CN1765929A - Contain the fusion rotein of peptide carrier and Urogastron and nucleic acid and uses thereof - Google Patents
Contain the fusion rotein of peptide carrier and Urogastron and nucleic acid and uses thereof Download PDFInfo
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Abstract
The present invention relates to human epidermal growth factor's modification.The invention discloses and comprise peptide carrier sequence (preferred protein transduction territory (PTD)) and fusion rotein, its coding nucleic acid of Urogastron (EGF), the composition that contains them and their purposes.Compound of the present invention and composition can be used as the activeconstituents or the additive of medicine, healthcare products and makeup.
Description
Technical field
The present invention relates to the modification of Urogastron.Particularly, the present invention relates to have modified Urogastron, its coding nucleic acid of strong penetrativity, the composition that contains them and their purposes.
Background technology
Urogastron is Cohen (Life Sci.1965 in 1962,4 (17): 1625-33) in mouse submandibular gland, find, extract and separate, single chain polypeptide by 53 amino-acid residues are formed does not have L-Ala in the polypeptide, phenylalanine and Methionin.It has three disulfide linkage, and these disulfide linkage are that its biologically active is necessary.EGF causes a series of biochemical variations in the cell by conducted signal, starts the gene relevant with cell fission, makes resting cell enter cell division cycle, thereby makes cell proliferation.Denier can promote the division and the growth of cell strongly.EGF is difficult for transdermal, also is difficult for penetrating mucous membrane, so only suitable wound and mucomembranous surface have limited the possibility of its deep tissues and systemic administration.The present invention will have the peptide carrier combination with it of extremely strong penetrativity, and clonal expression goes out people EGF fusion rotein, has solved this problem dexterously.
The peptide carrier is a kind of protein functional domain of people's discovered in recent years, it can lead its protein that carries to pass serous coat, and in cell, accumulate, so they are called protein transduction domain (protein transduction domain, PTD) (Schwarze SR, DowdySF, Trends Pharmacol Sci.2000,21 (2): 45-8, Fawell, Proc NatlAcad Sci USA.1994 Jan 18; 91 (2): 664-8.).Have been found that multiple PTD now, transcribe albumin A ntp (Derossi D from the homology abnormal shape of fruit bat respectively, et al.TheJournal of Biological chemistry.1996,27 (30): 18188-18193.), structural protein VP22 (the Elliott C of hsv, Ohare P.Cell.1997,88:223-233) and transcription activating protein (the Trans-activator transcription of human I type immunodeficiency virus, TAT) (Fawell S., Proc Natl AcadSci USA.1994 Jan 18; 91 (2): 664-8.) and glutathione-S-transferase (glutathione-S-transferase, GST) (Shigeyuki Namiki etc, Biochemical and Biophysical Research Communication 2003,305:592-7).The trans-activator TAT of human immunodeficiency virus coding (86 amino-acid residues are formed) structurally can be divided into five functional zone: (1) N end regions (amino acid/11-21); (2) be rich in the zone (amino acid 22-37) of halfcystine; (3) nucleus (amino acid 38-48); (4) base area (amino acid 48-57); (5) C of variable-length end.TAT is discovered by the transduction of cytolemma one is rich in basic aminoacids in the TAT molecule, have the polypeptide fragment of more positive charge relevant, thereby be called as PTD, be i.e. nexin transduction domain with spanning transduction membrane.
Target protein and PTD just can bring target protein in the cell at an easy rate and go after merging.The effect of PTD has following characteristics: (1) speed is fast; (2) thermal adaptability is wide; (3) the target protein quantity that enters cell depends on the concentration of fusion rotein in the nutrient solution; (4) fusion rotein almost can enter all culturing cells; (5) fusion rotein is injected into the abdominal cavity of animal, can in very short time, arrives all tissues and organ; (6) it is various in style to be brought into the material of cell and body by PTD, small molecules chemical substance, polypeptide, the molecular weight polypeptide from several thousand all can be entered cell by its guiding to the protein of hundreds of thousands of and nucleic acid etc., even the liposome of the iron shot of diameter 40 nanometers and diameter 200 nanometers etc. also can be brought into cell (Steven RS, Alan Ho, Adamina V et al.Science, 1999,285:1569-1572).
PTD mediation pass through cytolemma mechanism and acceptor, all it doesn't matter for translocator.PTD at first interacts by the positive charge of himself and the negative charge of surface of cell membrane, and fusion rotein is attached on the cytolemma, and the effect of passing through by alpha-helix makes fusion rotein enter cell then.The molecule of being brought into cell by PTD still keeps its original character.The PTD fusion molecule can be passed through skin, mucous membrane even hemato encephalic barrier.
Summary of the invention
One aspect of the present invention relates to the fusion rotein that comprises peptide carrier sequence and Urogastron (EGF).Preferably, peptide carrier sequence is protein transduction domain (PTD) or glutathione-S-transferase (GST).
