CN1556114A - Human dry cell factor macrophage clong irritant factor bifunction protein and its preparation - Google Patents
Human dry cell factor macrophage clong irritant factor bifunction protein and its preparation Download PDFInfo
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Abstract
A dual-function protein human stem cell factor-macrophage colony stimulating factor (hSCF/M-CSF) and a process for preparing its recombinant one (rhSCF/M-CSF) by efficient secretion expression in insect are disclosed. It can be used for rebuilding and restoring the hematopoietic and immune functions of a patient after radiotherapy, chemicotherapy and bone marrow transplantation.
Description
One. technical field
The invention belongs to genetically engineered and prepare the polypeptide drugs technical field.
Two. background technology
STEM CELL FACTOR (SCF, also be the Kit part, the Steel factor, mast cell growth factor etc.) be a kind of multifunctional cytokine, can promote the propagation of early stage hemopoietic stem cell and the not differentiation of inducing cell, can be in survival and the propagation of multistage hematopoiesis level with other cytokines synergy promotion hematopoietic stem and various hematopoietic cells.Its acceptor is distributed widely in the hematopoietic cell of different series, different generations, comprises erythron, monokaryon grain giant cells system and megakaryocytic series etc.SCF can strengthen the multiplication capacity of these cells and activate these cells makes it more responsive to the stimulation of corresponding various hematopoietic cytokines.Clinically be mainly used in radiotherapy, the reconstruction of hematopoiesis and immunologic function and recovery after chemotherapy and the bone marrow transplantation.SCF is to also having application prospect in bone marrow stem cell amplification in vitro and stem cell transplantation and the gene therapy in addition.SCF can promote the growth of mastocyte separately.And can activate sophisticated mastocyte, and promote it to take off particle, discharge histamine.So the itch rubella can appear in the injection site of most patient when using, abnormal sample reaction also can take place in a few patients.This side effects limit the clinical application of SCF.
Natural SCF is a kind of glycoprotein, exists with monomeric form more than 90% under normal physiological conditions, can form the ionic homodimer when high local concentrations, and the high 10-20 of dimeric forms SCF specific activity monomer doubly.
Macrophage colony stimulating factor (M-CSF) is a kind of multi-functional cytokine, also is a kind of immunomodulator.It can stimulate bone marrow stem cell to be divided into mononuclear macrophage and promote its propagation and maturation.It can also promote mononuclear macrophage to engulf bacterium, fungi and tumour cell specifically, and stimulates multiple hematopoietic cytokines such as scavenger cell secretion G-CSF, GM-CSF, TNF, IL-6, IL-8.In addition, it also is the somatomedin of marrow stromal cell, participates in hematopoieticmicroenviron-ment and marrow-reconstitution.Clinically, M-CSF can effectively improve the immunologic function behind bone marrow transplantation and the tumour patient chemicotherapy, significantly improves the survival rate of these patient infection lethality Candida albicanss and aspergillus tubigensis.But long-term and heavy dose of M-CSF that uses can suppress hematopoiesis, and cause the damage to multiple organ.Natural M-CSF is a kind of glycoprotein by the covalently bound homodimer form of interchain disulfide bond.
SCF and M-CSF have synergy, and especially under the serum-free culture condition, its synergy is more obvious.
Utilize M-CSF under natural condition, can form the covalency homodimer by cys31-cys31, and SCF and M-CSF have these characteristics of synergy, make up and at the STEM CELL FACTOR-macrophage colony stimulating factor bifunctional protein (SCF/M-CSF) of expressed in insect cells homodimer form by gene recombination technology, to significantly improve the activity of SCF and M-CSF, overcome the side effect that causes when using SCF and M-CSF because of heavy dose clinically.
Three. summary of the invention
The patent technical issues that need to address of the present invention are to have designed to guarantee SCF and the M-CSF impregnable flexible peptide linker of natural radioactivity separately, make up a kind of STEM CELL FACTOR-macrophage colony stimulating factor bifunctional protein gene (rhSCF/M-CSF) by genetic engineering technique, make it efficient secretory expression in Sf9 cell (Invitrogen), and from nutrient solution separation and purification rhSCF/M-CSF, but to reach application level.Becoming a kind of performance better recovers hematopoiesis and regulates the medicine of immunologic function than SCF and M-CSF monomeric protein.
Technology contents of the present invention comprises: 1. the design and the structure of rhMGF-macrophage colony stimulating factor bifunctional protein gene (rhSCF/M-CSF).2. the structure of the rhSCF/M-CSF of efficient secretory expression reorganization clover californica nuclear polyhedrosis virus AcNPV-SCF/M-CSF.3.rhSCF/M-CSF secreting, expressing in the Sf9 cell and separation and purification thereof and evaluation.
