1st day, and be normal after 6 weeks.Do in the marrow and progenitor cell decline in the 1st day, rising in the 14th day reaches 10 times, and is normal after 6 weeks.Show that initial peripheral blood is done and progenitor cell raises is by mobilizing peripheral blood (Bodine DM etc., Blood, 1996,88 (1): 89) in the marrow.(Blood, 1995,86 (12): 4674) report SCF and IL-11 share increases long-term bone marrow proliferation cell (LTMRC) from bone marrow mobilization to the spleen of not cutting the spleen mouse with cut the blood of spleen mouse to Mauch etc.(Blood, 1997,89 (6): 1915) think that SCF can not keep stem cell population external separately, effect is SCF and other cytokine results of interaction to Yonemura etc. in the body.

In external SCF and the collaborative promotion of IL-7 precursor B hyperplasia.Takeda etc. (Blood, 1997,89 (2): 518) think that the interior B cell development of body is not that acceptor c-kit and SCF interact, and another receptor type tyrosine kinase (FLK ₂) more important than c-kit to the B cell development.

SCF plays a crucial role in mastocyte is grown and survived.The genetically deficient of mouse SCF causes reticular tissue and mucomembranous surface mastocyte to lack.Because SCF causes the mastocyte threshing, be cost generally during application to reduce dosage.(Blood, 1997,90 (10): 3874) find that the dimer SCF that disulphide interrelates is stronger 10～20 times than common SCF irritation cell hyperplasia such as Nocka.But it is strong unlike common SCF to the mastocyte threshing.

The existing chemical excitability of SCF also has chemotaxis.Film mating type SCF promotes hemopoietic progenitor cell to get back to marrow.Venoclysis kit ⁺Its gradient along SCF moves to marrow after the hemopoietic progenitor cell.This is to cause (Okumura N etc., Blood, 1996,87 (10): 4100) by the SCF that kit adheres to the marrow stromal cell surface.(Blood, 1998,91 (1): 100) think that the stroma cell source factor-1 (SDF-1) has only chemotaxis, it suppresses hemopoietic progenitor cell as the anti-shifter factor of physiology and shifts out marrow Kim etc.

Application SCF, short PDGF (TPO), IL-12, IL-3 handle freezing bone marrow cell transplantation and give mouse, it recovers thrombocyte and neutrophil leucocyte compares with Zao 3～6 days (RatajczakMZ etc. of untreated bone marrow transplantation, Blood, 1998,91 (1): 353).In mouse model, the receptor before using 5-FU and after give SCF injection, can make stem cell enter the cell cycle from stationary phase.Stem cell is easy to kill to the 5-FU sensitivity like this, provides stable interior environment for donor marrow moves into the receptor, help bone marrow transplantation success (Van os R, etc., Blood, 1997,89 (7): 2376).

Luciferase gene is connected on the plasmid, and this gene is covalently bound with polylysine (PL) and streptavidin (SA), and the SCF of vitamin H acyl groupization is connected with SA with vitamin H, and adenovirus and PL are covalently bound, with this carrier transfection people MBO ₂With MO-7e cell (both all express c-kit), hatch and combined transfection efficiency with c-kit by SCF in 2 hours and can reach 90% (Schwarzenberger.Blood, 1996,87 (2): 472).But Fielding etc. (Blood, 1998,91 (5): 1802) the report retroviral vector makes SCF and the c-kit adhesion of hematopoietic cell surface by connecting SCF, and then virus can not the transfection hematopoietic cell, but can transfection to the non-hematopoietic cell of not expressing c-kit.

Once that serum SCF is low in the reason that causes hematopoietic disorder.It is reported that patient's serum SCF level is low after the bone marrow transplantation at barrier again, in the myelodysplastic syndrome.(Blood, 1996,87 (9): 4017) detected the pure red systems of 34 examples and hindered patients serum SCF and normal people's comparison, not statistically signigicant again such as Abkowitz.Think serum SCF level may with clinical no dependency.But serum SCF is solubility SCF, and SCF still can't detect as for film mating type.