In the present invention, Urogastron (EGF) can be naturally occurring, also can be that reorganization produces or synthetic the generation.In one embodiment, EGF is people EGF, for example recombinant human EGF.In a specific embodiments, Urogastron has the sequence shown in the SEQ ID NO:2.
EGF of the present invention comprises allele variant and species homologue, also comprises mutant and varient, and they keep the EGF activity.
Those skilled in the art can understand, can suitably modify EGF sequence and peptide carrier sequence sequence for example disclosed herein, for example increase, replace, lack and one or morely (as be no more than 20, preferably be no more than 15, more preferably no more than 10,8,5, even more preferably no more than 4,3,2,1) amino acid, and still keep its activity.Therefore, the present invention includes the varient of these modifications interior.For example, EGF of the present invention and peptide carrier comprise having the varient that has at least 70% identity (identity) with SEQ ID NO:2 or 4 or 6 and keep its active aminoacid sequence.Preferably, aminoacid sequence and SEQ ID NO:2 or 4 or 6 have at least 80% identity, more preferably at least 90% identity, at least 95% even 98%, 99% identity shown in.More preferably, EGF of the present invention has the sequence of SEQ ID NO:2.Equally, preferably, peptide carrier sequence of the present invention has the sequence of SEQ ID NO:4 or 6.
In fusion rotein of the present invention, peptide carrier sequence preference is positioned at the N-end of EGF, also can be positioned at the C-end of EGF or other position of protein molecular.Peptide carrier sequence preference next-door neighbour EGF also can link to each other with EGF by the interval aminoacid sequence.In preferred embodiments, the front and back of peptide carrier sequence and EGF order makes peptide carrier sequence to promote EGF to pass through cytolemma effectively respectively with mode of connection (directly link to each other or link to each other by the interval aminoacid sequence).At interval the selection of aminoacid sequence is as the criterion with the function of not damaging or reduce peptide carrier sequence and EGF, is well known by persons skilled in the art therefore.For example, aminoacid sequence is generally used for two compositions of fusion rotein separately making their non-interference spatially at interval.The length of aminoacid sequence can be 1-20 amino acid or longer at interval, for example 1-10.
Peptide carrier of the present invention comprises any aminoacid sequence that can lead its protein that carries to pass serous coat, and it can be natural, also can be synthetic.
In one embodiment, the peptide carrier sequence homology abnormal shape that is selected from fruit bat transcribe the PTD of transcription activating protein (TAT) of structural protein VP22, human I type immunodeficiency virus of albumin A ntp, hsv or the paddy Guang sweet-the S-transferring enzyme; The transcription activating protein (TAT) of preferred human I type immunodeficiency virus and the PTD of GST especially, have the sequence shown in the SEQ ID NO:4.
In one embodiment, EGF is people EGF, for example recombinant human EGF.
Fusion rotein of the present invention can also contain other sequence, and intervening sequence for example helps the appended sequence of purifying, for example contains the label of 6 Histidines at N end or C end.
In a specific embodiments, fusion rotein of the present invention has the sequence shown in the SEQ ID NO:8.
On the other hand, the present invention relates to nucleic acid molecule, it has the sequence that is selected from following nucleotide sequence: the nucleotide sequence of the above-mentioned fusion rotein of (1) coding; (2) with the nucleotide sequence complementary nucleotide sequence of (1).
Nucleic acid molecule of the present invention comprises the nucleotide sequence of encoded peptide carrier and Urogastron.The nucleotide sequence that can also comprise aminoacid sequences such as encoded interval sequence, appended sequence.
The nucleotide sequence of encoded peptide carrier of the present invention and Urogastron can be that any can coding has the nucleotide sequence of active peptide carrier and Urogastron, comprises native sequences and composition sequence.Comprise allele variant and species homologue, also comprise mutant and varient.Those skilled in the art can understand, can suitably modify the encoding sequence of EGF and peptide carrier sequence for example disclosed herein, for example increase, replace, lack and one or morely (as be no more than 60, preferably be no more than 45, more preferably no more than 30,10,5,4,3,2,1) Nucleotide, and still keep its code capacity.Therefore, the present invention includes the varient of these modifications interior.For example, encoding sequence of the present invention comprises having the varient that the nucleotide sequence of at least 70% identity (identity) and encode EGF or peptide carrier is arranged with SEQ ID NO:1 or 3 or 5.Preferably, nucleotide sequence and SEQ ID NO:1 or 3 or 5 have at least 80% identity, more preferably at least 90% identity, at least 95% even 98%, 99% identity shown in.In one embodiment, EGF encoding sequence of the present invention has the sequence of SEQ ID NO:1.In another embodiment, peptide vector encoded sequence of the present invention has the sequence of SEQ ID NO:3 or 5.
Preferably, described nucleic acid molecule comprises nucleotide sequence or its complementary sequence of the coding EGF of the codon design of having a preference for according to host cell.More preferably, described nucleic acid molecule comprises according to prokaryotic organism for example nucleotide sequence or its complementary sequence of the coding EGF of the codon design of intestinal bacteria preferences, perhaps according to plant for example nucleotide sequence or its complementary sequence of the coding EGF of the codon design of tomato preference.
