CN101077887A - Human accelerated haematogenous cell proliferated cytokine, preparation method and use thereof - Google Patents

Abstract

The present invention discloses one kind of human cytokine for promoting hematopoietic cell proliferation and its preparation process and use. The cytokine consists of protein with amino acid sequence of SEQ ID No. 2, codes cDNA molecule of p NH, and has all or segment of nucleotides in SEQ ID No. 1. It is PCR amplified with pf u DNA polyase, and pNH segment amplifying upstream primer is 5' -GCCATG GATGCCACCTGCAACTGTGAT-3' and the downstream primer is 5' -gCCTCgAgAGTTGTG ACCTTGAAGTCACCAT-3' . It is cloned through two cleavage sites, Nco I and Xho I, to protonucleus expression vector, and expressed efficiently in engineering bacterium, and the expressed and purified target protein provides technological support for researching SMB structure domain. The cytokine as experiment reagent may be prepared in large scale for clinical application in treating hematopoietic disorder diseases, and has the advantages of small molecular weight and weak immunogenicity.

Description

Human accelerated haematogenous cell proliferated cytokine and its production and use

Technical field

The present invention relates to a kind of cytokine, especially a kind of human accelerated haematogenous cell proliferated cytokine and its production and use.

Background technology

Genome times afterwards comprehensively are study periods of protein structure and function.Along with the life science theory constantly perfect, the continuous development of experimental technique, increasing structural albumen is found in and is bringing into play very important physiological function in the body.PNH is a different spliceosome form of structural protein glycoprotein 4 (PRG4 claims SZP again, synovium of joint albumen), is made up of part the 2nd exon of PRG4, complete the 3rd exon and part the 6th exon; Two exons of cDNA sequence 5 ' end, two the short cells of encoding respectively generate plain B (SMB) structural domain, 3 ' end exons coding PTT/XP mucoitin sample tumor-necrosis factor glycoproteins.Wherein 15 amino acid of first SMB structural domain N end disappearance comprise a pair of disulfide linkage in this sequence.Serum somatomedin that depends on tethelin performance function in 1972 be named as short cell generate plain B (Somatomedin B, SMB).It can excite nerve synthesizing of spongiocyte DNA.In human plasma and hemofiltrate, can both detect free solubility SMB albumen.Thereafter, different research groups finds to contain in many albumen and SMB homologous sequence, is called the short cell of people and generates plain B spline structure territory (SMB like domain), contains 39 to 51 amino-acid residues and does not wait.

The ox cartilage monolayer cell of Flannery in 1999 from cultivating, calendar year 2001 Gregory etc. obtain the full length product of SZP the 4th, the 5th Exon deletion during RT-PCR SZP sequence from person joint's synovia inoblast and articular chondrocytes, as identifying usefulness, still do not carry out work such as further expression activity evaluation.

The SMB domain C ys site of SZP/MSF and vitronectin (Vitronectin, Vn) the SBM structural domain has homology, but the flanking sequence of Cys is different fully.Both this structural domains have very big difference on function, the latter can be in conjunction with PAI-1, and MSF then can not; Both difference on function have been shown.Be research disulfide linkage topological framework, Kamikubo in 2002 had once expressed the SMB structural domain of people Vn.

Present hemopoietic stem cell cultured and amplified in vitro technology fully matured not as yet.Stem cell population deficiency in the culturing process, problems such as easy differentiation have to be solved.In culture system, add certain cytokine and become one of means.Such as the IL-3 of the amplification of the progenitor cell that can stimulate directed differentiation, bite the GM-CSF that progenitor cell has hormesis to huge, TPO acts on megakaryoblast, and SCF promotes hematopoietic stem cell expansion.In treating hematopathy drug research process, erythropoietin (Erythropoietin, EPO), the granulocyte clone forms stimulating factor (granulocyte colony-stimulating factor, G-CSF) and granulocyte-megakaryocyte stimulating factor (granulocyte-macrophage stimulatingfactor, GM-CSF) be three cytokines that have been approved for oncotherapy, effect is remarkable in anaemia after the treatment chemotherapy and the neutropenia shape.Human interleukin-13 is subjected to the ligand analogs of height affinity, (Synthokine), human interleukin-13 and G-CSF receptor activators (myelopoietin MPO), can stimulate the propagation of the hemopoietic progenitor cell of many differentiation potentials with the promegapoietin (PMP) that stimulates people IL-3, c-mpl acceptor, accept clinical experiment.Wherein, EPO is the first relevant somatomedin of hematopoiesis of utilizing the clone technology dna recombinant expression, is used to regulate the relevant anaemia of tumour.Finding and study new cell growth factor, to be applied to the blood cell culture system be one of current research emphasis.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of human accelerated haematogenous cell proliferated cytokine and its production and use, i.e. pNH.Comprise the clone of pNH cDNA and the expression in protokaryon, the preparation purifying of recombinant protein, the evaluation of physics-chem characteristic and biological function.