(Blood, 1996,88 (9): 3323) gave 3g (m on the 1st day such as Weaver with 48 routine epithelium ovarian cancer patients ²) ^-1The endoxan infusion was totally lost in 4 hours, mesna 6g (m ²) ^-1Infusion 12 hours.48 examples are divided into 4 groups at random then, and everyone all injects 5 μ gkgd ^-1G-CSF has in every group 9 examples to add SCF with recombinant human.Give 5 μ gkg respectively by group ^-1D ^-1, 10 μ gkg ^-1D ^-1, 15 μ gkg ^-1D ^-1, 20 μ gkg ^-1D ^-148 hours begin to use after the chemotherapy, up to peripheral blood WBC 〉=4.0 * 10 ⁹L ^-1At this moment carry out the collection of peripheral blood list composition.Found that long-term culture-initiating cell (LTC-IC) is at SCF 20 μ gkg ^-1D ^-1Group increases by 5.8 times, CD than single with the G-CSF group ₃₄ ⁺Cell increases by 3 times, CD ₃₄ ⁺CD ₃₃ ^-Cell increases by 64 times.(Blood, 1997,90 (8): such as Glaspy 2939) with random packet after the 215 example high-risk period patients with mastocarcinoma chemotherapy, single G-CSF 10mgkg that uses ^-1D ^-1Reach 7d, G-CSF 10 μ gkg ^-1D ^-1With recombinant human scf 5,10,15,20,25,30 μ gkg ^-1D ^-1Drug combination reaches 7,10,13d.The last 3d of every kind of therapy carries out the single composition collection of peripheral blood leucocyte, found that and uses 20 μ gkg ^-1D ^-1SCF and 10 μ gkg ^-1D ^-1Behind the G-CSF, began on the 5th day to carry out that the peripheral blood list adopts is the dose,optimum and the preferred plan of mobilizing the peripheral blood progenitor cell.(Blood, 1997,90 (9): 3378) random packet before the 62 routine early-stage breast cancer patient chemotherapy is accepted 12 μ gkg such as Begley ^-1D ^-1G-CSF and add rhSCF 5,10,15 μ gkg with dosage G-CSF ^-1D ^-1Reach 7 days, and with 10d 10 μ gkg ^-1D ^-1SCF and added with G-CSF in the 4th day and reach 7 days.Found that and use the heaven-made pretreat of SCF3 earlier that use the two combination therapy group again, the peripheral blood hematopoietic progenitor cell raises more obvious.SCF is generally subcutaneous injection.The most general side reaction is that injection site skin has Mild edema, has a circle red and swollen outward.Generally, continue 24～48 hours, recover normal later in injection beginning in back 4 hours.Idol has the anaphylaxis report, can give the antianaphylaxis prevention before using.

In sum, human stem cell growth has application widely.Yet the activity of at present commercially available human stem cell growth goods still needs further raising.And, at present come the expression amount of method of expressing human stem cell factor not high enough, and protein need renaturation when purifying with intestinal bacteria.Therefore, still need the higher human stem cell growth of a kind of biological activity and expression amount in the art.

Summary of the invention

The purpose of this invention is to provide a kind of isolating human stem cell growth mutain.Another object of the present invention provides the proteic dna molecular of coding said mutation.Another object of the present invention provides a kind of proteic pharmaceutical composition of said mutation that contains.A further object of the invention provides the proteic method of preparation said mutation.

In order to satisfy the foregoing invention purpose, one aspect of the invention provides a kind of isolating human stem cell growth mutain, and this mutain contains Arg23Gly, Tyr110Ser and Pro288Ala sudden change.In a preferable example, described mutain has the aminoacid sequence shown in the SEQ ID NO:2.

Another aspect of the present invention provides a kind of separated DNA sequence, and this dna sequence dna comprises above-mentioned human stem cell growth mutain encoding sequence.In a preferred embodiments, this dna sequence dna has the nucleotide sequence shown in the SEQ ID NO:4.

Another aspect of the present invention provides a kind of expression vector, and this expression vector contains the dna sequence dna of the above-mentioned human stem cell growth mutain of encoding and the expression regulation sequence that links to each other with this dna sequence dna operability.

A further aspect of the invention provides a kind of host cell, and this host cell is transformed by above-mentioned expression vector.In preferable example, this host cell is intestinal bacteria and Bacillus subtilus.

Another aspect of the present invention provides a kind of pharmaceutical composition, and this pharmaceutical composition contains the pharmaceutically above-mentioned human stem cell growth mutain and the pharmaceutically acceptable carrier of significant quantity.

A further aspect of the invention provides a kind of method for preparing above-mentioned human stem cell growth mutain, and this method comprises the following steps:

A) provide an expression vector, the expression regulation sequence that this expression vector contains the above-mentioned human stem cell growth mutain dna sequence dna of coding and links to each other with this dna sequence dna operability;

B) with the expression vector transformed host cell in the step a);

C) host cell of gained culturing step b under the condition that is fit to the described mutain of expression); With

D) separation and purification obtains the expressed mutain that goes out.

The invention has the advantages that the natural stem cell factor of the biological activity ratio of above-mentioned human stem cell growth mutain is high 10 times, and its expression amount in host cells such as intestinal bacteria is improved largely also.Other purpose of the present invention and advantage can be learnt by following detailed.

The accompanying drawing summary

Fig. 1 has shown the nucleotide sequence of multiple clone site in the collection of illustrative plates of pUC18 expression vector used herein and the pUC18 expression vector.

Fig. 2 has shown the SCF determination of activity graphic representation made from Microsoft Excel.