Nucleic acid molecule of the present invention can be DNA or RNA, and for example synthetic DNA comprises the DNA analogue.
In a specific embodiments, nucleic acid molecule of the present invention is selected from: the nucleic acid molecule with encoding said fusion protein of sequence shown in the SEQ ID NO:7, in SEQ ID NO:7, increase, replace, disappearance is one or more (as is no more than 60, preferably be no more than 45, more preferably no more than 30,10,5,4,3,2,1) Nucleotide and still keep the nucleic acid molecule of its code capacity, at least 70% identity (identity) is arranged and still keep the varient of its code capacity and their complementary sequence with SEQ ID NO:7.Preferably, described nucleic acid molecule and SEQ ID NO:1 or 3 or 5 have at least 80% identity, more preferably at least 90% identity, at least 95% even 98%, 99% identity.
On the other hand, the invention still further relates to the carrier that contains said nucleic acid molecule.This carrier can be a cloning vector, also can be expression vector, perhaps shuttle vectors.
Preferably, carrier of the present invention contains and is suitable for controlling element that express, that be operably connected with described nucleic acid molecule in the described fusion rotein purpose host cell.
On the other hand, the invention still further relates to the host cell that contains above-mentioned carrier, for example eukaryotic host cell such as zooblast (preferred mammal is people's cell for example) or insect cell or fungal cell, perhaps prokaryotic host cell such as Bacillus coli cells.The invention still further relates to the organism that contains this cell.
On the other hand, the invention still further relates to composition, it contains fusion rotein of the present invention or nucleic acid molecule or carrier or its one or more any combination.Randomly, described composition also contains vehicle, thinner or auxiliary substance.Preferably, described composition is pharmaceutical composition (being used for prevention or treatment EGF relative disease), Halth-care composition, perhaps make-up composition.
On the other hand, the invention still further relates to the purposes of peptide carrier sequence (preferred protein transduction territory (PTD)) in preparation fusion rotein of the present invention or nucleic acid molecule.
On the other hand, the invention still further relates to fusion rotein of the present invention or nucleic acid molecule or the carrier purposes in preparation medicine, healthcare products or makeup.
On the other hand, the invention still further relates to fusion rotein of the present invention or nucleic acid molecule or the carrier purposes in any purposes of EGF.Fusion rotein of the present invention or nucleic acid molecule or carrier can be used for suitably substituting EGF in any purposes of EGF.
The invention still further relates to nucleic acid molecule, it has nucleotide sequence or its complementary sequence according to the coding EGF of the codon design of host cell preference.In a preferred embodiment, described nucleic acid molecule has: according to the plant nucleotide sequence of the coding EGF of the codon design of tomato preference for example, or its complementary sequence, the sequence shown in the SEQ ID NO:1 for example is perhaps according to the prokaryotic organism nucleotide sequence of the coding EGF of the codon design of intestinal bacteria preference for example.
The present invention also provides nucleic acid molecule, and its nucleotide sequence and SEQ ID NO:1 or 3 or 5 or 7 have at least 80% identity, more preferably at least 90% identity, most preferably at least 95% even 98%, 99% identity.Preferably, described nucleic acid molecule has the sequence of SEQ IDNO:1 or 3 or 5 or 7.
The present invention also provides polypeptide on the other hand, and its aminoacid sequence and SEQ ID NO:2 or 4 or 6 or 8 have at least 80% identity, more preferably at least 90% identity, most preferably at least 95% even 98%, 99% identity.Preferably, described polypeptide has the aminoacid sequence of SEQ ID NO:2 or 4 or 6 or 8.
The accompanying drawing summary
Fig. 1: expression vector pPTD-hEGF structural representation.
The T7P:T7 promotor
PTD: protein transduction domain
EGF: Urogastron
Term: terminator
Fig. 2: SDS-PAGE analyzes PTD-hEGF and expresses
M is the low molecular weight protein (LMWP) standard, Control: control plasmid is expressed, PTD-hEGF: recombinant plasmid amalgamation and expression.
Fig. 3: SDS-PAGE determines the existence form of expressing protein
M: the protein molecular quality standard, the supernatant liquor of 1 cellular lysate liquid, the precipitation of 2-5 cellular lysate liquid is respectively with 2,4,6 and 8M urea handle back supernatant, the sedimentary scavenging solution of 6 lysates, 7 lysate throw outs.
Fig. 4: SDS-PAGE analyzes the PTD-hEGF fusion rotein of purifying
M is the protein molecular quality standard, and 1 is the fusion rotein with 150mM imidazoles elutriant purifying.
Fig. 5: the enzyme of recombinant plasmid pGEX-hEGF is cut evaluation
M:DNA molecular mass standard; 1. recombinant plasmid.
