CN102242124A - Modified Keratinocyte growth factor gene and its expression in yeast - Google Patents
Modified Keratinocyte growth factor gene and its expression in yeast Download PDFInfo
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- CN102242124A CN102242124A CN2010101663615A CN201010166361A CN102242124A CN 102242124 A CN102242124 A CN 102242124A CN 2010101663615 A CN2010101663615 A CN 2010101663615A CN 201010166361 A CN201010166361 A CN 201010166361A CN 102242124 A CN102242124 A CN 102242124A
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Abstract
The invention discloses a Keratinocyte growth factor gene, vectors with the Keratinocyte growth factor gene and an application of the Keratinocyte growth factor gene, and belongs to the technical field of genetic engineering. The invention is characterized by comprising a gene segment which codes a Keratinocyte growth factor and has a nucleotide sequence shown in the formula SEQ ID NO.1, expression vectors with the gene segment and a system of expressing the Keratinocyte growth factor in pichia yeast through the expression vectors. Through the system of expressing a Keratinocyte growth factor in pichia yeast, the advantages of low cost, short cycle, easy realization of a technical scheme, and favorable expression level reaching to an industrialized production level are realized.
Description
Technical field
The present invention relates to a kind of keratinocyte growth factor expressing gene and expression thereof and application, belong to gene engineering technology field.
Background technology
(Keratinocyte growth factor, (fibroblast growth factor FGF), claims FGF7 again to keratinocyte growth factor KGF) to belong to fibroblast growth family.KGF is in 1989, separate and purifying obtains through ultrafiltration, affinity chromatography and anti-phase high-pressure liquid phase (RP-HPLC) from the conditioned medium of embryo lung fibroblastic growth by Rubin, this molecule has very strong heparin affinity, the characteristics of cell growth that can influence nearly all mesoderm origin with other fibroblast growth factor are different, the activity of KGF mainly is limited to epithelial cell, have and promote the especially mitotic effect of keratinocyte of epithelial cell specifically, so the called after keratinocyte growth factor.
The expression of KGF only limits to mesenchymal cell, and its acceptor then is to express in epithelial cell, and its biologic activity is to act on epithelial cell, bears the signal transfer function between mesenchymal cell and epithelial cell.For relying on the interactional organ of mesenchymal cell-epithelial cell, tissue, as skin, respiratory tract, gi tract, urogenital system etc., in their mesenchymal cell, all can detect the expression of KGF mRNA, and in the epithelial cell of correspondence, can detect the KGF receptor mRNA.The biological action of KGF be by with its target cell membrane on receptors bind finish, the biologic activity of performance comprises: promote epithelial propagation, in developmental effect, effect in injury repairing, other there are some researches show the confidential relation that has of KGF and cancer.
Complete KGF protein has 194 amino acid (comprising the front end signal peptide sequence), contains 5 halfcystines in the peptide, and wherein 4 halfcystines form 2 pairs of disulfide linkage, and another halfcystine is folded in the protein.Ripe KGF is the single chain polypeptide of 163 amino-acid residues, and proteinic aminoterminal has a glycosylation site, and its apparent molecular weight is about 26～28KD.1993, Dina etc. have made up some KGF mutant, and in intestinal bacteria, express, be purified into after the associated protein mechanism of action in order to research albumen and cell surface receptor, found that preceding 23 amino acid whose disappearances of peptide chain have improved mitogenic activity and the thermostability of KGF, this has played positive effect to the exploitation of KGF pharmaceutical protein afterwards.
U.S. Amgen company has developed at 23 amino acid whose recombinant human horny cell growth factor-2 mutant Palifermin (Kepivance of expression in escherichia coli N-terminal disappearance
TM), be mainly used in the oral mucositis that treatment is put, chemotherapy causes.Clinical trial proves, Kepivance
TMObtaining better curative effect aspect the treatment bone marrow transplantation cancer patients oral mucositis, and side effect is little.Present this product is being used for the treatment of the research of the oral mucositis behind head and neck cancer, leukemia, the colorectal carcinoma patient chemicotherapy, in January, 2005, this medicine of drugs approved by FDA was used to treat the oral mucositis that malignant tumor patient accepts to have the treatment of bone marrow toxicity to cause, and authorized it and be rare medicine qualification.