Detailed Description Of The Invention

The invention provides the human stem cell growth mutain that separates of a kind of Arg23Gly of containing, Tyr110Ser and Pro288Ala sudden change.

Term used herein " stem cell factor " can be the human stem cell growth albumen with wild type sequence, also can be derivative type or the recombinant human stem cell factor albumen with mutant nucleotide sequence, as long as do not comprise Arg23Gly, Tyr110Ser and Pro288Ala sudden change in this mutant nucleotide sequence. The coded sequence of natural human stem cell growth albumen and amino acid sequence thereof are shown in SEQ ID NO:3 and SEQ ID NO:1.

The present inventor finds by research, by 23 arginine (Arg) of this protein amino acid sequence, 110 tyrosine (Tyr) and 288 proline (Pro) are mutated into respectively glycine (Gly), serine (Ser) and alanine (Ala), the biologically active of mutain is increased substantially. Term used herein " Arg23Gly " represents that namely the 23rd amino acid becomes Gly by Arg. Term " Tyr110Ser " represents that namely the 110th amino acid becomes Ser by Tyr. Term " Pro288Ala " represents that namely the 288th amino acid becomes Ala by Pro.

Term used herein " separation " is when being used for nucleic acid or protein, and expression nucleic acid or protein are substantially gone up and do not contained other cell component relevant under native state, and it preferably is homogeneous state, but also can be do or the aqueous solution. The common available analyses chemical method of purity and homogeneity such as polyacrylamide gel electrophoresis or high effective liquid chromatography for measuring.

In another preferred embodiment, also can make mutain of the present invention and another polypeptide, preferably with signal peptide sequence or other polypeptide amalgamation and expression in specific cleavage site is arranged at mature polypeptide N end. Usually, this signal coding sequence can be an assembly of carrier, and perhaps it can be the part of the polypeptid DNA of insertion vector. The function of this signal peptide is that the guiding expression product shifts (secretion) in matrix with the form of activity, so that later purifying and avoid renaturation process. The signal peptide sequence that example is colon bacillus OmpA: KKTAIAIAVALAGFATVAQA (20 amino acid that the present invention hereinafter adopts among the embodiment, SEQ ID NO:6) (GenBank ACCESSION Number:U40577), its coded sequence is: aaaaagacagctatcgcgattgcagtggcactggctggtttcgctaccg tagcgcaggcc (60bp, SEQ ID NO:5) (GenBank Accession Number:U40577). Certainly, those skilled in the art also can adopt other signal peptide sequence of knowing.

The present invention also provides a kind of code book to invent the dna sequence dna of above-mentioned human stem cell growth mutain. In a better example, described dna sequence dna has the nucleotide sequence shown in the SEQ ID NO:4. Certainly, those skilled in the art also can utilize the degeneracy of the genetic code of knowing in this area to obtain all other nucleotide sequences of the above-mentioned amino acid sequence of coding. In addition, also can carry out the codon preference design for this dna sequence dna and the optional signal coding sequence that inserts, in order to further improve its expression in host cell.

Mutain of the present invention can be prepared by the following method. At first, provide the nucleotide sequence that contains code book invention mutain and the expression vector of the expression regulation sequence that links to each other with this series of operations.

Term used herein " expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed. Expression regulation sequence comprises promoter and the termination signal that links to each other with target nucleotide sequence operability. They also comprise the suitably required sequence of translation of nucleotide sequence usually. " operability links to each other " refers to that some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence. For example, if promoter or enhancer have increased transcribing of coded sequence, then it is that operability links to each other with coded sequence.

The dna sequence dna of above-mentioned encoding mutant type stem cell factor albumen can obtain with conventional means well known to those skilled in the art, for example, come synthetic primer to carry out the amplification of PCR method according to the dna sequence dna of human stem cell growth albumen disclosed herein, or use such as methods such as direct mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis and carry out mutagenesis.

Behind the dna sequence dna after having obtained to have rite-directed mutagenesis, just can it be connected in the suitable expression vector by the whole bag of tricks well known in the art. Used expression vector is various commercially available expression vector well known by persons skilled in the art among the present invention, for example available from the expression vector of Qiagen and Promega company, such as pUC18.

Then, the expression vector with above-mentioned acquisition transforms suitable host cell. In the present invention, term " host cell " comprises prokaryotic and eukaryotic. The example of prokaryotic host cell commonly used comprises Escherichia coli, hay bacillus etc. Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell. Better host cell is Escherichia coli and Bacillus subtilis.

At last, under the condition that is fit to mutain expression of the present invention, cultivate the host cell that transforms gained, obtain mutain of the present invention with conventional separation and purification means purifying well known to those skilled in the art such as ion-exchange chromatography, hydrophobic chromatography and sieve chromatographies then.