In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of human accelerated haematogenous cell proliferated cytokine, described human accelerated haematogenous cell proliferated cytokine pNH is made up of the protein of the listed aminoacid sequence of SEQ ID NO.2.

The cDNA molecule of coding pNH is by the whole or fragment of the listed nucleotide sequence of SEQ ID NO.1.

The cloning process of the cDNA of a kind of pNH that encodes may further comprise the steps:

1), infers and the corresponding nucleotide sequence of these amino acid with known genetic code according to the listed aminoacid sequence of SEQ ID NO.2;

2) design Auele Specific Primer, carry out pcr amplification with the pfu archaeal dna polymerase, described Auele Specific Primer is: the segmental upstream primer of amplification pNH is 5 '-GCCATGGATGCCACCTGCAACTGTGAT-3 ', and downstream primer is 5 '-gCCTCgAgAGTTGTGACCTTGAAGTCACCAT-3 '.

3) with specificity cDNA amplified production as probe, filter out the cDNA of pNH from the human cDNA library, described specificity cDNA amplified production is coding 1) Nucleotide of described pNH aminoacid sequence.

The described human cDNA library embryonic liver cell cdna library of behaving.

A kind of plasmid of expressing pNH contains the resulting cDNA of aforesaid method.

Described plasmid constitutes with pET32c (+) or pET22b (+).

The purification process of the pNH of the listed aminoacid sequence of a kind of SEQ ID NO.2 comprises with Ni ²⁺Chelating chromatography column purified fusion protein, the enteropeptidase enzyme is cut fusion rotein, ResourceQ anion-exchange chromatography purifying, or with Ni ²⁺The purifying secreted type albumen of chelating chromatography column, Superdex75 gel-filtration purifying.

A kind of pharmaceutical composition comprises the pNH albumen of the listed aminoacid sequence of SEQ ID NO.2.

A kind of reagent that is used to study hematopoietic cell proliferation pNH comprises the pNH albumen of the listed aminoacid sequence of SEQ ID NO.2.

Be used to prepare the mono-clonal of anti-pNH or the antigen of polyclonal antibody, comprise the pNH albumen of the listed aminoacid sequence of SEQ ID NO.2.

The invention has the beneficial effects as follows:

1, the pNH fragment of utilizing the present invention to clone makes up the engineering bacterium expression system, and the expression and purification target protein is for research SMB structural domain function provides technical support.

2, pNH albumen can be used as the somatomedin of hematopoietic cell proliferation, can be used for the mechanism Journal of Sex Research of hematopoietic cell proliferation differentiation cell and factor interaction, also can be used as experiment reagent scale operation.

3, pNH and each structural domain polypeptide fragment thereof can be used as the disease that the lower molecular weight biotechnological formulation is applied to clinical hematopoietic disorder, have the weak advantage of molecular weight reduced immunogenicity.

4, pNH sequence fragment moderate length can be used for making up all kinds of gene therapy vectors, treatment hematopoietic disorder class disease.

5, there is a lot of dispute in SMB structural domain 3D structure elucidation result between each research group.PNH albumen is that the parsing of SMB structural domain space structure provides the foundation, and by the albumin crystal growth, the X-Ray diffraction obtains space structure information and disulfide linkage topological framework.

6, pNH and polypeptide thereof can be used for preparing polyclonal antibody and monoclonal antibody, are used for the ELISA experiment.

Description of drawings

Figure 1A is the structure of pET32c (+)-reorganization pNH protein expression vector.

Figure 1B is the structure of pET22b (+)-reorganization pNH protein expression vector.

Fig. 2 A is the abduction delivering pNH expressing fusion protein band that SDS-PAGE analyzes pNH, wherein: 1. do not induce pET32c (+)-pNH-BL21,2. induce back pET32c (+)-pNH-BL21,3. the supernatant after the carrying out ultrasonic bacteria breaking, 4. nickel post washing, 5. nickel post wash-out fusion rotein, M.Marker (kD of unit).

Fig. 2 B is the pNH albumen after SDS-PAGE analyzes reduction-non-reduced electrophoresis purifying of pNH, wherein: 1. reduced state, 2. non-reduced state, M.Marker (kD of unit).