The KGF of approval listing at present is by escherichia coli expression production, and the albumen existence that intestinal bacteria produce becomes, the difficulty of renaturation; Shi Lei etc. disclose and have utilized pichia spp heterogenous expression KGF at document " cDNA of recombinant human KGF clone and the expression in pichia spp ", lowly can't carry out industrialization but all cross because of expression amount.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of new keratinocyte growth factor expressing gene that can efficiently express in pichia spp and the carrier and the application of structure thereof are provided.
The present invention relates to:
[1] a kind of gene fragment of the keratinocyte growth factor of encoding, it has the nucleotide sequence shown in SEQ ID NO.1.
The gene fragment of keratinocyte growth factor of the present invention is by mainly being to obtain by following transformation: (a) leave out 69 Nucleotide of keratinocyte growth factor gene 5 ' end; (b) on the basis that does not change aminoacid sequence, be optimized according to the pichia spp codon preference; (c) the synthetic keratinocyte growth factor sequence of full gene.
In addition, the present invention relates to:
[2] a kind of expression vector that contains the nucleotide sequence shown in the SEQ ID NO.1.
In addition, the present invention relates to:
[3] a kind of pichia spp, this pichia spp contains the expression vector described in [2].
And, the present invention relates to:
[4] construction process of pichia spp in a kind of [3], this method comprises: (1) utilizes XhoI and EcoRI double digestion synthetic keratinocyte growth factor, construction recombination plasmid X-KGF; (2) transform the pichia spp competent cell, obtain positive transformant by the resistant panel screening; (3) utilize shake flask fermentation that positive transformant is further screened;
Recombinant plasmid X-KGF in the described step (1), the expression plasmid that the X representative is selected.
[5] construction process of the pichia spp of basis [4] record, the described construction recombination plasmid X-KGF of step (1) is meant and utilizes nucleic acid restriction endonuclease XhoI and EcoRI double digestion synthetic keratinocyte growth factor, be cloned on the expression plasmid X that same enzyme cuts, carry out sequence verification then.
[6] construction process of the pichia spp of basis [4] record, step (2) is recombinant plasmid X-KGF and the blank plasmid that makes up in the step (1), transform the pichia spp competent cell, according to antibiotics resistance or hereditary defect type, obtain corresponding positive pichia spp transformant by plate screening.
[7] construction process of the pichia spp of basis [4] record, the described method of step (3) are that the positive transformant that will obtain in the step (2) carries out the shake flask fermentation screening, the acquisition expression cell line in substratum.
[8] construction process of the pichia spp of basis [4] record, the described recombinant plasmid X-KGF of step (3), wherein expression plasmid X is: pGAPZ α A/B/C, pPICZ α A/B/C, pPIC9 or pPIC9K.
[9] construction process of the pichia spp of basis [4] record, the described pichia spp competent cell of step (2) is the competent cell of pichia pastoris engineered strain GS115, KM71, SMD1168 or X-33.
[10] construction process of the pichia spp of basis [4] record, the substratum of the described shake flask fermentation of step (3) is BMMY, BMGY, YPD or FBS.
And further, the present invention relates to:
[11] utilize pichia spp to carry out the keratinocyte growth factor of high density fermentation expression in the application aspect the preparation cancer therapy drug according to [4] record.
Expression plasmid pGAPZ α A/B/C, pPICZ α A/B/C, pPIC9 or the pPIC9K that the present invention relates to are the commercially available prod, the expression plasmid that can adopt Invitrogen company to produce.The concrete operations step of construction recombination plasmid X-KGF can be operated with reference to the working instructions of the said products.
Pichia pastoris engineered strain GS115, KM71, SMD1168 or the X-33 that the present invention relates to are the commercially available prod, can buy by Invitrogen company.Pichia pastoris engineered strain is handled acquisition pichia spp competent cell can adopt this area common method, as: electric shock conversion method etc.The working instructions of the above-mentioned engineering strain of those skilled in the art's reference can carry out shake flask fermentation positive transformant is further screened.
