CN1435426A - Recombinant human dyad stem cell factor and preparing thereof - Google Patents

Abstract

The invention belongs to the technical field of polypeptide medicine prepared by genetic engineering in biotechnology. Its purpose is to construct a new type of fusion doublet stem cell factor gene, and efficiently secrete, express and prepare recombinant human doublet stem cell factor (rhdSCF) with higher biological activity and less side effects in insect cells. The rhdSCF gene is a DNA fragment sequentially containing the 1-165th amino acid codon of human SCF, 12 amino acid connecting peptides, the 1-145th codon of human SCF and the stop codon. The specific activity of rhdSCF prepared from Sf9 cell adherence or suspension culture medium is 2.8-2.9×10 ⁶ units/mg. The specific activity is about 6 times higher than that of monomeric rhSCF produced by E.coli. It can be used to promote the reconstruction and recovery of hematopoietic function and immune function in patients after radiotherapy, chemotherapy and bone marrow transplantation, and can also be used for in vitro expansion research of stem cells.

Description

RhdSCF and preparation thereof

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 be in the survival and the propagation of multistage hematopoiesis level and other cytokines synergy promotion hematopoietic stem and various hemocytes, can also keep melanocyte, the survival of protoblast and mastocyte, and promote its proliferation and differentiation.Its target cell comprises the multipotency hematopoietic stem, erythron, megalokaryocyte, mononuclear macrophage, part lymphoid precursor cell, mastocyte, melanocyte and protoblast.

Natural SCF is a kind of glycoprotein, and mainly by stroma cell, inoblast and endotheliocyte are expressed, and the proportional difference that film mating type SCF and solvable type SCF express in the different tissues is very big.Can detect solubility SCF mean concns in the normal human serum is 3.3ng/mL, and wherein the SCF more than 90% exists with monomeric form.Under physiological condition, can form the ionic homodimer during high local concentrations.

The biologic activity of SCF mainly is and other cytokines such as TPO, FL, EPO, G-CSF, M-CSF, GM-CSF, IL-1, IL-2, IL3, synergies such as IL-7 are kept the survival of original hematopoietic stem, promote the propagation of original hematopoietic stem and multiple differentiation blood cell line.Clinically be mainly used in radiotherapy, the reconstruction of hematopoiesis and immunologic function and recovery after chemotherapy and the bone-marrow transplantation.SCF also has application prospect to the bone marrow stem cell amplification in vitro in addition.SCF can promote the growth of mastocyte separately.And can activate ripe mast cell, 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 solubility SCF mainly exists with monomeric form under physiological condition, can form the ionic homodimer during high local concentrations, and it must be by inducing the dimerization of acceptor when playing a role.Making up two covalently bound disjunctor STEM CELL FACTOR of pattern of fusion and can more effectively induce the dimerization of acceptor, is to improve its biological activity, reduces the effective ways of using dosage and side effect.

Three. summary of the invention

The purpose of patent of the present invention is to make up a kind of novel rhdSCF (rhdSCF) gene, and employing and glutathione-s-transferase gene amalgamation and expression form efficient secretory expression in Sf9 cell (Invitrogen), separation and purification rhdSCF from nutrient solution, but make it to reach application level.Become a kind of performance and better recover the medicine of hematopoiesis and immunologic function than monomer SCF.

Technology contents of the present invention comprises: 1. the design of rhdSCF's gene rhdSCF and structure.2. the structure of the reorganization clover californica nuclear polyhedrosis virus pAcSecG2T-rhdSCF of efficient secretory expression pattern of fusion GST-rhdSCF.3.rhdSCF secreting, expressing in the Sf9 cell and separation and purification thereof and evaluation.