The present invention also provides a kind of pharmaceutical composition, and said composition contains the pharmaceutically mutain of the present invention and the pharmaceutically acceptable carrier of significant quantity.Term used herein " pharmaceutically acceptable " is meant that they can not produce disadvantageous, hypersensitive or other untoward reaction when molecule body and composition suitably give the animal or human." pharmaceutically acceptable carrier " used herein should be compatible with mutain of the present invention, can not reduce the effect of pharmaceutical composition with its blend under normal conditions significantly.The object lesson that can be used as some materials of pharmaceutically acceptable carrier or its component is a carbohydrate, as lactose, dextrose plus saccharose; Starch is as W-Gum and potato starch; Mierocrystalline cellulose and derivative thereof are as Xylo-Mucine, ethyl cellulose and methylcellulose gum; The tragakanta powder; Fructus Hordei Germinatus; Gelatin; Talcum; Solid lubricant is as stearic acid and Magnesium Stearate; Calcium sulfate; Vegetables oil is as peanut oil, Oleum Gossypii semen, sesame oil, sweet oil, Semen Maydis oil and theobroma oil; Polyvalent alcohol is as propylene glycol, glycerine, Sorbitol Powder, mannitol and polyoxyethylene glycol; Lalgine; Emulsifying agent is as Tween ; Wetting agent is as Sodium Lauryl Sulphate BP/USP; Tinting material; Seasonings; Tablet agent, stablizer; Antioxidant; Sanitas; Apirogen water; Deng oozing salts solution; With phosphate buffered saline buffer etc.

Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode determine the useful dosage of patient is used.

Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.

Embodiment 1 gene is synthetic

At first, provide a mutant human stem cell factor encoding sequence (shown in SEQ ID NO:4) with colon bacillus OmpA signal coding sequence and the design of codon preference.In this sequence, the 124th bit base becomes G by C, makes the 42nd (corresponding to the 23rd that removes behind the signal peptide sequence) amino acid become glycine by arginine; The 386th bit base becomes C by A, and the 387th bit base becomes T by C, makes the 129th of its coding (corresponding to the 110th that removes behind the signal peptide sequence) amino acid become Serine by tyrosine; The 919th bit base becomes G by C, and the 921st bit base becomes T by G, makes the 307th of its coding (corresponding to the 288th that removes behind the signal peptide sequence) amino acid become L-Ala by proline(Pro).This gene structure note is made SCF/ompA.5 ' end in this structure during synthetic gene adds an EcoRI point of contact, adds a HindIII point of contact at 3 '-end.

According to above-mentioned SCF/ompA gene order, according to the principle of conventional design of primers above-mentioned sequence and complementary strand thereof are split into 26 length and be about the oligonucleotide fragment of 83～88b length and entrust Shanghai to give birth to worker company synthetic.Then, utilize PCR method to obtain each SCF/ompA gene fragment, concrete operations are as follows:

Reaction system: each material below in 0.2 milliliter of PCR pipe, adding.

The component volume

10 * Pfu PCR damping fluid, 5 microlitres

Each few fragment (100 nanogram(ng)s/microlitre) (every kind) 1 microlitre

50 * dNTP mixture, 1 microlitre

50 * Pfu archaeal dna polymerase, 1 microlitre

PCR level water to 40 microlitre

Cumulative volume 50 microlitres

(2) reaction conditions

Pre-sex change: 94 ℃ 2 minutes;

Major cycle: 95 ℃ 1 minute, 60 ℃ 40 seconds, 72 ℃ 1 minute, circulate 35 times;

Extend the back: 72 ℃ 10 minutes.

(3) electrophoresis observation result and glue reclaim hSCF/ompA full-length gene fragment.

The gene fragment that glue is reclaimed clones according to ordinary method that (Promega company, Madison WI) go up and carry out gene sequencing in plasmid pGEM-T Easy.Examining order is entrusted upward, and sea base Kanggong department carries out.Determined a right-on clone of sequence according to sequencing result, note is made pSCF/ompA.

The structure of embodiment 2. expression vectors

Present embodiment adopts the pUC18 carrier as expression vector, and this expression vector is available from (Promega company has demonstrated the collection of illustrative plates of this carrier among Fig. 1).The DNA of above-mentioned right-on pSCF/ompA is cut with EcoRI and HindIII enzyme, on 1% agarose electrophoresis, separate, with the fragment about the about 1kb of glue recovery test kit recovery length of PROMEGA company.Then with the connection test kit of MBI company the SCF/ompA gene fragment be connected in prokaryotic expression carrier pUC18 carrier on, constitute the pUC18-SCF/ompA expression vector.The detail operations process is as follows:

1) cuts expression plasmid pUC18 with EcoRI and HindIII enzyme.Each material below in Eppendorf tube, adding:

PUC18 plasmid DNA 3 micrograms

10 * restriction enzyme damping fluid, 3 microlitres

EcoRI and HindIII 15U

1 mg/ml acetylize BSA, 3 microlitres

Water to 40 microlitre of nuclease free

Hatched 4 hours for 37 ℃, carry out electrophoresis in contrast with dna marker, glue reclaims the plasmid pUC18 that enzyme is cut, and it is stand-by to put-20 ℃ of preservations.