The substratum that the present invention relates to is the general substratum in this area if no special instructions; The reagent that relates to is the commercially available prod; Working method all adopts the general working method in this area if no special instructions.
Can obtain to efficiently express in pichia spp through improved keratinocyte growth factor expressing gene, under conditions of flask fermentation, its expression amount reaches 30mg/L, and under 5L fermentor tank middle-high density fermentation condition, its expression amount reaches 110mg/L.Obtain the pure product of KGF through purifying, studies show that: it has consistent biologic activity and pharmacological characteristic with the product K epivance that goes on the market at present.Employing Pichia anomala expression keratinocyte growth factor cost of the present invention is low, the cycle short, technical easy realization, and the expression amount that carries out the high density fermentation expression has reached the suitability for industrialized production level.
Description of drawings
Fig. 1, recombinant plasmid pPIC9-KGF enzyme are cut checking
Swimming lane M:DNA Marker wherein, DL15000,
Swimming lane 1: recombinant plasmid pPIC9-KGF-1,
Swimming lane 2: recombinant plasmid pPIC9-KGF-2;
Fig. 2, KGF transformant fermented liquid SDS-PAGE detect figure
Swimming lane M wherein: protein Marker,
Swimming lane 1:KGF transformant fermented liquid,
Swimming lane 2: blank bacterial strain GS115 fermented liquid;
KGF SDS-PAGE is quantitative in Fig. 3, the fermented liquid
Swimming lane M wherein: protein Marker,
Swimming lane 1:KGF transformant fermented liquid,
Swimming lane 2:BSA 12.5 μ g/mL,
Swimming lane 3:BSA 25 μ g/mL,
Swimming lane 4:BSA 50 μ g/mL,
Swimming lane 5:BSA 100 μ g/mL,
Swimming lane 6:BSA 200 μ g/mL;
The determination of activity of the rhKGF of Fig. 4, purifying and the comparison diagram of reference substance;
Fig. 5, rhesus monkey single vein give serum drug-time curve (n=4) behind 90 μ g/kg rHuKGF or the Kepivance.
Embodiment
The embodiment of form is described in further detail foregoing of the present invention by the following examples, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Used instrument explanation among the embodiment:
Used Genepulster II electricity conversion instrument, Bio-rad company product, electric shock transforms parameter and is: voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω, discharge time 4.0ms.
FC-2002 methyl alcohol detects stream and adds controller: available from Shanghai Su Bo Information Technology Co., Ltd.
5L fermentor tank: Switzerland Bioengineering company product.
Raw materials used explanation among the embodiment:
The keratinocyte growth factor complete genome sequence is synthetic by Bo Shang Bioisystech Co., Ltd; The pUC57-KGF plasmid is also provided by the said firm.
Expression plasmid pGAPZ α A, pPICZ α A, pPIC9 are available from Invitrogen company;
Used pichia pastoris engineered strain GS115, KM71, SMD1168 or X-33 is Invitrogen company product.
The prescription of MD flat board is: 13.4g/L does not have amino acid yeast nitrogen (YNB); 0.4mg/L vitamin H; 20g/L D-glucose; Add water to 1L.
The prescription of YPD substratum is: the 10g/L yeast extract; The 20g/L peptone; 20g/L D-glucose; Add water to 1L.
The prescription of BMGY substratum is: the 10g/L yeast extract; The 20g/L peptone; 100mM phosphoric acid buffer (pH 6.0); 13.4g/L there is not amino acid yeast nitrogen (YNB); 0.4mg/L vitamin H; 10g/L glycerine; Add water to 1L.
The prescription of BMMY substratum is: the 10g/L yeast extract; The 20g/L peptone; 100mM phosphoric acid buffer (pH 6.0); 13.4g/L there is not amino acid yeast nitrogen (YNB); 0.4mg/L vitamin H; 5g/L methyl alcohol; Add water to 1L.