The rhSCF/M-CSF bifunctional protein gene of our design is to contain 25 amino acid whose complete signal peptides of SCF and 1-165 amino acids, 15 amino acid whose connection peptides and the 1-149 amino acids encoding sequence of M-CSF and the dna fragmentation of terminator codon.
ATG?AAG?AAG?ACA?CAA?-61
Met?Lys?Lys?Thr?Gln?-21
ACT?TGG?ATT?CTC?ACT?TGC?ATT?TAT?CTT?CAG?CTG?CTC?CTA?TTT?AAT?CCT?CTC?GTC?AAA?ACT -1
Thr?Trp?Ile?Leu?Thr?Cys?Ile?Tyr?Leu?Gln?Leu?Leu?Leu?Phe?Asn?Pro?Leu?Val?Lys?Thr -1
GAA?GGG?ATC?TGC?AGG?AAT?CGT?GTG?ACT?AAT?AAT?GTA?AAA?GAC?GTC?ACT?AAA?TTG?GTG?GCA 60
Glu?Gly?Ile?Cys?Arg?Asn?Arg?Val?Thr?Asn?Asn?Val?Lys?Asp?Val?Thr?Lys?Leu?Val?Ala 20
AAT?CTT?CCA?AAA?GAC?TAC?ATG?ATA?ACC?CTC?AAA?TAT?GTC?CCC?GGG?ATG?GAT?GTT?TTG?CCA?120
Asn?Leu?Pro?Lys?Asp?Tyr?Met?Ile?Thr?Leu?Lys?Tyr?Val?Pro?Gly?Met?Asp?Val?Leu?Pro 40
AGT?CAT?TGT?TGG?ATA?AGC?GAG?ATG?GTA?GTA?CAA?TTG?TCA?GAC?AGC?TTG?ACT?GAT?CTT?CTG?180
Ser?His?Cys?Trp?Ile?Ser?Glu?Met?Val?Val?Gln?Leu?Ser?Asp?Ser?Leu?Thr?Asp?Leu?Leu 60
GAC?AAG?TTT?TCA?AAT?ATT?TCT?GAA?GGC?TTG?AGT?AAT?TAT?TCC?ATC?ATA?GAC?AAA?CTT?GTG?240
Asp?Lys?Phe?Ser?Asn?Ile?Ser?Glu?Gly?Leu?Ser?Asn?Tyr?Ser?Ile?Ile?Asp?Lys?Leu?Val 80
AAT?ATA?GTG?GAT?GAC?CTT?GTG?GAG?TGC?GTG?AAA?GAA?AAC?TCA?TCT?AAG?GAT?CTA?AAA?AAA?300
Asn?Ile?Val?Asp?Asp?Leu?Val?Glu?Cys?Val?Lys?Glu?Asn?Ser?Ser?Lys?Asp?Leu?Lys?Lys?100
TCA?TTC?AAG?AGC?CCA?GAA?CCC?AGG?CTC?TTT?ACT?CCT?GAA?GAA?TTC?TTT?AGA?ATT?TTT?AAT?360
Ser?Phe?Lys?Ser?Pro?Glu?Pro?Arg?Leu?Phe?Thr?Pro?Glu?Glu?Phe?Phe?Arg?Ile?Phe?Asn?120
AGA?TCC?ATT?GAT?GCC?TTC?AAG?GAC?TTT?GTA?GTG?GCA?TCT?GAA?ACT?AGT?GAT?TGT?GTG?GTT?420
Arg?Ser?Ile?Asp?Ala?Phe?Lys?Asp?Phe?Val?Val?Ala?Ser?Glu?Thr?Ser?Asp?Cys?Val?Val?140
TCT?TCA?ACA?TTA?AGT?CCT?GAG?AAA?GAT?TCC?AGA?GTC?AGT?GTC?ACA?AAA?CCA?TTT?ATG?TTA?480
Ser?Ser?Thr?Leu?Ser?Pro?Glu?Lys?Asp?Ser?Arg?Val?Ser?Val?Thr?Lys?Pro?Phe?Met?Leu?160
CCC?CCT?GTT?GCA?GCC
540
Pro?Pro?Val?Ala?Ala
180
GAG?GAG?GTG?TCG?GAG?TAC?TGT?AGC?CAC?ATG?ATT?GGG?AGT?GGA?CAC?CTG?CAG?TCT?CTG?CAG?600
Glu?Glu?VaL?Ser?Glu?Tyr?Cys?Ser?His?Met?Ile?Gly?Ser?Gly?His?Leu?Gln?Ser?Leu?Gln?200
CGG?CTG?ATT?GAC?AGT?CAG?ATG?GAG?ACC?TCG?TGC?CAA?ATT?ACA?TTT?GAG?TTT?GTA?GAC?CAG?660
Leu?Arg?Ile?Asp?Ser?Gln?Met?Glu?Thr?Ser?Cys?Gln?Ile?Thr?Phe?Glu?Phe?Val?Asp?Gln?220
GAA?CAG?TTG?AAA?GAT?CCA?GTG?TGC?TAC?CTT?AAG?AAG?GCA?TTT?CTC?CTG?GTA?CAA?GAC?ATA?720