Fig. 6 SDS-PAGE analyzes the expression of gst fusion protein
M: protein molecular quality standard (97.4,66,42,31,14.4) kD; 1.pGEX-hEGF full bacterium lysate; 2. contrast the full bacterium lysate of bacterium pGEX-4T-1.
The Western blot of Fig. 7 expression product analyzes
1.BL21/pGEX-4T-1 lysate; 2.BL21/pGEX-hEGF lysate supernatant; 3.BL21/pGEX-hEGF lysate precipitation.
Determining and the solubleness in different concns urea of Fig. 8 GST-hEGF expression-form
1. cellular lysate liquid precipitate; 2. cellular lysate liquid supernatant liquor; 3-6. the precipitation of cellular lysate liquid is respectively with 2,4,6 and 8M urea handle the back supernatant; M. protein molecular quality standard (97.4,66,42,31,14.4) kD.
Fig. 9 SDS-PAGE analyzes the GST-hEGF fusion rotein of purifying
M: protein molecular quality standard (97.4,66,42,31,14.4) kD; 1. the GST-hEGF albumen of purifying
Figure 10: the biological activity of MTS method bioassay standard hEGF and reorganization PTD-hEGF
The biological activity of Figure 11 MTS method bioassay standard hEGF and reorganization GST-hEGF
Embodiment
The present invention relates to the recombinate nucleic acid molecule of Urogastron (EGF) of a kind of coding people who relies on the codon design of plant-preference aspect concrete at one; The structure of the nucleotide sequence structure of this molecule and people EGF Nucleotide is incomplete same, but coded protein sequence is identical.
The invention still further relates to the application of peptide carrier core nucleotide sequence.Peptide carrier core nucleotide sequence can be placed 5 ' end of human epidermis factor nucleic sequence, constitute the gene structure sequence of an amalgamation and expression.
The invention still further relates to the expression vector pPTD-hEGF and the pGEX-hEGF that contain described nucleic acid molecule; In e. coli bl21, given expression to the human epidermal growth factor of containing peptide carrier sequence, Expression of Fusion Protein is not limited only to BL21, also should expand to other all bio-reactors that all help expressing fusion protein, as Bacillus subtilus, small peptide bacillus and other the prokaryotic organism of protokaryon; Eukaryote such as yeast (yeast saccharomyces cerevisiae, pichia spp and saccharomyces hansenii etc.), animal and plant cells etc.
The invention still further relates to the application of recombinant human EGF fusion rotein.Peptide carrier people EGF fusion rotein is widely used, and as medicine, prevents and treats respiratory tract injury etc. as preventing and treat oral cavity and digestive tract ulcer, treatment skin physical property, chemical and inflammatory damage, spraying or sucking aspect disease prevention and treatment; Can activate glycolysis to increase respiratory metabolism aspect health care, physical strength reinforcing improves immunologic function and anti-ageing waiting for a long time; On cosmetic applications, can go deep into deep skin, improve the nutrition of skin cells, old and feeble skin is in time come off, and dispelling stain keeps skin ruddy, smooth, fine and smooth flexible lastingly, simultaneously can remove wrinkle, reach the purpose of skin-protecting face nursing, and can strengthen the skin defensive ability/resistance ability, prevent external irritant, repair damaged skin, accelerating wound healing.
Peptide carrier people EGF fusion rotein can multiple formulation be used, as lozenge, paster, spray, drops and liquid infusion agent etc.In a word, except that above-mentioned formulation, the EGF fusion rotein can also various other dosage forms easily be applied to clinical, health care and cosmetic industry.
Fusion rotein of the present invention can be used to be similar to the proteic dosage of EGF.Because the biological activity of fusion rotein of the present invention is higher than EGF, therefore, preferably, the application dosage of fusion rotein of the present invention is lower than the application dosage of conventional EGF.For example, the application dosage of fusion rotein of the present invention be no more than conventional EGF application dosage 80%, preferably be no more than 70%, 60% or 50%, more preferably no more than 40%, 30%, 20% even 10% of the application dosage of conventional EGF.
The peptide carrier that the present invention uses is PTD sequence or the GST sequence of the trans-activator TAT of human immunodeficiency virus coding.The guiding of EGF will be not limited only to above-mentioned PTD sequence, also should expand to the peptide carrier sequence that all possess this function, comprising the peptide carrier structure of synthetic.