1. natural secretor type solubility SCF is 165 amino acid. the rhdSCF gene of our design is the 1-165 amino acids that contains SCF, 12 amino acid whose connection peptides and the 1-145 amino acids codon of SCF and the dna fragmentation of termination codon.DNA：GAA GGG ATC TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA 60Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 20AAT CTT CCA AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA 120Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro 40AGT CAT TGT TGG ATA AGC GAG ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG 180Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu 60GAC AAG TTT TCA AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG 240Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val 80AAT ATA GTG GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA 300Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys 100TCA TTC AAG AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT 360Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn 120AGA TCC ATT GAT GCC TTC AAG GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT 420Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 140TCT TCA ACA TTA AGT CCT GAG AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA 480Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu 160

TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA AAT CTT CCA 600Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 200AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA AGT CAT TGT 660Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys 220TGG ATA AGC GAG ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG GAC AAG TTT 720Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe 240TCA AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA GTG 780Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val 260GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA TCA TTC AAG 840Asp Asp Leu Val Glu Cys Val Lys Glu Ash Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 280AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT AGA TCC ATT 900Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile 300GAT GCC TTC AAG GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT TCT TCA ACA 960Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr 320TTA AGT TAG TAALeu SerpUC18-SCF； With primer 1; 2 through synthetic 5 ' end band XbaI and the BglII restriction enzyme site of PCR method; 3 ' end disappearance terminator codon but with the genetic fragment that contains SCF 1-165 amino acids codon in flexible peptide (GGGGS) coded sequence and BamHI site is cloned between the XbaI of pBluscript and the BamHI site and obtains pBluscript-5 ' hSCF. In addition with primer 3,4 through being with BamHI site and flexible peptide (GSGGGGSGG) coded sequence with the synthetic 5 ' end of PCR method, the genetic fragment that contains SCF 1-145 amino acids codon in the two terminator codons of 3 ' end band and HindIII site, be cloned between the BamHI of pBluscript-5 ' hSCF and the HindIII site pBluscript-dhSCF.In full accord through sequencing result with design. (gene construction method is seen accompanying drawing 1, and sequencing result is seen accompanying drawing 2,3) primer 1:5 ' GGTCTAGATCTGAAGGGATCTGCAGGAAT 3 ' primer 2:

Primer 3:

Primer 4:

2. after pBluscript-dhSCF being cut with the HindIII enzyme and filling with the Klenow enzyme, again with the BglII enzyme cut the dhSCF gene segment, and be cloned between the BamHI of baculovirus transfer vector pAcSecG2T (PharMingenInternational) and the SamlI site pAcSecG2T-dhSCF.Recombinant transfer vector pAcSecG2T-hdSCF and wild-type clover californica nuclear polyhedrosis virus AcNPV DNA are coveted frugiperda cell Sf9 with liposome mediated-method cotransfection meadow, and homologous recombination and 4 is taken turns limited sparse method screening and is surveyed the recombinant virus AcNPV-hdSCF that selects the purified efficient secretory expression GST-rhdSCF that does not contain wild-type virus alive in conjunction with TF-1 cell mtt assay in born of the same parents.

3. recombinant virus is selected suitable infection multiplicity (MOI) to infect the good Sf9 cell of growth conditions, at serum free medium Sf900 or do not add among the TMN-FH of foetal calf serum and cultivate, express and collect nutrient solution after 72 hours and obtain GST-rhdSCF through Glutathione Sephrose 4B post affinity chromatography (Amersham Pharmacia Biotech Inc) separation and purification.Through cutting, cross Glutathione Sephrose 4B post affinity chromatography once more and get the rhdSCF elaboration with the zymoplasm enzyme.

The rdhSCF that the present invention makes up is connected to form novel two disjunctor STEM CELL FACTOR genes with 12 peptides (GGGGSGGGGSGG) of flexibility in the mode of being end-to-end by the SCF of brachymemma (1-145 amino acids) with a complete SCF monomer gene (1-165 amino acids) and one.Product in expressed in insect cells is surveyed the analysis of living through TF-1 cell mtt assay, and is higher 6 times than living than the 98% sterling rhSCF (Bio Lab) that expresses among the E.coli.Therefore can when clinical practice, reduce consumption, reduce its side effect.

The recombinant virus AcNPV-rhdSCF that the present invention makes up, wherein the rhdSCF gene is positioned under the polyhedrosis gene promotor control of AcNPV, merge the glutathione-s-transferase gene that 3 ' end has the zymoplasm recognition site in the front of rhdSCF gene, made separation and purification very convenient.Fusion rotein has the gp67 protein signal peptide of AcNPV, makes secernment efficiency very high, and expression product all is secreted into outside the born of the same parents more than 90%.

The recombinant virus that makes up infects individual layer Sf9 cell by the method for the invention, the collecting cell supernatant liquor, and cumulative rhdSCF reaches 6000units/mL in the supernatant liquor, and fusion protein expression is about 6mg/L.