Glu?Gln?Leu?Lys?Asp?Pro?Val?Cys?Tyr?Leu?Lys?Lys?Ala?Phe?Leu?Leu?Val?Gln?Asp?Ile?240
ATG?GAG?GAC?ACC?ATG?CGC?TTC?AGA?GAT?AAC?ACC?CCC?AAT?GCC?ATC?GCC?ATC?GTG?CAG?CTG?780
Met?Glu?Asp?Thr?Met?Arg?Phe?Arg?Asp?Asn?Thr?Pro?Asn?Ala?Ile?Ala?Ile?Val?Gln?Leu?260
CAG?GAA?CTC?TCT?TTG?AGG?CTG?AAG?AGC?TGC?TTC?ACC?AAG?GAT?TAT?GAA?GAG?CAT?GAC?AAG?840
Gln?Glu?Leu?Ser?Leu?Arg?Leu?Lys?Ser?Cys?Phe?Thr?Lys?Asp?Tyr?Glu?Glu?His?Asp?Lys?280
GCC?TGC?GTC?CGA?ACT?TTC?TAT?GAG?ACA?CCT?CTC?CAG?TTG?CTG?GAG?AAG?GTC?AAG?AAT?GTC?900
Ala?Cys?Val?Arg?Thr?Phe?Tyr?Glu?Thr?Pro?Leu?Gln?Leu?Leu?Glu?Lys?Val?Lys?Asn?Val?300
TTT?AAT?GAA?ACA?AAG?AAT?CTC?CTT?GAC?AAG?GAC?TGG?AAT?ATT?TTC?AGC?AAG?AAC?TGC?AAC?960
Phe?Asn?Glu?Thr?Lys?Asn?Leu?Leu?Asp?Lys?Asp?Trp?Asn?Ile?Phe?Ser?Lys?Asn?Cys?Asn?320
AAC?AGC?TTT?GCT?GAA?TGC?TCC?AGC?CAA?TAA?TAG
Asn?Ser?Phe?Ala?Glu?Cys?Ser?Ser?Gln
1.1.SCF/M-CSF structure:
A. with pUC18-SCF template, with primer 1,2 through synthetic 5 ' the end band Xba I restriction enzyme site of PCR method, 3 ' end disappearance terminator codon but band flexible peptide linker (GGGGS) encoding sequence and BamH I site contain SCF25 amino acid whose complete signal peptide and 1-165 amino acids encoding sequence, and it is cloned in the pUC18 plasmid vector (commercially available), obtain pUC18-5 ' SCF plasmid
Primer 1:5 ' GG
TCTAGATGAAGAAGACACAAACT-3 '
Primer 2: 5 ' CC
GGATCCTCCTCCTCCGGCTGCAACAGGGGGTAA-3 '
B. with pUC18-M-CSF template, with primer 3,4 through synthetic 5 ' the end band BamH I restriction enzyme site of PCR method, what flexible peptide linker (GSGGGGSGGSIT) encoding sequence and 3 ' end had two continuous terminator codons and a KpnI site contains M-CSF 1-149 amino acids encoding sequence, obtains pUC18-3 ' M-CSF plasmid
Primer 3:5 '-GG
GGATCCGGAGGAGGAGGCTCCGGCGGCAGTATCACCGAGGAGGTGTCG-3 '
Primer 4:5 '-GC
GGTACCGGGCTATTATTGGCTGGAGCATTC-3 '
C. use Xba I and BamH I from pUC18-5 '-SCF plasmid, to downcut the SCF gene fragment, insert between the Xba I and BamH I of pUC18-3 ' M-CSF plasmid, obtain recombinant plasmid pUC18-SCF/M-CSF.In this recombinant plasmid, the encoding sequence of SCF signal peptide and N end 1-165aa and M-CSF N end 1-149aa encoding sequence and two terminator codons link together by this section of 15 amino acid (GGGGSGGGGSGGSIT) flexible peptide linker, thereby obtain SCF/M-CSF bifunctional protein gene, total length 1068bp.