In a specific embodiments, the present invention relates to a kind of nucleic acid molecule of encoding human epidermal growth factor of the codon design according to plant-preference.Contriver's synthetic the complete nucleotide sequence of this nucleic acid, this sequence and natural human epidermal growth factor's nucleotide sequence are also incomplete same, but coded protein sequence is identical.Human epidermal growth factor's aminoacid sequence of the nucleotide sequence of the nucleic acid of above-mentioned encoding human epidermal growth factor and correspondence is as follows:
atg?aat?agc?gat?tct?gaa?tgt?cca?ctt?tcc?cac?gac?gga?tac?tgc
M N S D S E C P L S H D G Y C
ttg?cat?gat?ggt?gtt?tgc?atg?tac?att?gag?gct?ttg?gac?aag?tat
L H D G V C M Y I E A L D K Y
gca?tgc?aac?tgt?gtt?gtg?ggt?tac?atc?gga?gag?aga?tgt?caa?tac
A C N C V V G Y I G E R C Q Y
cgt?gac?ctt?aaa?tgg?tgg?gaa?ctt?cgt?taa?tag (SEQ?ID?NO：1)
R D L K W W E L R * *?(SEQID?NO：2)
Another aspect of the present invention relates to the utilization of PTD.Preferentially relate to HIV-ITAT albumen PTD and GST.The core sequence of HIV-ITAT albumen PTD is made up of 11 amino acid, and its sequence is YGRKKRRQRRR.The nucleotide sequence that we utilize the codon of intestinal bacteria preferences to design this aminoacid sequence pedestrian worker that goes forward side by side is complete synthesis.Exemplary coding nucleotide sequence and the corresponding amino acid sequence of HIV-ITAT albumen PTD are as follows:
5’tac?ggc?cgc?aag?aaa?cgc?cgc?cag?cgc?cgc?cgc3’(SEQ?ID?NO：3)
Y G R K K R R Q R R R (SEQ?ID?NO：4)
Nucleotide sequence and the corresponding amino acid sequence of an exemplary coding GST are as follows:
1 atg?tcc?cct?ata?cta?ggt?tat?tgg?aaa?att?aag?ggc?ctt?gtg?caa 45
1 Met?Ser?Pro?Ile?Leu?Gly?Tyr?Trp?Lys?Ile?Lys?Gly?Leu?Val?Gln 15
46 ccc act cga ctt?ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag 90
16 Pro Thr Arg Leu?Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu 30
91 cat ttg tat gag?cgc gat gaa ggt gat aaa tgg cga aac aaa aag 135
31 His Leu Tyr Glu?Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys 45
136 ttt gaa ttg ggt?ttg gag ttt ccc aat ctt cct tat tat att gat 80
46 Phe Glu Leu Gly?Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp 60
181 ggt gat gtt aaa?tta aca cag tct atg gcc atc ata cgt tat ata 225
61 Gly Asp Val Lys?Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile 75
226 gct gac aag cac?aac atg ttg ggt ggt tgt cca aaa gag cgt gca 270
76 Ala Asp Lys His?Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala 90
271 gag att tea atg?ctt gaa gga gcg gtt ttg gat att aga tac ggt 315
91 Glu Ile Ser Met?Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly 105
316 gtt tcg aga att?gca tat agt aaa gac ttt gaa act ctc aaa gtt 360
106 Val Ser Arg Ile?Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val 120
361 gat ttt ctt agc?aag cta cct gaa atg ctg aaa atg ttc gaa gat 405
121 Asp Phe Leu Ser?Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp 135
406 cgt tta tgt cat?aaa aca tat tta aat ggt gat cat gta acc cat 450
136 Arg Leu Cys His?Lys Thr Tyr Leu Asn Gly Asp His Val Thr His 150
451 cct gac ttc atg?ttg tat gac gct ctt gat gtt gtt tta tac atg 495
151 Pro Asp Phe Met?Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met 165
496 gac cca atg tgc?ctg gat gcg ttc cca aaa tta gtt tgt ttt aaa 540
166 Asp Pro Met Cys?Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys 180
541 aaa cgt att gaa?gct atc cca caa att gat aag tac ttg aaa tcc 585
181 Lys Arg Ile Glu?Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser 195
586 agc aag tat ata?gca tgg cct ttg cag ggc tgg caa gcc acg ttt 630
196 Ser Lys Tyr Ile?Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe 210
Place 5 ' of people EGF gene to hold the nucleotide sequence of PTD, made up the expression vector pPTD-hEGF and the pGEX-hEGF that can in e. coli bl21, efficiently express.The pPTD-hEGF carrier is to be made up by pET series, contains the expression that the T7 promotor starts fusion gene, 5 ' of fusion rotein hold contain 6 histidine-tagged, and some additional structures that help affinitive layer purification.