The prescription of FBS substratum is: phosphoric acid (85%) 26.7mL/L; Calcium sulfate 0.93g/L; Vitriolate of tartar 18.2g/L; Magnesium sulfate heptahydrate 14.9g/L; Magnesium sulfide 4.13g/L; Glycerine 40.0g/L; Add water to 1L.
1, gene is synthetic
The KGF gene order is from Genebank, accession number NM_002009, and its sequence is carried out following transformation: (1) leaves out 69 Nucleotide of gene 5 ' end; (2) on the basis that does not change aminoacid sequence, be optimized according to the pichia spp codon preference; (3) the synthetic keratinocyte growth factor sequence of full gene.
2, construction of recombinant plasmid
Utilize the two pUC57-KGF plasmids of cutting of restriction enzyme XhoI and EcoRI, glue reclaims the KGF target fragment, cuts the pPIC9 plasmid with same enzyme then and is connected.To connect product transformed into escherichia coli JM109 competent cell, at the enterprising row filter of LB flat board (containing Amp), picking bacterial strain extracting plasmid carries out XhoI/EcoRI double digestion checking (the results are shown in accompanying drawing 1) at random, the correct laggard pacing preface checking of advancing of checking.
3, Pichi strain GS115 transforms and plate screening
Utilize restriction enzyme SalI enzyme to cut recombinant plasmid pPIC9-KGF and blank plasmid pPIC9, get 20 μ g plasmids electric shock and transform pichia spp GS115 competent cell.Used Genepulster II electricity conversion instrument (parameter CA) is for Bio-Rad laboratories Inc., Hercules: voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω, discharge time 4.0ms.
Add ice-cold (0 ℃) the 1.0M sorbyl alcohol of 1.0mL after the electric shock immediately, be coated with MD flat board (His rapidly
-), 28 ℃ of-30 ℃ of cultivations, screening His
+Transformant.
4, KGF transformant shake flask fermentation screening
Each picking 3 strain KGF recombinant conversion sons and 3 strain blank bacterial strains at random are inoculated in the 10mL BMGY liquid nutrient medium on the MD flat board, and 30 ℃, 200r/min are cultured to cell density OD
600=8-12,5000r/min, centrifugal 5min.With BMMY substratum re-suspended cell to OD
600=2.0, shift the 30mL suspension in the 150mL triangular flask.28 ℃, 250r/min were cultivated 7 days, added the methyl alcohol of 0.5% (v/v) every day.Utilize every day SDS-PAGE to detect KGF expression amount in the fermented liquid, detection of biological amount and fermented liquid pH serve as that KGF expression amount mensuration is carried out in contrast with concentration known BSA simultaneously.The sub-protein expression amount of three strain KGF recombinant conversion indifference is got a wherein strain and is further analyzed as a result, its fermented liquid is carried out the SDS-PAGE detected result see accompanying drawing 2, and the protein expression amount is carried out quantitative assay, the results are shown in accompanying drawing 3.
5, KGF transformant 5L fermentor tank high density fermentation
Utilize BMGY medium preparation seed liquor, be inoculated in then in the FBS substratum (adding 4.34mL PTM1 trace element solution in every liter of substratum) that contains 4wt% glycerine and spread cultivation, culture condition is: 28 ℃ of temperature, pH6.0, dissolved oxygen 20% (saturation ratio per-cent), mixing speed, air input (air or pure oxygen) and dissolved oxygen coupling.Behind the about 20h of cultured continuously, when glycerine exhausts, add the 50wt% glycerine that contains 1.2wt%PTM1, add in the fermentor tank with the speed of the initial fermented liquid of 50mL/h/L, it is the initial fermented liquid of 80mL/L that 50wt% glycerine is added total amount, and the feed supplement duration is about 4-6h.When glycerine exhausts once more, add the methyl alcohol that contains 1.2wt%PTM1, and culture temperature is transferred to 20 ℃.It is the initial fermented liquid of 3.6mL/h/L that methyl alcohol is added initial rate, to engineering bacteria adaptation methyl alcohol, methyl alcohol is added speed and is adjusted to the initial fermented liquid of 7.2mL/h/L, behind the 2h methyl alcohol is added speed and is adjusted to the initial fermented liquid of 10mL/h/L, keep behind the 3h in the jar methanol concentration at 5-10g/L until fermentation ends.After engineering bacteria adapted to methyl alcohol, every 24h added nitrogenous source supplemented medium (20wt% peptone solution) 150mL.Beginning to induce in the fermenting process, every 12h sampling 1 time detects cell density A
600, induce fermentation 60h after, finish fermentation and also detect the KGF expressing quantity.In the fermenting process, utilize FC-2002 methyl alcohol to detect stream and add controller detection methanol concentration.Cell density A in the final fermenting process
600Can reach 500, reorganization KGF expression amount is about 110mg/L.