1.2. the structure of recombinant transfer vector and the purifying of recombinant virus:
A. the SCF/M-CSF bifunctional protein gene fragment of from the pUC18-SCF/M-CSF plasmid, downcutting the band signal peptide with Xba I and KpnI, insert between the Xba I and KpnI site of AcNPV transfer vector pVL1392 (Invitrogen), obtain recombinant transfer vector pVL1392-SCF/M-CSF.
B. with recombinant transfer vector pVL1392-SCF/M-CSF and wild-type clover californica nuclear polyhedrosis virus DNA liposome mediated-method cotransfection fall army worm cell Sf9, homologous recombination and 4 is taken turns limited sparse method screening and is surveyed the recombinant virus AcNPV-SCF/M-CSF that filters out efficient secretory expression SCF/M-CSF alive in conjunction with TF-1 cell mtt assay in born of the same parents.
2. the SCF/M-CSF virus of will recombinating infects the good Sf9 cell of growth conditions with suitable infection multiplicity (MOI), infect the back at serum free medium Sf900 or do not cultivate among the TMN-FH of increase serum, expresses that the collection nutrient solution obtains the SCF/M-CSF bifunctional protein through the separation and purification of M-CSF antibody column affinity chromatography after 72 hours.
The rhSCF/M-CSF gene that the present invention makes up is for being connected the Novel Human STEM CELL FACTOR-macrophage colony stimulating factor bifunctional protein gene that forms with a SCF monomer gene (25 amino acid signal peptide and 1-165 amino acids) and a M-CSF monomer gene (1-149 amino acids) with 15 peptides (GGGGSGGGGSGGSIT) encoding sequence of flexibility in the mode of being end-to-end.Product in expressed in insect cells forms (CFU-M through TF-1 cell mtt assay (SCF activity) and people's bone marrow macrophage colony, the M-CSF activity) surveys the analysis of living, the result shows, SCF is active detect in the monomer rhSCF (purity＞98%) that expresses in than E.coli of rhSCF/M-CSF higher 5.3 times than living, and short people's bone marrow macrophage colony form activity (M-CSF activity) detect in the pure product rhM-CSF that expresses in than silkworm of rhSCF/M-CSF exceed 5 times.Therefore can when clinical application, reduce consumption, reduce its side effect.
The recombinant virus that makes up infects individual layer Sf9 cell by the method for the invention, the collecting cell supernatant liquor, and cumulative expression of recombinant proteins amount is about 1mg/L in the supernatant liquor.
The SCF/M-CSF that can obtain from the Sf9 cell culture fluid by separation purification method provided by the invention is 1.0 * 10 than work
6Units/mg.Non-reduced condition SDS-PAGE is after coomassie brilliant blue staining detects, and rhSCF/M-CSF is the band of the about 84KD of molecular weight, and SDS-PAGE is after coomassie brilliant blue staining detects under the reductive condition, and rhSCF/M-CSF is the band of the about 42KD of molecular weight.Purity is about 90%.
Human stem cell factor macrophage colony stimulating factor bifunctional protein of the present invention can be used for promoting hematopoiesis and immunity function restructuring and recovery after patient's radiotherapy, chemotherapy and the bone marrow transplantation, and strengthen the ability of its anti-fungal infection, also can be used for the amplification in vitro research of stem cell.
Four. description of drawings
The structure schema of Fig. 1 rhSCF/M-CS gene
The sequencing result figure of Fig. 2 rhSCF/M-CSF gene (5 ' end sequencing result)
The sequencing result figure of Fig. 3 rhSCF/M-CSF gene (3 ' end sequencing result)
The monomeric active comparison diagram of Fig. 4 rhSCF/M-CSF and rhSCF
Fig. 5 rhSCF/M-CSF SDS-PAGE and Westen-blot collection of illustrative plates
Five. embodiment
1.1. with pUC18-SCF is template, with primer 1,2 are cloned into the pUC18 plasmid vector through the synthetic SCF gene fragment of PCR method and with it, obtain pUC18-5 ' SCF plasmid, with pUC18-M-CSF is template, synthesizes the M-CSF gene fragment with primer 3,4 through the PCR method, and it is cloned into the pUC18 plasmid vector obtains pUC18-3 ' M-CSF plasmid, the SCF gene fragment clone is got pUC18-SCF/M-CSF to pUC18-3 ' M-CSF.
1.2. with the pUC18-SCF/M-CSF enzyme cut the SCF/M-CSF gene fragment, and insert between the Xba I and KpnI site of AcNPV transfer vector, obtain recombinant transfer vector pVL1392-M-CSF/SCF.With pVL1392-SCF/M-CSF and AcNPV DNA cotransfection Sf9, survey the recombinant virus AcNPV-SCF/M-CSF that does not contain wild-type virus that selects alive through screening and in conjunction with the TF-1 cell.