2) cut the plasmid pGEM-T Easy that carries goal gene with EcoRI and HindIII enzyme, downcut goal gene and glue and reclaim.Method is the same.

3) ligase enzyme is cut expression plasmid pUC18 and target gene fragment.Each thing below in 0.2 milliliter of PCR pipe, adding:

10 * ligase enzyme damping fluid, 2 microlitres

100mM DTT 2 microlitres

10mM ATP 1 microlitre

PUC18 carrier 2 microlitres that 50 nanogram(ng)s/microlitre makes

T4 ligase enzyme (0.2-0.4Weise unit/microlitre) 1 microlitre

Goal gene (0.2pmol) 1 microlitre of preparation

Water 11 microlitres of nuclease free

Cumulative volume 20 microlitres

4 ℃ of connections are spent the night.

4) plasmid pUC18 that connection is built is transformed into and expresses among the engineering bacteria DH5a.

5) cut evaluation in order to following method enzyme and transform plasmid:

Single expression engineering bacteria bacterium colony carries out amplification cultivation on the picking penbritin culture dish, and the extracting plasmid is cut evaluation with EcoRI and HindIII enzyme, and method is the same.Enzyme is cut product and is identified on 1% agarose electrophoresis, has determined that tentatively 11 clones have the purpose insertion sequence.Choose wherein 2 correct clones and check order, confirm that according to sequencing result 1 clone's insertion sequence is right-on, note is made pUC18-SCF/ompA.

Embodiment 3. induces destination gene expression

Bacterium liquid 100 microlitres of getting correct clone are inoculated into respectively in 5 milliliters of LB liquid nutrient mediums that contain penbritin (100 mcg/ml), 37 ℃, 260rpm concussion is cultured to OD value and reaches 0.4～1.0, adding isopropylthiogalactoside (IPTG) to final concentration 1 mmole/liter.Cultivated 3 hours, and from every kind of culture, took out 1 milliliter and transfer in the centrifuge tube, centrifugal, remove supernatant; The cracking thalline carries out the SDS protein electrophoresis, sds polyacrylamide is dyeed the proteic expression of testing goal with Xylene Brilliant Cyanine G.The result shows, the purpose product that this bacterial strain is expressed accounts for 68% of thalline soluble proteins, than the expression amount that method obtained 15% (the Chen W that directly natural gene is expressed that has abroad delivered, Di X, Li J, Song F, Chen S.cDNA cloning of human stem cell factor and itshigh level expression in E.coli, Chinese Academy of Medical Sciences's journal, 1997 Feb; 19 (1): 29-34) exceed at least 4 times.This high expression level result is significant for the industrialization that realizes this gene.

The purifying of embodiment 4. target proteins and active detection the thereof

In the LB substratum, induce this genetic expression according to the method described above and obtained purity at this recombinant protein more than 95% through behind ion exchange chromatography, hydrophobic chromatography and the sieve chromatography.

With following ELISA method the expression amount of above-mentioned bacterial strains is studied.The ELISA method is carried out with the MO-7E cell strain.Containing RPMI1640 substratum, the 5%CO of 8% calf serum ₂Cultivate 24 hours MO-7E cell 500RPM centrifugal collecting cell in the incubator, be mixed with 5 * 10 with the DEME substratum that contains 10% calf serum ⁴The suspension of cells/ml.

Get 96 orifice plates, add 100 microlitre cell suspensions, SCF testing sample of 100 microlitre serial dilutions (200 millimicro grams per milliliter) or standard substance (recombinant natural people SCF is available from Pharmacia) (200 millimicro grams per milliliter), 37 ℃ of 5%CO in every hole ₂Cultivated 48 hours in the incubator.Not add SCF as negative control.3 repeating holes of every processing.

Learnt from else's experience 0.1 milliliter in the cell of above-mentioned processing, adding 1/10 volumetric concentration is the MTT solution of 5 mg/ml, cultivates the photoabsorption of reading the 570nm place after 30 minutes on microplate reader for 37 ℃.With the mean number of three repeating hole readings as calculating and foundation relatively.The result shows that the cell of handling through SCF obviously improves than undressed contrast photoabsorption, and read-around ratio contrasts high by 36%～670%.The comparative result of testing sample and positive control is presented in the following table 1.