PTM1 trace element solution: copper sulfate 5H
2O 6.0g, sodium iodide 0.08g, manganous sulfate H
2O 3.0g, Sodium orthomolybdate 2H
2O 0.2g, boric acid 0.02g, cobalt chloride 0.5g, zinc chloride 20.1g, ferric sulfate 7H
2O 65.0g, vitamin H 0.2g, sulfuric acid 5.0mL adds sterilized water to 1L.
1, gene is synthetic
With embodiment 1.
2, construction of recombinant plasmid
Utilize the two pUC57-KGF plasmids of cutting of restriction enzyme XhoI and EcoRI, glue reclaims the KGF target fragment, cuts pGAPZ α A plasmid with same enzyme then and is connected, and obtains recombinant plasmid pGAPZ α A-KGF.With recombinant plasmid pGAPZ α A-KGF transformed into escherichia coli JM109 competent cell, carry out checking of plate screening, double digestion and sequence verification then, carry out next step experiment after correct.
3, Pichi strain GS115 transforms and plate screening
Utilize restriction enzyme SacI enzyme to cut recombinant plasmid pGAPZ α A-KGF and blank plasmid pGAPZ α A, get 5 μ g-20 μ g plasmids electric shock and transform pichia spp GS115 competent cell, concrete grammar is with embodiment 1.
4, KGF transformant shake flask fermentation screening
On the YPDS flat board, carry out the screening of transformed clone, utilize the shake flask fermentation screening of in the YPD substratum, fermenting then, concrete fermentation parameter is with embodiment 1, because pGAPZ α A is a constitutive expression, so need not add methyl alcohol induces, finally its expression amount is measured, reorganization KGF expression amount is about 25mg/L.
1, gene is synthetic
With embodiment 1.
2, construction of recombinant plasmid
Utilize the two pUC57-KGF plasmids of cutting of restriction enzyme XhoI and EcoRI, glue reclaims the KGF target fragment, cuts pPICZ α A plasmid with same enzyme then and is connected, and obtains recombinant plasmid pPICZ alpha A-KGF.With recombinant plasmid pPICZ alpha A-KGF transformed into escherichia coli JM109 competent cell, containing checking of the antibiotic LB plate screening of Zeocin, double digestion and sequence verification then, carry out next step experiment after correct.
3, Pichi strain GS115 transforms and plate screening
Utilize restriction enzyme SacI enzyme to cut recombinant plasmid pPICZ alpha A-KGF and blank plasmid pPICZ α A, get 5 μ g-20 μ g plasmids electric shock and transform pichia spp GS115 competent cell, concrete grammar is containing the dull and stereotyped enterprising row filter of the antibiotic YPDS of Zeocin then with embodiment 1.
4, KGF transformant shake flask fermentation screening
Obtain positive transformant at plate screening, utilize shake flask fermentation to screen then, concrete grammar is finally measured its expression amount with embodiment 2, and reorganization KGF expression amount is about 30mg/L.