2. getting the doubling time is 21 hours, and viable cell accounts for 90%, and density is 1 * 10
6Cell/mLSf9 cell infects recombinant virus after 1 hour by infection multiplicity (MOI)=10, and the venom of preventing or cure a disease that inclines changes the TMN-FH substratum of serum-free into.Cultivate after 72 hours for 27 ℃, survey nutrient solution recombinant protein SCF/M-CSF with the TF-1 cell, the activity that obtains SCF/M-CSF expression supernatant liquor is 1000units/mL.After the cell freeze thawing that low-speed centrifugal is collected in the extraction liquid of cell not excretory recombinant protein activity only be 50units/6 * 10
6Cells illustrates the recombinant protein secretion fully.Expression amount is about 1mg/L.To 1 * PBS (pH7.4) dialysis 12 hours, with the M-CSF antibody mab post affinity column of sample on the 1mL/min flow velocity to 0.5mL, chromatography column was used 1 * PBS balance in advance with the 40mL cell culture supernatant.Behind the last sample, be washed till baseline with 1 * PBS earlier, again with the 1 * PBS washing that contains 0.8%Triton X-100, after using 1 * PBS to be washed till baseline once more, (pH 8.0,50mM Tris-Cl with elutriant, 0.5Mol/L NaCl), collect elution peak 15mL with the flow velocity wash-out of 2mL/min.Elutriant obtains SCF/M-CSF bifunctional protein elaboration to distill water dialysis and freeze-drying.Measure protein content and activity.The result shows that rhSCF/M-CSF is 1.0 * 10 than work in the active detection of SCF
6Units/mg is higher by 6 than living than the monomer rhSCF that expresses among the E.coli (purity＞98%); Short people's bone marrow macrophage colony form activity (M-CSF activity) detect in rhSCF/M-CSF be 7 * 10 than work
6CFU/mg exceeds 5 times than the pure product of the rhM-CSF that expresses in the silkworm.SDS-PAGE under the non-reduced condition is after coomassie brilliant blue staining detects, and rhSCF/M-CSF is the band of the about 84KD of molecular weight, and SDS-PAGE is after coomassie brilliant blue staining detects under the reductive condition, and rhSCF/M-CSF is the band of the about 42KD of molecular weight.Purity is about 90%.Immunoblotting (Westernblot) analytical results consistent with it (seeing accompanying drawing 5).
Human stem cell factor macrophage colony stimulating factor bifunctional protein gene order table .txt
Human stem cell factor macrophage colony stimulating factor bifunctional protein gene order table
＜110〉Nanjing University
＜120〉human stem cell factor macrophage colony stimulating factor bifunctional protein and preparation
<210>1
<211>1068
<212>DNA
＜213〉people (Homo sapiens)
<400>1
atgaagaaga?cacaaacttg?gattctcact?tgcatttatc?ttcagctgct?cctatttaat 60
cctctcgtca?aaactgaagg?gatctgcagg?aatcgtgtga?ctaataatgt?aaaagacgtc 120