Fig. 3 A is that the proteic Western Blot of recombinant human pNH identifies.

Fig. 3 B is that pNH iso-electric point and purity are measured in isoelectrofocusing, 1.pNH albumen wherein, M.pI standard Marker.

Fig. 4 A and PBS contrast, pNH is to the influence of the U937 growth and proliferation of cell that is in logarithmic phase.

Fig. 4 B and PBS contrast, pNH stimulates 3 days the influence of breaking up adherent U937 growth and proliferation of cell to PMA.

Fig. 5 A and PBS contrast, pNH is to the influence of the K562 growth and proliferation of cell that is in logarithmic phase.

Fig. 5 B and PBS contrast, pNH stimulates the influence of 3 days K562 growth and proliferation of cell to PMA.

Embodiment

Below in conjunction with the drawings and specific embodiments the present invention is described in further detail: but the present invention not merely limits to following examples.

The clone and the construction of prokaryotic expression vector of embodiment 1 people pNH gene

1.1 the clone of people pNH gene

Get 5 months big aborted fetus hepatic tissue 100mg, tissue is placed in the mortar, and add a small amount of liquid nitrogen, clay into power, add 1ml Trizol (available from Invitrogen company) then, change the suspension in this mortar the centrifuge tube of 1.5ml over to, add the chloroform of 0.2ml then, cover tight centrifuge tube, acutely swayed centrifuge tube 15 seconds with hand; Get supernatant liquid in a new centrifuge tube, add the 0.5ml Virahol, mixing, room temperature was placed 20 minutes, centrifugal 15 minutes of 12000g; Abandoning supernatant adds 80% ethanol of 1ml, centrifugal 5 minutes of 4 ℃ of following 7500g; Careful abandoning supernatant, room temperature or vacuum-drying 10-15 minute then, then that RNA is soluble in water, use spectrophotometric determination A260/A280, calculate rna content, and confirm that through the 12g/L agarose gel electrophoresis RNA is complete.20 μ l reverse transcription reaction systems contain the total RNA of 2 μ g, 4 μ l RT damping fluids, 50pmol/L oligo (dT) 16,1 μ mol/L dNTPs, 0.1 μ mol/L DTT, 15U RNA enzyme inhibitors (Takara), 200U M-MLV (available from Invitrogen company) is in 65 ℃ of water-bath 5min, ice bath 5min, 37 ℃ of water-bath 60min, 70 ℃ of 15min termination reactions, gained cDNA-20 ℃ of preservation is standby.Utilize gene runner software design primer according to people PRG4 gene order (NM_005807), add NcoI antisense strand 5 ' end respectively at positive-sense strand 5 ' end and add Xho I restriction enzyme site, pNH positive-sense strand primer: 5 '-G CCATGGATGGCATGGAAAACACTTCCCA-3 '; Antisense strand primer: 5 '-GG CTCGAGCTAAGGACAGTTGTACCAGACTTTGG-3 ', PCR product total length 3937bp estimates PCR fragment length 3952bp.Use Pyrobest archaeal dna polymerase (available from Takala company) amplification people pNH gene, PCR reaction conditions: 95 ℃ of pre-sex change 5min, 94 ℃ of 45s, 68 ℃ of 5min, 32 circulations, 72 ℃ of 15min.PCR product cloning behind the recovery purifying is gone in the pMD18-T carrier (available from Takala company) called after pMD18-PRG4 Δ.

1.2 pNH modifier construction of prokaryotic expression vector

Above-mentioned cytokine can prepare by several different methods.Comprising being built into prokaryotic expression system in the cDNA sequence is packed into pET32c (+) and pET22b (+) prokaryotic expression carrier.