The purifying of 1 reorganization KGF
The preparation fermented liquid, 10000r/min, centrifugal 30min collects supernatant after the centrifugal end.Sample is to the good Sephadex G-25 Coarse desalination chromatography column of 20mmol/L Tris-HCl (buffer A) balance with pH7.2 on the fermented liquid supernatant.Last sample finishes, and with buffer A elution chromatography post, first elution peak is and contains the rhKGF protein peak, collects this elution peak.
The desalination chromatography is collected on the liquid sample to using in the buffer A equilibrated CM-Sepharose Fast Flow chromatography column.Last sample finishes, and washes chromatography column with buffer A, with the washing fluid wash-out foreign protein of buffer A+0.3mol/L NaCl, uses buffer A+0.8M NaCl wash-out at last.
Fermented liquid is exchanged for the buffering system of supernatant by ultrafiltration the 20mMTris-HCl damping fluid of pH7.2 through the centrifugal fermented liquid supernatant that obtains.Be further purified through a step cation-exchange chromatography, obtained electrophoresis purity and be higher than 95% rhKGF.
Embodiment 5
1 reorganization KGF biologic activity is checked
Utilize mink lung epithelial cell (Mv-1-Lu), with the rhKGF mutant Kepivence of U.S.'s list marketing
TMBe contrast, (Amgen company product) adopts mtt assay to detect the rhKGF activity.
The Mv-1-Lu cell strain with complete culture solution in 37 ℃, the CO of 5% volumetric concentration
2In the incubator, the back of going down to posterity is cultivated 24～36h and is used for Determination of biological activity.Testing sample is carried out serial dilution according to the method for doubling dilution, the Tissue Culture Plate of preparation is discarded and keeps liquid, add the good sample solution of dilution, put 37 ℃, the CO of 5% volumetric concentration
2Cultivate 64～72h in the incubator.
Every hole adds MTT solution 20 μ L, in 37 ℃, the CO of 5% volumetric concentration
2Cultivate 5h in the incubator.After discarding the liquid in the culture plate, adding dimethyl sulfoxide (DMSO) (DMSO) 100 μ L in every hole, on microplate reader, is reference wavelength with 630nm behind the mixing, measures absorbancy in wavelength 570nm place, and the record measurement result the results are shown in accompanying drawing 4.
1 reorganization KGF drug efficacy study
For investigating biologic activity in the reorganization KGF body, set up Fluracil (50mg/kg respectively, continuous 4 days) and methotrexate (5mg/kg, continuous 4d) the mouse stomatitis model that brings out after the chemotherapy, that utilizes embodiment 1 preparation carries out corresponding observation after the reorganization KGF behind embodiment 4 purifying is with prevention and the administration of treatment dual mode.Result of study shows, index changing conditions (table 1～7) such as mouse food-intake, body weight, oral mucositis incidence, small intestine coefficient and small intestine's morphological change after the 5-Fu chemotherapy, reorganization KGF has certain prevention and therapeutic action to oral mucositis due to 5 FU 5 fluorouracil (5-Fu) chemotherapy, can reduce animal oral mucositis incidence, postpone the stomatitis time of occurrence, shorten the stomatitis course of disease, reduce Inflammation of Radioactive Oral Cavity degree of damage and mortality ratio thereof, drug action is identical or close with same dose positive drug Kepivance.(rhKGF representative reorganization KGF in the table)
Table 1 respectively organize food-intake after the mouse 5-Fu chemotherapy variation (
N=16)
Annotate: ###P＜0.001, compare with control group; * P＜0.05, * * P＜0.01, * * * P＜0.001, with model group relatively
Table 2 respectively organize body weight after the mouse 5-Fu chemotherapy variation (
N=16)
Annotate: ##P＜0.001, compare with control group; * P＜0.05, * * P＜0.01, * * * P＜0.001, with model group relatively