actaaattgg?tggcaaatct?tccaaaagac?tacatgataa?ccctcaaata?tgtccccggg 180
atggatgttt?tgccaagtca?ttgttggata?agcgagatgg?tagtacaatt?gtcagacagc 240
ttgactgatc?ttctggacaa?gttttcaaat?atttctgaag?gcttgagtaa?ttattccatc 300
atagacaaac?ttgtgaatat?agtggatgac?cttgtggagt?gcgtgaaaga?aaactcatct 360
aaggatctaa?aaaaatcatt?caagagccca?gaacccaggc?tctttactcc?tgaagaattc 420
tttagaattt?ttaatagatc?cattgatgcc?ttcaaggact?ttgtagtggc?atctgaaact 480
agtgattgtg?tggtttcttc?aacattaagt?cctgagaaag?attccagagt?cagtgtcaca 540
aaaccattta?tgttaccccc?tgttgcagcc?ggaggaggag?gatccggagg?aggaggctcc 600
ggcggcagta?tcaccgagga?ggtgtcggag?tactgtagcc?acatgattgg?gagtggacac 660
ctgcagtctc?tgcagcggct?gattgacagt?cagatggaga?cctcgtgcca?aattacattt 720
gagtttgtag?accaggaaca?gttgaaagat?ccagtgtgct?accttaagaa?ggcatttctc 780
ctggtacaag?acataatgga?ggacaccatg?cgcttcagag?ataacacccc?caatgccatc 840
gccatcgtgc?agctgcagga?actctctttg?aggctgaaga?gctgcttcac?caaggattat 900
gaagagcatg?acaaggcctg?cgtccgaact?ttctatgaga?cacctctcca?gttgctggag 960
aaggtcaaga?atgtctttaa?tgaaacaaag?aatctccttg?acaaggactg?gaatattttc?1020
agcaagaact?gcaacaacag?ctttgctgaa?tgctccagcc?aataatag
Human stem cell factor macrophage colony stimulating factor bifunctional protein gene DNA sequence
Met?Lys?Lys?Thr?Gln?Thr?Trp?Ile?Leu?Thr?Cys?Ile?Tyr?Leu?Gln?Leu?Leu?Leu?Phe?Asn
5 10 15 20
Pro?Leu?Val?Lys?Thr?Glu?Gly?Ile?Cys?Arg?Asn?Arg?Val?Thr?Asn?Asn?Val?Lys?Asp?Val
25 30 35 40
Thr?Lys?Leu?Val?Ala?Asn?Leu?Pro?Lys?Asp?Tyr?Met?Ile?Thr?Leu?Lys?Tyr?Val?Pro?Gly
45 50 55 60
Met?Asp?Val?Leu?Pro?Ser?His?Cys?Trp?Ile?Ser?Glu?Met?Val?Val?Gln?Leu?Ser?Asp?Ser
65 70 75 80
Leu?Thr?Asp?Leu?Leu?Asp?Lys?Phe?Ser?Asn?Ile?Ser?Glu?Gly?Leu?Ser?Asn?Tyr?Ser?Ile
85 90 95 100
Ile?Asp?Lys?Leu?Val?Asn?Ile?Val?Asp?Asp?Leu?Val?Glu?Cys?Val?Lys?Glu?Asn?Ser?Ser
105 110 115 120
Lys?Asp?Leu?Lys?Lys?Ser?Phe?Lys?Ser?Pro?Glu?Pro?Arg?Leu?Phe?Thr?Pro?Glu?Glu?Phe
125 130 135 140
Phe?Arg?Ile?Phe?Asn?Arg?Ser?Ile?Asp?Ala?Phe?Lys?Asp?Phe?Val?Val?Ala?Ser?Glu?Thr
145 150 155 160
Ser?Asp?Cys?Val?Val?Ser?Ser?Thr?Leu?Ser?Pro?Glu?Lys?Asp?Ser?Arg?Val?Ser?Val?Thr
165 170 175 180
Lys?Pro?Phe?Met?Leu?Pro?Pro?Val?Ala?Ala?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
185 190 195 200
Gly?Gly?Ser?Ile?Thr?Glu?Glu?VaL?Ser?Glu?Tyr?Cys?Ser?His?Met?Ile?Gly?Ser?Gly?His
205 210 215 220
Leu?Gln?Ser?Leu?Gln?Leu?Arg?Ile?Asp?Ser?Gln?Met?Glu?Thr?Ser?Cys?Gln?Ile?Thr?Phe
225 230 235 240
Glu?Phe?Val?Asp?Gln?Glu?Gln?Leu?Lys?Asp?Pro?Val?Cys?Tyr?Leu?Lys?Lys?Ala?Phe?Leu
245 250 255 260
Leu?Val?Gln?Asp?Ile?Met?Glu?Asp?Thr?Met?Arg?Phe?Arg?Asp?Asn?Thr?Pro?Asn?Ala?Ile
265 270 275 280
Ala?Ile?Val?Gln?Leu?Gln?Glu?Leu?Ser?Leu?Arg?Leu?Lys?Ser?Cys?Phe?Thr?Lys?Asp?Tyr
285 290 295 300
Glu?Glu?His?Asp?Lys?Ala?Cys?Val?Arg?Thr?Phe?Tyr?Glu?Thr?Pro?Leu?Gln?Leu?Leu?Glu
305 310 315 320
Lys?Val?Lys?Asn?Val?Phe?Asn?Glu?Thr?Lys?Asn?Leu?Leu?Asp?Lys?Asp?Trp?Asn?Ile?Phe