The pMD18-PRG4 Δ is identified correct through order-checking, be template with it, uses upstream primer: 5 '-G C CATGGATGCCACCTGCAACTGTGAT-3 ' and downstream primer: 5 '-gC CTCgAgAGTTGTGACCTTGAAGTCACCAT-3 ' upstream and downstream restriction enzyme site is respectively Nco I and Xho I.Carry out pcr amplification with pfu archaeal dna polymerase (giving birth to the worker available from Shanghai), reaction conditions is 95 ℃ of pre-sex change 5min, 94 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 50s, 25 circulations are extended 15min for back 72 ℃, circulation finish 72 ℃ extend 2min after, add 1U Taq archaeal dna polymerase and continue to extend 13min.The fragment reading frame length of amplification is 336bp (SEQ ID NO.1).After the PCR product reclaimed test kit and reclaim with gel, connect in the pMD18-T carrier called after pMD18-pNH.Transformed E .coli DH5 α bacterial strain carries out blue hickie screening in the LB plate, the picking positive colony extracts plasmid with Nco I and Xho I double digestion, runs 1% agarose gel electrophoresis, reclaims test kit (available from vast Imtech) with glue and reclaims the purpose fragment.With through Nco I with after Xho I double digestion protokaryon fusion protein expression vector pET32c is connected, Transformed E .coli DH5 α bacterial strain is cut by enzyme and to be identified and obtain containing the segmental recombinant clone of purpose, and called after pET32c-pNH, sees Figure 1A.The pNH-1 that Nco I and Xho I enzyme are cut with through Nco I with after the Procaryon secreted protein expression vector pET22b of Xho I double digestion is connected, Transformed E .coli DH5 α bacterial strain, cut evaluation by enzyme and obtain containing the segmental recombinant clone of purpose, and called after pET22b-pNH, see Figure 1B.

Embodiment 2 recombinant human pNH induction expression of protein and purifying (method one)

2.1 the preparation of pNH fusion rotein

The pET32c-pNH that obtains among the embodiment 1.2 is transformed Rosetta (DE3) (available from Novagen company) prokaryotic expression bacterial strain, screening positive clone on acillin and the two resistance LB medium agar flat boards of paraxin.The single clone of picking is inoculated in the LB substratum that contains acillin (50mg/L) and paraxin (34mg/L), and 37 ℃ of shaking culture are to OD ₆₀₀Reach 0.6-0.8.Be inoculated in 1: 100 ratio and contain in the antibiotic LB substratum of same concentrations, 37 ℃ of shaking culture are to OD ₆₀₀After reaching 0.6-0.8, adding IPTG is that 1mmol/L carries out abduction delivering to final concentration.After 4 hours, 4 ℃ of centrifugal collection thalline.Be resuspended in an amount of 1 * SDS sample-loading buffer, put boiling water bath 3～5min, get 50 μ l and carry out the SDS-PAGE electrophoretic analysis.

2.2 the cracking of pNH-1 fusion rotein and purifying

3ml LB Amp ⁺Culture medium inoculated positive monoclonal bacterium colony, 37 ℃ of shaking table overnight incubation.Go into fresh culture with 1: 100 (v/v) voltage regulator tube, 37 ℃ of shaking tables are cultured to OD ₆₀₀=0.6, add IPTG to final concentration 1mM.Behind 25 ℃ of abduction delivering 7.5h, the centrifugal 30min of 8000rpm collects bacterial sediment.The 50mM Tris pH8.0 suspendible thalline that 1/10 bacteria liquid is long-pending, the centrifugal 30min of ultrasonication thalline .12000rpm collects supernatant, includes fusion rotein.

Ni ²⁺Chelating chromatography column purified fusion protein:

50mM TrisHCl pH8.0,0.5M NaCl goes up sample behind the 2mM imidazoles balance nickel post.

50mM TrisHCl pH8.0,0.5M NaCl, the washing of 80mM imidazoles,

50mM TrisHCl pH8.0,0.5M NaCl, 160mM imidazoles wash-out fusion rotein.

With the 50mM Tris pH8.0 damping fluid fusion rotein of fully dialysing, desalination.Ultrafiltration and concentration reads the 280nm ultraviolet absorptivity, utilize xPASy-ProtParam ( Http:// kr.expasy.org/tools/p Rotparam.html) optical extinction coefficient of software prediction converts, and carries out protein quantification.SDS-PAGE detects fusion rotein, sees Fig. 2 A.

Cut 16h with 4 ℃ of enzymes of 1mg albumen 3U enteropeptidase (Invitrogen company).Cut in the system to enzyme that to add 1M NaCl be that 0.5M. is with Resource Q anion-exchange column, 20mM TrispH8.0 0.5M-0M NaCl concentration gradient wash-out carrier of separating albumen and target protein and enteropeptidase to final concentration.Target protein is crossed the Superdex200 gel-filtration, is further purified and changes damping fluid to PBS.SDS-PAGE detects pure product albumen, sees Fig. 2 B.PNH albumen contains 112 amino-acid residues (SEQ ID NO.2), the separation and purification label that comprises 6 Histidines formations of two carrier amino acid of N-terminal and C-terminal, the albumen theoretical molecular is 12.9kD, and because of N end SMB structural domain is that halfcystine enriches sequence, and electrophoretic mobility is slow partially.