The recombinant virus that makes up infects suspension Sf9 cell by the method for the invention, the reducible 12500units/mL that reaches of cumulative rhdSCF in the cell culture fluid, and fusion protein expression is about 12.2mg/L.

Can from the Sf9 cell culture fluid, obtain to reach 2.9 * 10 by separation purification method provided by the invention than living ⁶The rdhSCF of units/mg.Be a band with the detection of SDS-PAGE argentation.Purity is about 90%.

Four. description of drawings

The structure schema of accompanying drawing 1 two disjunctor STEM CELL FACTOR genes

The sequencing result figure of accompanying drawing 2rdhSCF gene (3 ' end sequencing result)

The sequencing result figure of accompanying drawing 3rdhSCF gene (5 ' end sequencing result)

The active comparison diagram of accompanying drawing 4rhSCF monomer and rdhSCF

● rhdSCF (rdhSCF)

■ recombinant human monomer STEM CELL FACTOR (SCF) (E.coli)

Accompanying drawing 5 SDS-PAGE electrophoretograms

1.rdhSCF

2.rdhSCF fusion rotein with glutathione-S-transferase

3. the Sf9 cell expressing supernatant liquor of recombinant virus infection

4. molecular weight standard protein

Five. embodiment 1

1. with pUC18- SCF template primer 1,2 through the synthetic 5 ' hSCF of PCR, and be cloned into pBluscript to get pBluscript-5 ' hSCF. be template equally with pUC18-SCF with primer 3,4 through with the synthetic 3 ' SCF of PCR, be cloned into pBluscript-5 ' hSCF and get pBluscript-rhdSCF.In full accord through sequencing result with design.(flow process is seen accompanying drawing 1)

2. the pBluscript-rdhSCF enzyme is cut the rdhSCF gene fragment, and be cloned into pAcSecG2T and get pAcSecG2T-hdSCF.With pAcSecG2T-hdSCF and AcNPV DNA cotransfection Sf9, survey the net net weight papova AcNPV-rdhSCF that does not contain wild-type virus that selects alive through screening and in conjunction with the TF-1 cell.

3. be 21 hours with the doubling time, viable cell accounts for 90%, and density is 6 * 10 ⁵The individual layer Sf9 cell of cell/mL 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 ℃, surveying nutrient solution fusion rotein activity with the TF-1 cell is 6000units/mL (6 * 10 ⁵Cells/mL).The low-speed centrifugal collecting cell, the fusion activity of not secreting in the extraction liquid of cell after the freeze thawing only is 500units/6 * 10 ⁵Cells illustrates the fusion secretion fully.Expression amount is about 6mg/L.To 1 * PBS (pH7.4) dialysis 12 hours, with the Glutathione Sephrose 4B post of sample on the 2mL/min flow velocity to 2mL, pillar 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, contain 1 * PBS washing of 0.8%Triton X-100 again with 10mL, after using 1 * PBS to be washed till baseline once more, with 10mL elutriant (pH8.0,50Mm Tris-Cl, the 10mM reduced glutathion) with the flow velocity wash-out of 2mL/min, collects elution peak 13mL.Elutriant to distill water dialysis after, measure protein content and activity, fusion rotein GST-rhdSCF is 1.0 * 10 than work ⁶Units/mg.After the freeze-drying, be dissolved in 1 * PBS of 200 μ L, (Amersham Pharmacia Biotech Inc) cut 16 hours in 22 ℃ of enzymes with 2 units (10unit/mL) zymoplasm.With Glutathione Sephrose 4B post on the 2mL/min flow velocity, collect and pass peak 12mL again.After the distill water dialysis desalination, freeze-drying.Measuring rhdSCF is 2.9 * 10 than work ⁶Units/mg.The 98% purity rhSCF that the E.coli that its specific activity Bio Lab sells expresses is than live high 6 times (seeing accompanying drawing 2).SDS-PAGE silver dyes a band (seeing accompanying drawing 3) that detects to the about 40KDa of apparent molecular weight.Immunoblotting (Westen blotting) analytical results consistent with it (seeing accompanying drawing 4).Separation and purification process purification is more than 220 times.Active yield is 72.5%.