Proteic full length amino acid of exemplary fused and nucleotide sequence are as follows:
CAT?CAT?CAT?CAT?CAT?CATGGT?ATG?GCT?AGC?ATG?ACT?GGT?GGA?CAG
His?His?His?His?His?HisGly?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln
CAA?ATG?GGT?CGG?GAT?CTG?TAC?GAC?GAT?GAC?GAT?AAG?GAT?CGA?TGG
Gln?Met?Gly?Arg?Asp?Leu?Tyr?Asp?Asp?Asp?Asp?Lys?Asp?Arg?Trp
GGA?TCC?AAG?CTT?GGC?TAC?GGC?CGC?AAG?AAA?CGC?CGC?CAG?CGC?CGC
Gly?Ser?Lys?Leu?Gly?Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg
CGC?GGT?GGA?TCC?ACC?ATG?TCC?GGC?TAT?CCA?TAT?GAC?GTC?CCA?GAC
Arg?Gly?Gly?Ser?Thr?Met?Ser?Gly?Tyr?Pro?Tyr?Asp?Val?Pro?Asp
TAT?GCT?GGC?TCC?ATG?GCC?GGT?ACC?ATG?AAT?AGC?GAT?TCT?GAA?TGT
Tyr?Ala?Gly?Ser?Met?Ala?Gly?Thr?Met?Asn?Ser?Asp?Ser?Glu?Cys
CCA?CTT?TCC?CAC?GAC?GGA?TAC?TGC?TTG?CAT?GAT?GGT?GTT?TGC?ATG
Pro?Leu?Ser?His?Asp?Gly?Tyr?Gys?Leu?His?Asp?Gly?Val?Cys?Met
TAC?ATT?GAG?GCT?TTG?GAC?AAG?TAT?GCA?TGC?AAC?TGT?GTT?GTG?GGT
Tyr?Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys?Asn?Cys?Val?Val?Gly
TAC?ATC?GGA?GAG?AGA?TGT?CAA?TAC?CGT?GAC?CTT?AAA?TGG?TGG?GAA
Tyr?Ile?Gly?Glu?Arg?Cys?Gln?Tyr?Arg?Asp?Leu?Lys?Trp?Trp?Glu
CTT?CGT?TAA?TGA (SEQ?ID?NO：7)
Leu?Arg?End?End (SEQ?ID?NO：8)
Line place is 6 His sequences, and black matrix is the PTD sequence, and italic is according to the EGF encoding sequence of the codon design of tomato preference and expressed aminoacid sequence.Rest part is intervening sequence or modification sequence.
PGEX-hEGF derives from pGEX-4T-1 carrier (is Time Inc. available from sky, Beijing), holds at 5 ' of fusion rotein and contains the GST protein sequence.
This carrier imports in the e. coli bl21 by chemical transformation, is cultured to finite concentration in the LB liquid nutrient medium that contains penbritin, adds IPTG and induces the fusion rotein PTD-hEGF and the GST-hEGF that can obtain the high expression level amount.
Utilize the method for recombinant bacterial strain BL21/pPTD-hEGF of the present invention and BL21/pGEX-hEGF production recombinant human epidermal growth factor specific as follows:
1. seed liquor preparation
In the test tube that fills 3ml LB nutrient solution, 37 ℃ of overnight incubation (being generally about 16 hours) are worked as OD with the 10ul bacterial classification inoculation
600Can ferment when reaching 0.6 left and right sides, above-mentioned used seed liquor substratum adds the 100mg/L penbritin.
2. fermentation
Under aseptic condition, the inoculum size of above-mentioned cultured seed liquid by 30ml/L is inoculated on the fermention medium; The composition of fermention medium consists of (g/L): Trypt one (peptone) 10g, Yeast extract (yeast extract) 5g, NaCl 5g, penbritin 0.1g; Use the deionized water dissolving constant volume, autoclaving.Fermentation condition is: temperature 28-37 ℃, pH7.0 ferments to OD
600Add IPTG (iprotiazem generation-β-D-galactoside) 100mM when reaching 1.0 left and right sides and induce, centrifugal collection thalline after 3-4 hour, the thalline of collecting can be placed in-20 ℃ of refrigerators, purifying to be separated.
3. the separation and purification of recombinant human epidermal growth factor
(1) purifying of PTD-hEGF
The somatic cells of above-mentioned collection is washed 1-2 time with phosphoric acid buffer PBS (pH7.6);
Every 100ml inoculum suspends with 50ul bacterial lysate (pH7.6), adds N,O-Diacetylmuramidase 1mg/ml, RNase 20ug/ml, and ultrasonication, centrifugal 10 minutes of 10000rpm abandons supernatant liquor, gets crude extract with the urea dissolution precipitation of 8M;
Show that through the SDS-PAGE electrophoresis detection fusion rotein overwhelming majority that obtains with this law is an inclusion body, can obtain expression amount and reach fusion rotein more than 40%.Major part is dissolved in the urea of 8M.It is simple that this law is slightly carried rhEGF, and scale can arbitrarily enlarge, and variable factor is few, and repeatability is fine.
The fusion rotein of expressing can pass through Ni
2+The post single step purification is at last with the imidazoles elutriant wash-out that contains 150mM.
(2) purifying of GST-hEGF
Reset and add 100 μ l Glutathione Sepharose 4B in the cracking of every mL bacterium, room temperature 30min, the centrifugal 5min of 500g abandons supernatant, washes 3 times with PBS, adds the gsh damping fluid of 1 times of volume in the precipitation, and room temperature is stirred 10min gently, and supernatant is collected in centrifugal back.The Lowrry method is measured and is collected proteic content in the liquid.