Behind the pure product ultrafiltration and concentration, Bio-Rad ' s DC Protein Assay test kit carries out protein quantification, packing, and-80 ℃ are frozen.

Embodiment 3 recombinant human pNH induction expression of protein and purifying (method two)

3.1 the preparation of pNH-1 secreted protein

The pET22b-pNH-1 plasmid that obtains among the embodiment 1.2 is transformed Rosetta (DE3) prokaryotic expression bacterial strain, screening positive clone on acillin and the two resistance LB medium agar flat boards of paraxin.The single clone of picking is inoculated in the LB substratum that contains acillin (50mg/L) and paraxin (34mg/L), and 37 ℃ of shaking culture reach 0.6-0.8 to OD600.Be inoculated in 1: 100 ratio and contain in the antibiotic LB substratum of same concentrations, after 37 ℃ of shaking culture reached 0.6-0.8 to OD600, adding IPTG after cooling to 28 ℃ was that 1mmol/L carries out abduction delivering to final concentration.After 8 hours, 4 ℃ of centrifugal collection thalline.Be resuspended in an amount of 1 * SDS sample-loading buffer, put boiling water bath 3～5min, get 50 μ l and carry out the SDS-PAGE electrophoretic analysis.

3.2pNH the purifying of secreted protein

The centrifugal bacterium that obtains wetting of fermented liquid that high density fermentation (method reference molecule clone) obtains is weighed, and volume ratio adds pure water at 1: 20 by weight, and thalline is fully suspended, placed 30 minutes for 4 ℃, 12500 left the heart 30 minutes, got supernatant, crossed the nickel post and prepared the pNH-1 crude product.

50mM TrisHCl pH8.0,0.5M NaCl goes up sample behind the 2mM imidazoles balance nickel post,

50mM TrisHCl pH8.0,0.5M NaCl, the washing of 40mM imidazoles,

50mM TrisHCl pH8.0,0.5M NaCl, 160mM imidazoles wash-out target protein.Cross 1 * PBS damping fluid equilibrated Superdex, 75 gel-filtration columns behind the centrifugal concentrating sample of Vivaspin3000MWCO ultrafiltration and concentration, be further purified albumen.Collect the target protein peak according to retention time, behind the pure product albumen ultrafiltration and concentration, carry out protein quantification with the Lowry method, packing ,-80 ℃ are frozen.

The proteic evaluation of embodiment 4 recombinant human pNH

4.2 Western Blotting

It is 1mg/ml that the target protein of purifying concentrates back quantitative adjustment concentration, and Western Blot method is identified target protein.

1) SDS-PAGE electrophoresis

Carry out with reference to " molecular cloning " described method.Separation gel and concentrated gum concentration are respectively 15% and 5%.

	Separation gel (ml)	Concentrate glue (ml)
			DDW 30% Polyscrylamide liquid storage 10%SDS	7 15 0.3	6.8 1.7 0.1

10％AP Tris·HCl TEMED

0.3 7.5(pH8.8) 0.012

0.1 1.25(pH6.8) 0.01

60ul target protein solution adds 10ul6 * sample-loading buffer, and 100 ℃ are boiled 3min.Voltage 80V when concentrating gel electrophoresis, voltage is 120V during the separation gel electrophoresis.Electrophoresis finishes, and soaks several minutes with transfering buffering liquid.

2) Western blot reaction

Pvdf membrane is cut SDS-PAGE glue size, and pure methyl alcohol soaks 1min, and DDW flushing SDS-PAGE glue, Whatman filter paper, pvdf membrane soak 5min with pile liner in transfering buffering liquid.Transfer device is installed in order: positive plate, pile liner, Whatman filter paper, nitrocellulose filter, SDS-PAGE glue, Whatman filter paper, pile liner, negative plate clamp clip.

The interior 1 * electrophoretic buffer of electrophoresis chamber is put the ice chest of pre-freeze.Transfer device is vertically inserted electrophoresis chamber, and the logical negative electrode of one side of glue is arranged.

120V voltage 200mA electric current (8mM/cm ²) constant voltage transfer 1h

Shift and finish, remove filter paper, SDS-PAGE glue detects the albumen transfer mass with the dyeing of Xylene Brilliant Cyanine G dye liquor

20ml confining liquid sealing pvdf membrane 1h

Add one at 1: 1000 and resist, hatch 1h

20mlPBS-T washes film three times, each 10min

10mlPBS-T, adding in 1: 1,000 two is anti-, hatches 1h

20mlPBS-T washes film three times, each 10min

Colour developing liquid 10ml adds 50ulH2O2, puts the film colour developing, approximately 2-5min.