Embodiment 2

Step 1,2 with embodiment 1 in step 1,2 is identical

3. be 19 hours with the doubling time, viable cell accounts for 95%, and density is 1.5 * 10 ⁶The Sf9 suspension cell 40mL of the SF900II culture medium culturing that contains 4% new-born calf serum of cells/mL, through 500rpm centrifugal 10 minutes, collecting cell was resuspended in the 40mLSF900II serum free medium.Adding titre by infection multiplicity (MOI)=0.1 is 1.0 * 10 ⁷The recombinant virus AcNPV-rhdSCF storing solution 0.6ml. paddle rotating speed of/ml is 60 rev/mins, cultivates after 48 hours for 27 ℃, and 10000rpm abandoned cell harvesting supernatant liquor 40ml in centrifugal 5 minutes.Surveying and expressing liquid fusion rotein activity is 12500units/mL (1.5 * 10 ⁶Cells/mL).Freeze in the molten extraction liquid of cell activity and only be 900units/1.5 * 10 ⁶Cells.The excretory fusion protein expression is about 12.2mg/L in the nutrient solution.

4 with the 40mL culture supernatant to 1 * PBS (pH7.4) dialysis 12 hours, with the Glutathione Sephrose 4B post of 2mL on the 2mL/min flow velocity, pillar is used 1 * PBS balance in advance.Contain the 1 * PBS washing of 0.8%TritonX-100 with 10mL, be washed till baseline with 1 * PBS after, use the flow velocity wash-out of 10mL elutriant (pH8.0,50MmTris-Cl, 10mM reduced form glutathione) again, collection elution peak 27mL with 2mL/min.Behind distill water dialysis, measure protein content and activity respectively, be 1.1 * 10 than work ⁶Units/mg.Be dissolved in 1 * PBS of 300 μ L after the freeze-drying, (Amersham PharmaciaBiotech Inc) cut 16 hours in 22 ℃ of enzymes with 3 units (10unit/mL) zymoplasm.Pass through Glutathione Sephrose4B post with the 2mL/min flow velocity again, collect and pass peak 25mL.After the distill water dialysis desalination, freeze-drying.Measuring protein content and activity, is 2.8 * 10 than work ⁶Units/mg.It is the band of 40KDa for apparent molecular weight that SDS-PAGE silver dyes detection.Western blotting (Westen blotting) is analyzed consistent with it.Separation and purification process purification is more than 210 times.Active yield is 72%.

＜110〉＜120〉＜160〉1＜210〉1＜211〉972＜212〉cDNA＜213〉 ( homo sapiens )＜400〉gaa ggg atc tgc agg aat cgt gtg act aat aat gta aaa gac gtc act aaa ttg gtg gca 60Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 20 1 6 11 16aat ctt cca aaa gac tac atg ata acc ctc aaa tat gtc ccc ggg atg gat gtt ttg cca 120Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro 40 21 26 31 36agt cat tgt tgg ata agc gag atg gta gta caa ttg tca gac agc ttg act gat ctt ctg 180Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu 60 41 46 51 56gac aag ttt tca aat att tct gaa ggc ttg agt aat tat tcc atc ata gac aaa ctt gtg 240Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val 80 61 66 71 76aat ata gtg gat gac ctt gtg gag tgc gtg aaa gaa aac tca tct aag gat cta aaa aaa 300Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys 100 81 86 91 96tca ttc aag agc cca gaa ccc agg ctc ttt act cct gaa gaa ttc ttt aga att ttt aat 360Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn 120101 106 111 116aga tcc att gat gcc ttc aag gac ttt gta gtg gca tct gaa act agt gat tgt gtg gtt 420Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 140121 126 131 136tct tca aca tta agt cct gag aaa gat tcc aga gtc agt gtc aca aaa cca ttt atg tta 480Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu 160141 146 151 156ccc cct gtt gca gcc gga gga gga gga tcc gga gga gga ggc tcc ggc ggc gaa ggg atc 540Pro Pro Val Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Gly Ile 180161 166 171 176tgc agg aat cgt gtg act aat aat gta aaa gac gtc act aaa ttg gtg gca aat ctt cca 600Cys Arg Asn Arg Val Thr Ash Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 200181 186 191 196aaa gac tac atg ata acc ctc aaa tat gtc ccc ggg atg gat gtt ttg cca agt cat tgt 660Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys 220201 206 211 216tgg ata agc gag atg gta gta caa ttg tca gac agc ttg act gat ctt ctg gac aag ttt 720Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe 240221 226 231 236tca aat att tct gaa ggc ttg agt aat tat tcc atc ata gac aaa ctt gtg aat ata gtg 780Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val 260241 246 251 256gat gac ctt gtg gag tgc gtg aaa gaa aac tca tct aag gat cta aaa aaa tca ttc aag 840Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 280261 266 271 276agc cca gaa ccc agg ctc ttt act cct gaa gaa ttc ttt aga att ttt aat aga tcc att 900Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile 300281 286 291 296gat gcc ttc aag gac ttt gta gtg gca tct gaa act agt gat tgt gtg gtt tct tca aca 960Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr 320301 306 311 316tta agt tag taaLeu Ser321Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 20Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro 40Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu 60Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val 80Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys 100Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn 120Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 140Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu 160Pro Pro Val Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Gly Ile 180Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 200Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys 220Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe 240Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val 260Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 280Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile 300Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr 320Leu Ser