Table 1 SCF determination of activity result

Sample number into spectrum	1	2	?3	?4	?5	??6	?7	?8	?9	?10	?11	?12	?13	?14	?15	?16	?17
																		Concentration (ng/ml)	0.01	0.05	?0.1	?0.5	?1	??2.5	?5	?7.5	?10	?12.5	?15	?17.5	?20	?22.5	?25	?50	?100
Positive control	0.04	0.05	?0.05	?0.06	?0.07	??0.17	?0.41	?0.74	?0.99	?1.24	?1.42	?1.6	?1.72	?1.76	?1.79	?1.81	?1.82
																		Testing sample	0.14	0.21	?0.32	?0.56	?0.87	??1.16	?1.43	?1.59	?1.73	?1.78	?1.81	?1.83	?1.85	?1.87	?1.87	?1.89	?1.9

According to above-mentioned data, with the Excel (see figure 2) of mapping.

From table 1 and Fig. 2 as can be seen, the activity of testing sample of the present invention is apparently higher than the SCF of natural structure.The ED of natural structure ₅₀(promptly reaching the needed dosage of 50% maximum efficiency) is about 10 millimicro grams per milliliters, and the biological activity of SCF of the present invention significantly improves, its ED ₅₀Be approximately about the 1nm/ milliliter, the two approximately differs an order of magnitude.

Sequence table＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd＜120〉human stem cell growth mutain and method for making and pharmaceutical composition＜130〉020770＜160〉6＜170〉PatentIn version 3.0＜210〉1＜211〉323＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉1Met Gln Ala Ala Trp Leu Leu Gly Ala Leu Val Val Pro Gln Leu Leu1,5 10 15Gly Phe Gly His Gly Ala Arg Gly Ala Glu Arg Glu Trp Glu Gly Gly

20??????????????????25??????????????????30Trp?Gly?Gly?Ala?Gln?Glu?Glu?Glu?Arg?Glu?Arg?Glu?Ala?Leu?Met?Leu

35??????????????????40??????????????????45Lys?His?Leu?Gln?Glu?Ala?Leu?Gly?Leu?Pro?Ala?Gly?Arg?Gly?Asp?Glu

50??????????????????55??????????????????60Asn?Pro?Ala?Gly?Thr?Val?Glu?Gly?Lys?Glu?Asp?Trp?Glu?Met?Glu?Glu65??????????????????70??????????????????75??????????????????80Asp?Gln?Gly?Glu?Glu?Glu?Glu?Glu?Glu?Ala?Thr?Pro?Thr?Pro?Ser?Ser

85??????????????????90??????????????????95Gly?Pro?Ser?Pro?Ser?Pro?Thr?Pro?Glu?Asp?Ile?Val?Thr?Tyr?Ile?Leu

100?????????????????105?????????????????110Gly?Arg?Leu?Ala?Gly?Leu?Asp?Ala?Gly?Leu?His?Gln?Leu?His?Val?Arg

115?????????????????120?????????????????125Leu?His?Ala?Leu?Asp?Thr?Arg?Val?Val?Glu?Leu?Thr?Gln?Gly?Leu?Arg

130?????????????????135?????????????????140Gln?Leu?Arg?Asn?Ala?Ala?Gly?Asp?Thr?Arg?Asp?Ala?Val?Gln?Ala?Leu145?????????????????150?????????????????155?????????????????160Gln?Glu?Ala?Gln?Gly?Arg?Ala?Glu?Arg?Glu?His?Gly?Arg?Leu?Glu?Gly

165?????????????????170?????????????????175Cys?Leu?Lys?Gly?Leu?Arg?Leu?Gly?His?Lys?Cys?Phe?Leu?Leu?Ser?Arg

180?????????????????185?????????????????190Asp?Phe?Glu?Ala?Gln?Ala?Ala?Ala?Gln?Ala?Arg?Cys?Thr?Ala?Arg?Gly

195?????????????????200?????????????????205Gly?Ser?Leu?Ala?Gln?Pro?Ala?Asp?Arg?Gln?Gln?Met?Glu?Ala?Leu?Thr

2l0?????????????????215?????????????????220Arg?Tyr?Leu?Arg?Ala?Ala?Leu?Ala?Pro?Tyr?Asn?Trp?Pro?Val?Trp?Leu225?????????????????230?????????????????235?????????????????240Gly?Val?His?Asp?Arg?Arg?Ala?Glu?Gly?Leu?Tyr?Leu?Phe?Glu?Asn?Gly

245?????????????????250?????????????????255Gln?Arg?Val?Ser?Phe?Phe?Ala?Trp?His?Arg?Ser?Pro?Arg?Pro?Glu?Leu

260?????????????????265?????????????????270Gly?Ala?Gln?Pro?Ser?Ala?Ser?Pro?His?Pro?Leu?Ser?Pro?Asp?Gln?Pro

275?????????????????280?????????????????285?????Asn?Gly?Gly?Thr?Leu?Glu?Asn?Cys?Val?Ala?Gln?Ala?Ser?Asp?Asp?Gly