Treat that the protein band colour developing is clear, washing film dries, and keeps in Dark Place, and sees Fig. 3 A.

4.2 isoelectric focusing electrophoresis

The theoretical iso-electric point that xPASy-ProtParam (http://kr.expasy.org/tools/protparam.html) is used for predicted protein is 6.47.

The actual iso-electric point of isoelectrofocusing experimental identification target protein.

According to laboratory manual Instruction 1818-A operation (LKB-Produkter AB company)

0.55mm polyacrylamide thin layer glue, pH3.5-9.5 carrier ampholyte, Marker are wide region iso-electric point standard reagent box (pI 3.5-9.3) (Amersham Biosciences company).

The sample desalination is to water, and adjustment concentration is 1mg/ml.

After the glue upper flat plate was cleaned, the DDW flushing was dried; Evenly be coated with silane in the glass plate, dry; The DDW flushing is dried.Following dull and stereotyped shop hydrophobic membrane.Two plate hydrophobic surfaces are relative.

Join alkaline glue (T=5%C=3%): AB 2.5ml

LKB 0.9ml

DDW 11.5ml

TEMED 8ul

The 10min that bleeds adds AP 75ul

Encapsulating.40 ℃ of short coagulating of baking oven.

Stripping glue, the generous filter paper bar of 0.5cm, it is long that length slightly is shorter than glue; Add the electrode damping fluid, negative electrode NaOH anode H ₃PO ₄Filter paper slightly blots, and places glue the two poles of the earth.

Kerosene evenly is laid on the electrophoresis plate, puts glue, puts point sample filter paper.

Point sample 20ul, 1500V, 50mA behind the 20W prefocus 30min, removes point sample filter paper.

Electrophoresis to electric current is tending towards 0mA.

Fixing, Fig. 3 B is seen in the colour developing of Xylene Brilliant Cyanine G dye liquor.

According to the position of electrophoretic band and the iso-electric point data pH gradient curve that draws, measuring and calculating target protein pI

Embodiment 5 short cell proliferation experiment

5.1 external short U937 cell proliferation experiment

U937 is with IMDM for people's acute monocytic leukemia clone, 10%FCS, and 1% cultivates for culture system, is inoculated in 96 well culture plates, every hole inoculation 5 * 10 when best to growth conditions ³Individual cell, culture system adopts IMDM, 1%FCS, 1% penicillin and streptomycin.The pNH that adds different concns behind the 5h, every hole adds 5mg/mlMTT20ul behind the cultivation 72h, continues to cultivate 4h, the centrifugal 10min of 1000rpm, careful supernatant discarded, every hole adds DMSO150ul, concussion 10min, 490nM mensuration absorbance value A.

Calculate cell proliferation rate (%):

Cell proliferation rate (%)=[(experimental group A value-PBS group A value)/(PBS group A value-not celliferous control group A value)] * 100%.

The height of A value can reflect the quantity of viable cell, and experimental result is represented with the cell proliferation percentage.Experimental result proof pNH has the propagation promoter action to the U937 cell, and the cell growth increases 26%-31%, sees Fig. 4 A.

U937 is with IMDM for people's acute monocytic leukemia clone, 10%FCS, and 1% penicillin and streptomycin is that culture system is cultivated, be inoculated in 6 well culture plates during to growth conditions the best, culture system adopts IMDM, 15%FCS, 1% penicillin and streptomycin, every hole inoculation 1 * 10 ⁶Individual cell/5ml substratum.It is 162mM that Buddhist ripple ester (PMA, Sigma company) is dissolved into final concentration with DMSO, and it is 50nM that adding K5626 orifice plate culture system becomes final concentration.When cultivating 3d, abandoning supernatant, after PBS cleans the cell of adherent differentiation, is 0.25% trysinization with final concentration, and collecting cell is inoculated in 96 orifice plates, with every hole inoculation 5 * 10 ³Individual cell, culture system adopts IMDM, 1%FCS, 1% penicillin and streptomycin.The pNH that adds different concns behind the 5h, MTT detects viable cell quantity behind the cultivation 72h.

Calculate cell proliferation rate (%):

Cell proliferation rate (%)=1-[(PBS group A value-experimental group A value)/(PBS group A value-not celliferous control group A value)] * 100%.

The U937 cell that has broken up is slightly bred inhibition to experimental result proof pNH but difference is not obvious, sees Fig. 4 B.