Oral mucositis prophylactic effect after the table 3 pair mouse 5-Fu chemotherapy
Annotate: every group of n=16, * P＜0.05, * * P＜0.01, compare with model group * * * P＜0.01
Table 4 is respectively organized the small intestine index variation of chemotherapy mouse
Annotate: chemotherapy the 6th day (n=6), chemotherapy the 12nd day (n=5-10).* P＜0.05, * * P＜0.01, with model group relatively
Table 5 is respectively organized the 6th day small intestine's morphological change of mouse chemotherapy
Annotate: every group of n=6, * P＜0.05, * * P＜0.01, * * * P＜0.01; With model group ratio, ###*P＜0.01, compare with control group
Table 6 is respectively organized the 12nd day small intestine's morphological change of mouse chemotherapy
Annotate: every group of n=6, * P＜0.05, * * P＜0.01, * * * P＜0.01; With model group ratio, ###*P＜0.01, compare with control group
Table 7 is respectively organized the 6th, 12 days small intestine outward appearances of mouse chemotherapy and is changed
Annotate: every group of n=6, * P＜0.05, * * P＜0.01, * * * P＜0.01, with the model group ratio, ## * P＜0.01, with the control group ratio
2 reorganization KGF medicines are for research
Be the similarities and differences of thoroughly evaluating this product with the listing product, finished this product through single-dose toxicity test and the repeat administration toxicity test of intravenous administration, discovered that toxicity performance, duration of symptoms and the main target organ etc. of animal were all consistent with the product that goes on the market after laboratory animal was to the tolerance of this product, administration SD rat, KM mouse and rhesus monkey.In addition, this product and reference substance have been investigated the two pharmacokinetics behavior in the rhesus monkey body with the same dose administration, studies show that curve when the two has consistent medicine in rhesus monkey blood plasma, and main pharmacokinetic parameter is approaching, the results are shown in accompanying drawing 5, T
1/2Be respectively 3.1 ± 0.7h and 2.8 ± 0.6h, C
MaxBe respectively 368.5 ± 120.4ng/mL and 345.0 ± 81.9ng/mL, V
SsBe respectively 3581.1 ± 1280.8mL/kg and 3466.7 ± 921.3mL/kg, CL
sBe respectively 1535.4 ± 331.4mL/h/kg and 1410.8 ± 140.7mL/h/kg, AUC
0 → ∞Be respectively 64.4 ± 16.4ng/h/mL and 67.2 ± 6.4ng/h/mL.
SEQUENCE?LISTING
＜110〉Qilu Pharmaceutical Co., Ltd.
＜120〉keratinocyte growth factor gene of Gai Zaoing and the expression in yeast thereof
<160>1
<170>PatentIn?version?3.5
<210>1
<211>453
<212>DNA
<213>Pichia?pastoris
<400>1
ctcgagaaaa?gagaagctga?agcttcttac?gactacatgg?aaggtggtga?catcagagtc 60
agaagattgt?tctgtagaac?tcagtggtac?ttgagaatcg?acaagagagg?taaggtcaag 120
ggtactcaag?agatgaagaa?caactacaac?atcatggaga?tcagaactgt?tgctgttggt 180
atcgttgcta?tcaagggtgt?tgagtctgag?ttctacttgg?ctatgaacaa?ggaaggtaag 240
ttgtacgcta?agaaggagtg?taacgaagac?tgtaacttca?aggagttgat?cttggagaac 300
cactacaaca?cttacgcttc?tgctaagtgg?actcacaacg?gtggtgagat?gttcgttgcc 360
ttgaaccaga?agggtatccc?agttagaggt?aagaagacta?agaaggagca?gaagactgct 420
cacttcttgc?caatggctat?cacttaagaa?ttc 453
Claims (10)
1. the gene fragment of the keratinocyte growth factor of encoding, it has the nucleotide sequence shown in SEQ ID NO.1.
2. expression vector that contains the nucleotide sequence shown in the SEQ ID NO.1.
3. a pichia spp is characterized in that, this pichia spp contains expression vector as claimed in claim 2.
4. the construction process of a pichia spp as claimed in claim 3, this method comprises: (1) utilizes XhoI and EcoRI double digestion synthetic keratinocyte growth factor, construction recombination plasmid X-KGF; (2) transform the pichia spp competent cell, obtain positive transformant by the resistant panel screening; (3) utilize shake flask fermentation that positive transformant is further screened;
Recombinant plasmid X-KGF in the described step (1), the expression plasmid that the X representative is selected.