325 330 335 340
Ser?Lys?Asn?Cys?Asn Asn?Ser?Phe?Ala?Glu Cys?Ser?Ser?Gln
345 350
Human stem cell factor macrophage colony stimulating factor bifunctional protein aminoacid sequence
Claims (3)
1. STEM CELL FACTOR-macrophage colony stimulating factor bifunctional protein gene (rhSCF/M-CSF) is characterized in that designing and make up connection peptides and the 1-149 amino acids encoding sequence of M-CSF and the dna fragmentation of termination codon of 25 amino acid whose complete signal peptides containing SCF and 1-165 amino acids, 15 amino acid GGGGSGGGGSGGSIT.The nucleotide sequence and the aminoacid sequence of reorganization rhSCF/M-CSF bifunctional protein gene are:
ATG?AAG?AAG?ACA?CAA -61
Met?Lys?Lys?Thr?Gln -21
ACT?TGG?ATT?CTC?ACT?TGC?ATT?TAT?CTT?CAG?CTG?CTC?CTA?TTT?AAT?CCT?CTC?GTC?AAA?ACT -1
Thr?Trp?Ile?Leu?Thr?Cys?Ile?Tyr?Leu?Gln?Leu?Leu?Leu?Phe?Asn?Pro?Leu?Val?Lys?Thr -1
GAA?GGG?ATC?TGC?AGG?AAT?CGT?GTG?ACT?AAT?AAT?GTA?AAA?GAC?GTC?ACT?AAA?TTG?GTG?GCA 60
Glu?Gly?Ile?Cys?Arg?Asn?Arg?Val?Thr?Asn?Asn?Val?Lys?Asp?Val?Thr?Lys?Leu?Val?Ala 20
AAT?CTT?CCA?AAA?GAC?TAC?ATG?ATA?ACC?CTC?AAA?TAT?GTC?CCC?GGG?ATG?GAT?GTT?TTG?CCA 120
Asn?Leu?Pro?Lys?Asp?Tyr?Met?Ile?Thr?Leu?Lys?Tyr?Val?Pro?Gly?Met?Asp?Val?Leu?Pro 40
AGT?CAT?TGT?TGG?ATA?AGC?GAG?ATG?GTA?GTA?CAA?TTG?TCA?GAC?AGC?TTG?ACT?GAT?CTT?CTG 180
Ser?His?Cys?Trp?Ile?Ser?Glu?Met?Val?Val?Gln?Leu?Ser?Asp?Ser?Leu?Thr?Asp?Leu?Leu 60
GAC?AAG?TTT?TCA?AAT?ATT?TCT?GAA?GGC?TTG?AGT?AAT?TAT?TCC?ATC?ATA?GAC?AAA?CTT?GTG 240
Asp?Lys?Phe?Ser?Asn?Ile?Ser?Glu?Gly?Leu?Ser?Asn?Tyr?Ser?Ile?Ile?Asp?Lys?Leu?Val 80
AAT?ATA?GTG?GAT?GAC?CTT?GTG?GAG?TGC?GTG?AAA?GAA?AAC?TCA?TCT?AAG?GAT?CTA?AAA?AAA 300
Asn?Ile?Val?Asp?Asp?Leu?Val?Glu?Cys?Val?Lys?Glu?Asn?Ser?Ser?Lys?Asp?Leu?Lys?Lys 100
TCA?TTC?AAG?AGC?CCA?GAA?CCC?AGG?CTC?TTT?ACT?CCT?GAA?GAA?TTC?TTT?AGA?ATT?TTT?AAT 360
Ser?Phe?Lys?Ser?Pro?Glu?Pro?Arg?Leu?Phe?Thr?Pro?Glu?Glu?Phe?Phe?Arg?Ile?Phe?Asn 120
AGA?TCC?ATT?GAT?GCC?TTC?AAG?GAC?TTT?GTA?GTG?GCA?TCT?GAA?ACT?AGT?GAT?TGT?GTG?GTT 420
Arg?Ser?Ile?Asp?Ala?Phe?Lys?Asp?Phe?Val?Val?Ala?Ser?Glu?Thr?Ser?Asp?Cys?Val?Val 140
TCT?TCA?ACA?TTA?AGT?CCT?GAG?AAA?GAT?TCC?AGA?GTC?AGT?GTC?ACA?AAA?CCA?TTT?ATG?TTA 480
Ser?Ser?Thr?Leu?Ser?Pro?Glu?Lys?Asp?Ser?Arg?Val?Ser?Val?Thr?Lys?Pro?Phe?Met?Leu 160
CCC?CCT?GTT?GCA?GCC
540
Pro?Pro?Val?Ala?Ala
180
GAG?GAG?GTG?TCG?GAG?TAC?TGT?AGC?CAC?ATG?ATT?GGG?AGT?GGA?CAC?CTG?CAG?TCT?CTG?CAG 600
Glu?Glu?VaL?Ser?Glu?Tyr?Cys?Ser?His?Met?Ile?Gly?Ser?Gly?His?Leu?Gln?Ser?Leu?Gln 200
CGG?CTG?ATT?GAC?AGT?CAG?ATG?GAG?ACC?TCG?TGC?CAA?ATT?ACA?TTT?GAG?TTT?GTA?GAC?CAG 660
Leu?Arg?Ile?Asp?Ser?Gln?Met?Glu?Thr?Ser?Cys?Gln?Ile?Thr?Phe?Glu?Phe?Val?Asp?Gln 220
GAA?CAG?TTG?AAA?GAT?CCA?GTG?TGC?TAC?CTT?AAG?AAG?GCA?TTT?CTC?CTG?GTA?CAA?GAC?ATA 720
Glu?Gln?Leu?Lys?Asp?Pro?Val?Cys?Tyr?Leu?Lys?Lys?Ala?Phe?Leu?Leu?Val?Gln?Asp?Ile 240