5.2 external short K562 cell proliferation experiment

Human erythorleukemia cell line K562 is with IMDM, 10%FCS, and 1% penicillin and streptomycin is that culture system is cultivated, and is inoculated in 96 well culture plates, every hole inoculation 5 * 10 when best to growth conditions ³Individual cell, culture system adopts IMDM, 1%FCS, 1% penicillin and streptomycin.The pNH that adds different concns behind the 5h, every hole adds 5mg/mlMTT20ul behind the cultivation 72h, continues to cultivate 4h, the centrifugal 10min of 1000rpm, careful supernatant discarded, every hole adds DMSO150ul, concussion 10min, 490nM mensuration absorbance value A.

Calculate cell proliferation rate (%):

Cell proliferation rate (%)=[(experimental group A value-PBS group A value)/(PBS group A value-not celliferous control group A value)] * 100%.

The height of A value can reflect the quantity of viable cell, and experimental result is represented with the cell proliferation percentage.Experimental result proof pNH has the propagation promoter action to the K562 cell, and the cell growth increases 10%-26%, sees Fig. 5 A.

Human erythorleukemia cell line K562 is with IMDM, 10%FCS, and 1% penicillin and streptomycin is that culture system is cultivated, and is inoculated in 6 well culture plates to growth conditions when best, culture system adopts IMDM, 15%FCS, 1% penicillin and streptomycin, every hole inoculation 1 * 10 ⁶Individual cell/5ml substratum.It is 162mM that Buddhist ripple ester (PMA, Sigma company) is dissolved into final concentration with DMSO, and it is 50nM that adding K5626 orifice plate culture system becomes final concentration.The difference collecting cell is inoculated in 96 well culture plates behind cultivation 3d, the 5d behind the PBS washed cell, every hole inoculation 5 * 10 ³Individual cell, culture system adopts IMDM, 1%FCS, 1% penicillin and streptomycin.The PNH that adds different concns behind the 5h, every hole adds 5mg/mlMTT20ul behind the cultivation 72h, continues to cultivate 4h, the centrifugal 10min of 1000rpm, careful supernatant discarded, every hole adds DMSO150ul, concussion 10min, 490nM mensuration absorbance value A.

Calculate cell proliferation rate (%):

Cell proliferation rate (%)=1-[(PBS group A value-experimental group A value)/(PBS group A value-not celliferous control group A value)] * 100%.

The K562 cell that has broken up is slightly bred inhibition to experimental result proof pNH but difference is not obvious, sees Fig. 5 B.

Embodiment 6 recombinant human pNH Polyclonal Antibody Preparation

Only select the big sub-4-6 of New Zealand's large ear rabbit of 2 weeks of fine, adaptability is fed, is observed an anosis week.Routine is carried out the fundamental immunity rabbit with bacille Calmette-Guerin vaccine.A certain amount of antigen SDS-PAGE electrophoresis coomassie brilliant blue staining (or 250Mm KCl dyeing) downcuts the purpose band, glue is ground in mortar with liquid nitrogen, mixes with adjuvant (1: 1), continues to grind to form pasty state, immunizing rabbit.Time is fundamental immunity after one week, 1-2mg antigen/only.The glue of preparation is 1mg antigen/1g glue.

Booster immunization after two weeks (or after three weeks): 1-2mg antigen/only.

Booster immunization after one week: 1-2mg antigen/only.Available pair of expansion method monitoring has or not antibody to produce.

Carry out booster immunization for the third time after one week: 1-2mg antigen/only.

After 2 weeks, get rabbit auricular vein blood, detect anti-HAPO antibody with agar double immunodiffusion method.Measure antibody titer with the ELISA method.In the serum purpose antibody positive after, extract rabbit blood in a large number, 37 ℃ of the blood of collection were placed 1-2 hour, 4 ℃ are spent the night, under aseptic condition, centrifugal collection serum, packing (0.2ml) is stored in-70 ℃.

The foregoing description proves that the resulting pNH of the present invention has hormesis for the growth of the hemocyte of naivety, and does not influence the growth of the cell that has broken up.The present invention discovers that containing pNH albumen that short cell generates plain B spline structure territory and a small amount of mucoitin sample tumor-necrosis factor glycoproteins has the function that promotes propagation for the early stage blood cell of naivety, and do not influence the growth of noble cells, show the specificity of its function performance.Clinical hematopoiesis or the vascular function sexual dysfunction disease of having, the present invention provides platform for the ancillary drug that the pNH factor becomes this type of disease of treatment.