5. the construction process of pichia spp as claimed in claim 4, it is characterized in that, the described construction recombination plasmid X-KGF of step (1) is meant and utilizes nucleic acid restriction endonuclease XhoI and EcoRI double digestion synthetic keratinocyte growth factor, be cloned on the expression plasmid X that same enzyme cuts, carry out sequence verification then.
6. the construction process of pichia spp as claimed in claim 4, it is characterized in that, step (2) is recombinant plasmid X-KGF and the blank plasmid that makes up in the step (1), transform the pichia spp competent cell, according to antibiotics resistance or hereditary defect type, obtain corresponding positive pichia spp transformant by plate screening; Preferably, described pichia spp competent cell is the competent cell of pichia pastoris engineered strain GS115, KM71, SMD1168 or X-33.
7. the construction process of pichia spp as claimed in claim 4 is characterized in that, the described method of step (3) is that the positive transformant that will obtain in the step (2) carries out the shake flask fermentation screening in substratum, obtains expression cell line.
8. the construction process of pichia spp as claimed in claim 4 is characterized in that, the described recombinant plasmid X-KGF of step (3), and wherein expression plasmid X is: pGAPZ α A/B/C, pPICZ α A/B/C, pPIC9 or pPIC9K.
9. the construction process of pichia spp as claimed in claim 4 is characterized in that, the substratum of the described shake flask fermentation of step (3) is BMMY, BMGY, YPD or FBS.
10. utilize pichia spp as claimed in claim 3 to carry out the application of keratinocyte growth factor aspect the preparation cancer therapy drug that high density fermentation is expressed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436575A (en) * | 2013-08-15 | 2013-12-11 | 华南理工大学 | High-density fermentation medium and high-density fermentation method for pichia pastoris recombinant bacteria |
| WO2017219455A1 (en) * | 2016-06-22 | 2017-12-28 | 陕西慧康生物科技有限责任公司 | Gene for encoding truncated human keratinocyte growth factor, and preparation method therefor |
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| CN1168678A (en) * | 1994-10-13 | 1997-12-24 | 安姆根有限公司 | Method of treating diabetes mellitus using KGF |
| CN1169734A (en) * | 1994-10-13 | 1998-01-07 | 安姆根有限公司 | Method for purifying keratinocyte growth factors |
| CN1408872A (en) * | 2001-09-29 | 2003-04-09 | 上海汉殷药业有限公司 | Plasmid carrier of recombination human horny cell growth factor, its host and method for preparing recombination human horny cell growth factor |
| CN1766107A (en) * | 2005-07-22 | 2006-05-03 | 成都芝田生物工程有限公司 | Recombinant human keratinized cell growth factor production method |
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| CN1168678A (en) * | 1994-10-13 | 1997-12-24 | 安姆根有限公司 | Method of treating diabetes mellitus using KGF |
| CN1169734A (en) * | 1994-10-13 | 1998-01-07 | 安姆根有限公司 | Method for purifying keratinocyte growth factors |
| CN1408872A (en) * | 2001-09-29 | 2003-04-09 | 上海汉殷药业有限公司 | Plasmid carrier of recombination human horny cell growth factor, its host and method for preparing recombination human horny cell growth factor |
| CN1766107A (en) * | 2005-07-22 | 2006-05-03 | 成都芝田生物工程有限公司 | Recombinant human keratinized cell growth factor production method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103436575A (en) * | 2013-08-15 | 2013-12-11 | 华南理工大学 | High-density fermentation medium and high-density fermentation method for pichia pastoris recombinant bacteria |
| CN103436575B (en) * | 2013-08-15 | 2015-07-01 | 华南理工大学 | High-density fermentation medium and high-density fermentation method for pichia pastoris recombinant bacteria |
| WO2017219455A1 (en) * | 2016-06-22 | 2017-12-28 | 陕西慧康生物科技有限责任公司 | Gene for encoding truncated human keratinocyte growth factor, and preparation method therefor |
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