4. promoting growth of cell activity identification
In 96 orifice plate cultured human embryo kidney HEK-293 cells, add the PTD-hEGF and the GST-hEGF of purifying, be divided into 0.1,0.2,0.4,0.8,1.6,3.2ng/ml different concentration adds MTS and continues to cultivate 1-4 hour after 48 hours, the 492nm place measures optical density value on enzyme connection instrument.Compared good biological activity with standard EGF.
5. use
The raw material that can be used as makeup adds in the ordinary cosmetics; Control oral cavity and digestive tract ulcer; Promote the healing of various skin wounds; Multiple uses such as control respiratory tract injury.
Embodiment
The structure of expression vector pPTD-hEGF and evaluation
Synthetic EGF gene (SEQ ID NO:1) is connected on the pBS-T carrier (purchasing in sky, Beijing is Time Inc.), downcuts goal gene from the T carrier, reclaim purifying with Kpn I+EcoR I; Use Kpn I+EcoR I double digestion expression vector pPTD (nucleotide sequence (SEQ ID NO:3) of synthetic PTD to be connected to carrier pRSET (purchase in Invitrogen, San Diego is built on CA)), to reclaim the cmy vector fragment simultaneously; The target gene fragment and the purpose carrier segments that reclaim are got suitable proportion, at T
4Under the effect of dna ligase, 16 ℃ of connections are spent the night, and get suitable connection liquid transformed competence colibacillus cell TOP10 (purchasing in sky, Beijing is Time Inc.), select several culture identification of carrying out from clone's bacterial plaque that screening culture medium grows.
Extract the plasmid of reorganization bacterium colony, under the effect of Kpn I+EcoR I double digestion, occurred its intended purposes fragment in the electrophoretogram, promptly target gene fragment and carrier segments prove that with these recombinant plasmid order-checkings the expression vector that makes up is correct.Expression vector pPTD-hEGF structural representation is seen Fig. 1.
The PTD-hEGF Expression of Fusion Protein
Extract the correct expression vector transformed into escherichia coli BL21 (DE3) of above-mentioned structure, a plurality of clones of picking carry out abduction delivering, and Fig. 2 is one of them typical case clone's a expression, and as can be seen from the figure, this plasmid has carried out effectively expressing.Through the biosoftware analysis, expression amount accounts for 40% of total bacterial protein.
Determining of expression of recombinant proteins form
After the centrifugal collection of the thalline of abduction delivering, use the PBS buffer solution for cleaning, thalline after will cleaning again is dissolved in the suitable lysate, the ultrasonic disruption thalline, centrifugation supernatant liquor and precipitation, the SDS-PAGE analytical results shows, in the supernatant liquor target protein content seldom, major part all exists with the inclusion body of insoluble form.The lysate precipitation of getting equivalent respectively is dissolved in 2,4,6, in the 8M urea, electrophoretic analysis as can be known, this inclusion body is solubleness maximum (Fig. 3) in 8M urea.
The purifying of PTD-hEGF fusion rotein
With the expression that the PTD-hEGF fusion gene carries out, the albumen of solubility seldom, the overwhelming majority exists with insoluble inclusion body form, so we are dissolved in expression product in the urea of 8M, carries out Ni then
2+-NTA affinity chromatography.With the imidazoles elutriant wash-out of 10-150mM, impurity is more in the imidazoles elutriant of low concentration respectively, and the content of target protein seldom significantly target protein band occurred when concentration is elevated to 150mM.With the elutriant elution of bound that contains imidazoles 150mM the medium of fusion rotein, reclaim elutriant (Fig. 4).Ni
2+The fusion rotein purity that-NTA affinitive layer purification obtains reaches more than 99.5%
The structure of pGEX-hEGF expression vector and evaluation
Synthetic hEGF gene (SEQ NO:1) has added restriction enzyme BamH I restriction enzyme site and ATG atg start codon at its 5 ' end; Added terminator codon TAA and TGA at its 3 ' end, and then linked to each other with EcoR I restriction enzyme site with NOS terminator sequence.(available from the sky is Time Inc. for BamH I and EcoR I double digestion hEGF and pGEX-4T-1, this carrier comprises the GST coding region in the multiple clone site upstream, be used to express the fusion rotein that the N end has merged GST), reclaim target gene fragment and carrier segments, getting suitable proportion mixes, 16 ℃ of connections under the effect of T4DNA ligase enzyme, transformed competence colibacillus cell TOP10, identify that with BamH I and EcoR I double digestion the result has cut out the target gene fragment of 500bp and the carrier segments of 4900bp (Fig. 5) after extracting the positive colony plasmid.Recombinant vectors is delivered the order-checking of Bo Ya biotech firm, and the result proves that the nucleotide sequence of constructed expression vector gene is entirely true.