SEQUENCE LISTING

＜110〉Inst. of Hematology, Chinese Academy of Medical Sciences, dirt life think factory of Taida of Inst. of Hematology, Chinese Academy of Medical Sciences

＜120〉human accelerated haematogenous cell proliferated cytokine and its production and use

<130>

<160>2

<170>PatentIn version 3.3

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<211>336

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＜213〉human (Homo sapiens)

<400>1

gccatggatg ccacctgcaa ctgtgattat aactgtcaac actacatgga gtgctgccct 60

gatttcaaga gagtctgcac tgcggagctt tcctgtaaag gccgctgctt tgagtccttc 120

gagagaggga gggagtgtga ctgcgacgcc caatgtaaga agtatgacaa gtgctgtccc 180

gattatgaga gtttctgtgc agaagtaaaa gataacaaga agaacagaac taaaaagaaa 240

cctaccccca aaccaccagt tgtagatgaa gctggaagtg gattggacaa tggtgacttc 300

aaggtcacaa ctctcgagca ccaccaccac caccac 336

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<212>PRT

＜213〉human (Homo sapiens)

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Ala Met Asp Ala Thr Cys Asn Cys Asp Tyr Asn Cys Gln His Tyr Met

1 5 10 15

Glu Cys Cys Pro Asp Phe Lys Arg Val Cys Thr Ala Glu Leu Ser Cys

20 25 30

Lys Gly Arg Cys Phe Glu Ser Phe Glu Arg Gly Arg Glu Cys Asp Cys

35 40 45

Asp Ala Gln Cys Lys Lys Tyr Asp Lys Cys Cys Pro Asp Tyr Glu Ser

50 55 60

Phe Cys Ala Glu Val Lys Asp Asn Lys Lys Asn Arg Thr Lys Lys Lys

65 70 75 80

Pro Thr Pro Lys Pro Pro Val Val Asp Glu Ala Gly Ser Gly Leu Asp

85 90 95

Asn Gly Asp Phe Lys Val Thr Thr Leu Glu His His His His His His

100 105 110

Claims (10)

1, a kind of human accelerated haematogenous cell proliferated cytokine is characterized in that, described human accelerated haematogenous cell proliferated cytokine pNH is made up of the protein of the listed aminoacid sequence of SEQ ID NO.2.

2, the cDNA molecule of coding pNH is characterized in that, by the whole or fragment of the listed nucleotide sequence of SEQ ID NO.1.

3, the cloning process of the cDNA of the described coding of a kind of claim 2 pNH is characterized in that, may further comprise the steps:

1), infers and the corresponding nucleotide sequence of these amino acid with known genetic code according to the listed aminoacid sequence of SEQ ID NO.2;

2) design Auele Specific Primer, carry out pcr amplification with the pfu archaeal dna polymerase, described Auele Specific Primer is: the segmental upstream primer of amplification pNH is 5 '-GCCATGGATGCCACCTGCAACTGTGAT-3 ', and downstream primer is 5 '-gCCTCgAgAGTTGTGACCTTGAAGTCACCAT-3 '.

3) with specificity cDNA amplified production as probe, filter out the cDNA of pNH from the human cDNA library, described specificity cDNA amplified production is coding 1) Nucleotide of described pNH aminoacid sequence.

4, method according to claim 3 is characterized in that, the described human cDNA library embryonic liver cell cdna library of behaving.

5, a kind of plasmid of expressing pNH is characterized in that, contains right and requires the resulting cDNA of 3 methods.

According to the plasmid of the described expression of claim 5 pNH, it is characterized in that 6, described plasmid constitutes from pET32c (+) or pET22b (+).

7, the purification process of the pNH of the listed aminoacid sequence of a kind of SEQ ID NO.2 is characterized in that, comprises with Ni ²⁺Chelating chromatography column purified fusion protein, the enteropeptidase enzyme is cut fusion rotein, ResourceQ anion-exchange chromatography purifying target protein, or with Ni ²⁺The purifying secreted type albumen of chelating chromatography column, Superdex75 gel-filtration purifying target protein.

8, a kind of pharmaceutical composition is characterized in that, comprises the pNH albumen of the listed aminoacid sequence of SEQ ID NO.2.

9, a kind of reagent that is used to study hematopoietic cell proliferation pNH is characterized in that, comprises the pNH albumen of the listed aminoacid sequence of SEQ ID NO.2.

10, be used to prepare the mono-clonal of anti-pNH or the antigen of polyclonal antibody, it is characterized in that, comprise the pNH albumen of the listed aminoacid sequence of SEQ ID NO.2.

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