ATG?GAG?GAC?ACC?ATG?CGC?TTC?AGA?GAT?AAC?ACC?CCC?AAT?GCC?ATC?GCC?ATC?GTG?CAG?CTG 780
Met?Glu?Asp?Thr?Met?Arg?Phe?Arg?Asp?Asn?Thr?Pro?Asn?Ala?Ile?Ala?Ile?Val?Gln?Leu 260
CAG?GAA?CTC?TCT?TTG?AGG?CTG?AAG?AGC?TGC?TTC?ACC?AAG?GAT?TAT?GAA?GAG?CAT?GAC?AAG 840
Gln?Glu?Leu?Ser?Leu?Arg?Leu?Lys?Ser?Cys?Phe?Thr?Lys?Asp?Tyr?Glu?Glu?His?Asp?Lys 280
GCC?TGC?GTC?CGA?ACT?TTC?TAT?GAG?ACA?CCT?CTC?CAG?TTG?CTG?GAG?AAG?GTC?AAG?AAT?GTC 900
Ala?Cys?Val?Arg?Thr?Phe?Tyr?Glu?Thr?Pro?Leu?Gln?Leu?Leu?Glu?Lys?Val?Lys?Asn?Val 300
TTT?AAT?GAA?ACA?AAG?AAT?CTC?CTT?GAC?AAG?GAC?TGG?AAT?ATT?TTC?AGC?AAG?AAC?TGC?AAC 960
Phe?Asn?Glu?Thr?Lys?Asn?Leu?Leu?Asp?Lys?Asp?Trp?Asn?Ile?Phe?Ser?Lys?Asn?Cys?Asn 320
AAC?AGC?TTT?GCT?GAA?TGC?TCC?AGC?CAA?TAA?TAG
Asn?Ser?Phe?Ala?Glu?Cys?Ser?Ser?Gln
According to what claim 1 provided gene constructed a kind of can be in insect Sf9 the reorganization clover californica nuclear polyhedrosis virus (AcNPV-rhSCF/M-CSF) of efficient secretory expression.It is characterized in that the rhSCF/M-CSF gene clone between the BamHI and SmaI site of baculovirus transfer vector pVL1392, obtain recombinant transfer vector pVL1392rhSCF/M-CSF, whole fusion gene is under the polyhedrin promotor control of AcNPV.To recombinate pVL1392 rhSCF/M-CSF carrier DNA and wild-type AcNPV DNA cotransfection Sf9 cell are taken turns the limiting dilution assay purifying and in conjunction with the active determination in vitro of recombinant protein, are filtered out recombinant virus AcNPV-rhSCF/M-CSF through 4.
3. the preparation method of a STEM CELL FACTOR macrophage colony stimulating factor bifunctional protein (rhSCF/M-CSF).It is characterized in that with the constructed adherent Sf9 cell of recombinant virus infection of claim 1 expressed 72 hours for 27 ℃, collecting cell is expressed liquid in serum free medium.Can obtain the SCF/M-CSF bifunctional protein elaboration of purity about 90% through the separation and purification of M-CSF antibody mab post affinity chromatography.
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| CN100336907C (en) * | 2005-09-29 | 2007-09-12 | 南京大学 | Recombinant human platelet auxin/dry cell factor fusion protein and preparation thereof |
| CN105219750A (en) * | 2015-08-25 | 2016-01-06 | 长春恒晓生物科技有限责任公司 | Nature enzymic activity platelet-activating factor acetylhydro-lase and preparation process |
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| CN100336907C (en) * | 2005-09-29 | 2007-09-12 | 南京大学 | Recombinant human platelet auxin/dry cell factor fusion protein and preparation thereof |
| CN105219750A (en) * | 2015-08-25 | 2016-01-06 | 长春恒晓生物科技有限责任公司 | Nature enzymic activity platelet-activating factor acetylhydro-lase and preparation process |
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