290 295 300Ser Trp Trp Asp His Asp Cys Gln Arg Arg Leu Tyr Tyr Val Cys Glu305,310 315 320Phe Pro Phe＜210〉2＜211〉323＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉2Met Gln Ala Ala Trp Leu Leu Gly Ala Leu Val Val Pro Gln Leu Leu1,5 10 15Gly Phe Gly His Gly Ala Gly Gly Ala Glu Arg Glu Trp Glu Gly Gly

20??????????????????25??????????????????30Trp?Gly?Gly?Ala?Gln?Glu?Glu?Glu?Arg?Glu?Arg?Glu?Ala?Leu?Met?Leu

35??????????????????40??????????????????45Lys?His?Leu?Gln?Glu?Ala?Leu?Gly?Leu?Pro?Ala?Gly?Arg?Gly?Asp?Glu

50??????????????????55??????????????????60Asn?Pro?Ala?Gly?Thr?Val?Glu?Gly?Lys?Glu?Asp?Trp?Glu?Met?Glu?Glu65??????????????????70??????????????????75??????????????????80Asp?Gln?Gly?Glu?Glu?Glu?Glu?Glu?Glu?Ala?Thr?Pro?Thr?Pro?Ser?Ser

85??????????????????90??????????????????95Gly?Pro?Ser?Pro?Ser?Pro?Thr?Pro?Glu?Asp?Ile?Val?Thr?Ser?Ile?Leu

100?????????????????105?????????????????110Gly?Arg?Leu?Ala?Gly?Leu?Asp?Ala?Gly?Leu?His?Gln?Leu?His?Val?Arg

115?????????????????120?????????????????125Leu?His?Ala?Leu?Asp?Thr?Arg?Val?Val?Glu?Leu?Thr?Gln?Gly?Leu?Arg

130?????????????????135?????????????????140Gln?Leu?Arg?Asn?Ala?Ala?Gly?Asp?Thr?Arg?Asp?Ala?Val?Gln?Ala?Leu145?????????????????150?????????????????155?????????????????160Gln?Glu?Ala?Gln?Gly?Arg?Ala?Glu?Arg?Glu?His?Gly?Arg?Leu?Glu?Gly

165?????????????????170?????????????????175Cys?Leu?Lys?Gly?Leu?Arg?Leu?Gly?His?Lys?Cys?Phe?Leu?Leu?Ser?Arg

180?????????????????185?????????????????190Asp?Phe?Glu?Ala?Gln?Ala?Ala?Ala?Gln?Ala?Arg?Cys?Thr?Ala?Arg?Gly

195?????????????????200?????????????????205Gly?Ser?Leu?Ala?Gln?Pro?Ala?Asp?Arg?Gln?Gln?Met?Glu?Ala?Leu?Thr

210?????????????????215?????????????????220Arg?Tyr?Leu?Arg?Ala?Ala?Leu?Ala?Pro?Tyr?Asn?Trp?Pro?Val?Trp?Leu225?????????????????230?????????????????235?????????????????240Gly?Val?His?Asp?Arg?Arg?Ala?Glu?Gly?Leu?Tyr?Leu?Phe?Glu?Asn?Gly

245?????????????????250?????????????????255Gln?Arg?Val?Ser?Phe?Phe?Ala?Trp?His?Arg?Ser?Pro?Arg?Pro?Glu?Leu

260?????????????????265?????????????????270Gly?Ala?Gln?Pro?Ser?Ala?Ser?Pro?His?Pro?Leu?Ser?Pro?Asp?Gln?Ala

275?????????????????280?????????????????285Asn?Gly?Gly?Thr?Leu?Glu?Asn?Cys?Val?Ala?Gln?Ala?Ser?Asp?Asp?Gly