Claims (4)

1. a rhdSCF ( rhdSCF ) antigen-4 fusion protein gene. ( rhdSCF ) ：GAA GGG ATC TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA 60Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 20AAT CTT CCA AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA 120Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro 40AGT CAT TGT TGG ATA AGC GAG ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG 180Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu 60GAC AAG TTT TCA AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG 240Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val 80AAT ATA GTG GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA 300Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys 100TCA TTC AAG AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT 360Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn 120AGA TCC ATT GAT GCC TTC AAG GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT 420Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 140TCT TCA ACA TTA AGT CCT GAG AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA 480Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu 160

TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA AAT CTT CCA 600Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 200AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA AGT CAT TGT 660Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys 220TGG ATA AGC GAG ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG GAC AAG TTT 720Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe 240TCA AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA GTG 780Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val 260GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA TCA TTC AAG 840Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 280AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT AGA TCC ATT 900Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile 300GAT GCC TTC AAG GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT TCT TCA ACA 960Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr 320TTA AGT TAG TAALeu Ser

2. a rhdSCF (rhdSCF) antigen-4 fusion protein gene.It is characterized in that design and make up a coding region that contains soluble human STEM CELL FACTOR (hSCF1-165 amino acids), 12 amino acid GGGGSGGGGSGG flexibly connect the coding region and the covalently bound end to end successively dna fragmentation of termination codon of peptide-coding region and another hSCFl-145 amino acids.

3. claim 2 provided gene constructed a kind of can be in insect Sf9 the reorganization clover californica nuclear polyhedrosis virus (AcNPV-rhdSCF) of efficient secretory expression GST-rhdSCF fusion rotein.It is characterized in that the rhdSCF gene clone between the BamHI and SmaI site of baculovirus transfer vector pAcSerG2T (PharMingenInternational), obtain recombinant transfer vector pAcSerG2T-rhdSCF, wherein the dhSCF upstream region of gene has merged the natural gp67 protein signal peptide and the gst gene fragment of being with the zymoplasm cleavage site of AcNPV, and whole fusion gene is under the polyhedrin promotor control of AcNPV.With pAcSerG2T carrier DNA and wild-type AcNPV DNA cotransfection Sf9 cell, take turns the limiting dilution assay purifying and, filter out purified recombinant virus AcNPV-rhdSCF in conjunction with the active determination in vitro of recombinant protein through 4.

4. the preparation method of a rhdSCF (rhdSCF) fusion rotein.The Sf9 cell that it is characterized in that or suspension culture adherent with the constructed recombinant virus infection of claim 3, in serum free medium, expressed 72 hours for 27 ℃, the GST-rhdSCF fusion rotein of secreting, expressing has the SCF activity in the nutrient solution, and expression level reaches 6-12mg/L.Collecting cell is expressed liquid, and through Glutathione Sephrose4B post affinity chromatography, the rhdSCF elaboration that fibrin ferment cuts and chromatography can obtain purity about 90% again is silver through the SDS-PAGE analysis and dyes a band.Molecular weight is about 40KD, is 2.8-2.9 * 10 than living ⁶Units/mg.Activity recovery reaches more than 50%, the about 2-4mg/L of productive rate.

Images