290 295 300Ser Trp Trp Asp His Asp Cys Gln Arg Arg Leu Tyr Tyr Val Cys Glu305 310 315 320Phe Pro Phe＜210〉3＜211〉969＜212〉DNA＜213〉 ( Homo sapiens )＜400〉3atgcaggcag cctggctttt gggggctttg gtggtccccc agctcttggg ctttggccat 60ggggctcggg gagcagagag ggagtgggag ggaggctggg gaggtgccca ggaggaggag 120cgggagaggg aggccctgat gctgaagcat ctgcaggaag ccctaggact gcctgctggg 180aggggggatg agaatcctgc cggaactgtt gagggaaaag aggactggga gatggaggag 240gaccaggggg aggaagagga ggaggaagca acgccaaccc catcctccgg ccccagcccc 300tctcccaccc ctgaggacat cgtcacttac atcctgggcc gcctggccgg cctggacgca 360ggcctgcacc agctgcacgt ccgtctgcac gcgttggaca cccgcgtggt cgagctgacc 420caggggctgc ggcagctgcg gaacgcggca ggcgacaccc gcgatgccgt gcaagccctg 480caggaggcgc agggtcgcgc cgagcgcgag cacggccgct tggagggctg cctgaagggg 540ctgcgcctgg gccacaagtg cttcctgctc tcgcgcgact tcgaagctca ggcggcggcg 600caggcgcggt gcacggcgcg gggcgggagc ctggcgcagc cggcagaccg ccagcagatg 660gaggcgctca ctcggtacct gcgcgcggcg ctcgctccct acaactggcc cgtgtggctg 720ggcgtgcacg atcggcgcgc cgagggcctc tacctcttcg aaaacggcca gcgcgtgtcc 780ttcttcgcct ggcatcgctc accccgcccc gagctcggcg cccagcccag cgcctcgccg 840catccgctca gcccggacca gcccaacggt ggcacgctcg agaactgcgt ggcgcaggcc 900tctgacgacg gctcctggtg ggaccacgac tgccagcggc gtctctacta cgtctgcgag 960ttccccttc 969＜210〉4＜211〉1026＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜223〉＜400〉4aaaaaaaccg ctatcgctat cgctgttgct ctggctggtt tcgctaccgt tgctcaggct 60caggctgctt ggctgctggg tgctctggtt gttccgcagc tgctgggttt cggtcacggt 120gctggtggtg ctgaacgtga atgggaaggt ggttggggtg gtgctcagga agaagaacgt 180gaacgtgaag ctctgatgct gaaacacctg caggaagctc tgggtctgcc ggctggtcgt 240ggtgacgaaa acccggctgg taccgttgaa ggtaaagaag actgggaaat ggaagaagac 300cagggtgaag aagaagaaga agaagctacc ccgaccccgt cttctggtcc gtctccgtct 360ccgaccccgg aagacatcgt tacctctatc ctgggtcgtc tggctggtct ggacgctggt 420ctgcaccagc tgcacgttcg tctgcacgct ctggacaccc gtgttgttga actgacccag 480ggtctgcgtc agctgcgtaa cgctgctggt gacacccgtg acgctgttca ggctctgcag 540gaagctcagg gtcgtgctga acgtgaacac ggtcgtctgg aaggttgcct gaaaggtctg 600cgtctgggtc acaaatgctt cctgctgtct cgtgacttcg aagctcaggc tgctgctcag 660gctcgttgca ccgctcgtgg tggttctctg gctcagccgg ctgaccgtca gcagatggaa 720gctctgaccc gttacctgcg tgctgctctg gctccgtaca actggccggt ttggctgggt 780gttcacgacc gtcgtgctga aggtctgtac ctgttcgaaa acggtcagcg tgtttctttc 840ttcgcttggc accgttctcc gcgtccggaa ctgggtgctc agccgtctgc ttctccgcac 900ccgctgtctc cggaccaggc taacggtggt accctggaaa actgcgttgc tcaggcttct 960gacgacggtt cttggtggga ccacgactgc cagcgtcgtc tgtactacgt ttgcgaattc 1020ccgttc 1026＜210〉5＜211〉60＜212〉DNA＜213〉 ( Escherichia coli )＜400〉5aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt agcgcaggcc 60＜210〉6＜211〉20＜212〉PRT＜213〉 ( Escherichia coli )＜400〉6Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr1 5 10 15Val Ala Gln Ala

20

Claims (9)

1. an isolating human stem cell growth mutain is characterized in that, it contains Arg23Gly, Tyr110Ser and Pro288Ala sudden change.

2. mutain according to claim 1 is characterized in that described mutain has the aminoacid sequence shown in the SEQ IDNO:2.

3. a separated DNA sequence is characterized in that, it comprises the encoding sequence of the described human stem cell growth mutain of claim 1.

4. separated DNA sequence according to claim 3 is characterized in that, it has the nucleotide sequence shown in the SEQ ID NO:4.

5. an expression vector is characterized in that, it contains the dna sequence dna of the described human stem cell growth mutain of coding claim 1 and the expression regulation sequence that links to each other with this dna sequence dna operability.

6. a host cell is characterized in that, it is transformed by the described expression vector of claim 5.

7. host cell according to claim 6 is characterized in that described host cell is intestinal bacteria.

8. a pharmaceutical composition is characterized in that, it contains the pharmaceutically described human stem cell growth mutain of claim 1 and the pharmaceutically acceptable carrier of significant quantity.

9. a method for preparing the described human stem cell growth mutain of claim 1 is characterized in that, this method comprises:

A) provide an expression vector, this expression vector contains the dna sequence dna of the described human stem cell growth mutain of coding claim 1 and the expression regulation sequence that links to each other with this dna sequence dna operability;

B) with the expression vector transformed host cell in the step a);

C) host cell of gained culturing step b under the condition that is fit to the described mutain of expression); With

D) separation and purification obtains the expressed mutain that goes out.

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