The expression of pGEX-hEGF expression vector in e. coli bl21
Recombinant plasmid pGEX-hEGF transformed competence colibacillus e. coli bl21 (DE3) is at Amp
rSelect single colony inoculation on the agar plate in 3ml LB/amp nutrient solution, cultivate OD for 33 ℃
600During=0.5 left and right sides, add IPTG to final concentration 0.5mmol/L, continue to cultivate 2h, collect thalline, PBS washing 2 times adds the resuspended thalline of 50 μ l cell pyrolysis liquids by every mL inoculum, ultrasonic degradation, and centrifugal back is collected and is gone up cleer and peaceful bacterial chip.SDS-PAGE detects, and a tangible protein band occurred in the 33kD place, and a protein band has appearred in contrast bacterium (containing empty plasmid pEGX-4T-1) at the 27kD place.The synthetic human epidermal growth factor is contained 54 amino acid, and the molecular weight size is 6kD, and the GST size is 27kD, so the albumen at 33kD place should be the fusion rotein of GST-hEGF.Proof GST-hEGF fusion rotein has obtained expression in intestinal bacteria, analyze expression amount through biosoftware and account for 34% of total protein content.The expression of SDS-PAGE analysis fusioning protein is seen Fig. 6.
Bacterial strain carries out SDS-PAGE to the last cleer and peaceful precipitation of its lysate respectively and analyzes behind the IPTG abduction delivering, goes to then on the nylon membrane, and be one anti-with the anti-hEGF of rabbit, goat anti-rabbit antibody is that the two anti-Western traces that carry out detect, and the results are shown in shown in Figure 7.Cleer and peaceful precipitation swimming lane has all demonstrated special band on the lysate of this bacterium, the protein expression band position consistency of its position and coomassie brilliant blue staining, and the swimming lane of contrast bacterium does not show any band, illustrates that expression product is the fusion rotein of GST-hEGF.
Embodiment 7
Determining of GST-hEGF expression-form
Be to determine the expression-form of hEGF in E.coli, behind the thalline usefulness PBS thorough washing with centrifugal collection, ultrasonic disruption (or liquid nitrogen freezing fragmentation) centrifugation supernatant and precipitation.Precipitation is respectively with containing 2M, 4M, 6M, the dissolving of the PBS damping fluid of 8M urea is got 10 μ l and isopyknic sample-loading buffer mixing after centrifugal, 95 ℃ of sex change 5min, the SDS-PAGE electrophoresis all contains fusion rotein in precipitation and the supernatant liquor as can be seen, and the form of the existing solubility of expressed proteins is described, insoluble inclusion body is also arranged, and the amount that contains in the inclusion body is more more.Urea dissolving proof GST-hEGF solubleness maximum (Fig. 8) in the urea of 8M.
Embodiment 8
The purifying of GST-hEGF fusion rotein
Reset and add 100 μ l Glutathione Sepharose 4B in the cracking of every mL bacterium, room temperature 30min, the centrifugal 5min of 500g abandons supernatant, washes 3 times with PBS, adds the gsh damping fluid of 1 times of volume in the precipitation, and room temperature is stirred 10min gently, and supernatant is collected in centrifugal back.Albumen supernatant behind the purifying is surveyed protein concentration through the Lowry method.Every 100ml bacterium liquid finally can obtain the GST-hEGF fusion rotein of 6.3mg.The SDS-PAGE electrophoresis observation only stays size and is the band (Fig. 9) of 33kD.
Embodiment 9
Short cell-proliferation activity analysis
With the fusion rotein (shared ratiometric conversion in fusion rotein becomes the actual content of hEGF according to hEGF) behind standard rhEGF and the purifying, degerming after filtration is diluted to 0.1,0.2,0.4,0.8,1.6,3.2,6.4ng/ml with the RPMI 1640 that contains 0.5%FBS.Getting one of aseptic 96 orifice plate, is blank with No. 1 hole, and No. 2 the hole is contrast, adds the RPMI 1640 substratum 50ul that contain 0.5%FBS.Since adding above-mentioned half-and-half each 50ul of nutrient solution of dilution for No. 3 to the right successively, each sample is established 3 parallel group.Select one bottle in logarithmic phase cell, trysinization is adjusted cell with the substratum that contains 0.5%FBS behind the cell harvesting and is counted to desired concn (every hole adds 5000 left and right sides cells), and every hole adds cell suspension 50ul.37 ℃, 5%CO
2Incubator was cultivated 48 hours.Every empty 20ul MTS solution, 37 ℃, 5%CO of adding
2Incubator continues to cultivate 1-4 hour, surveys OD value, λ=492nm with enzyme-linked immunosorbent assay instrument.
(1) the short cell-proliferation activity of reorganization PTD-hEGF fusion rotein
Do experiment with human embryo kidney (HEK) 293 cells, standard substance rhEGF and PTD-hEGF have shown has tangible biological activity to culturing cell, rising with concentration in the 0.1-0.4ng/ml scope strengthens, but PTD-hEGF shows the character different with standard again, reaches maximum activity value consumption littler (Figure 10).
The fusion rotein PTD-hEGF of prokaryotic expression has mainly formed inclusion body, inclusion body is the aggregate of denatured protein, do not have biological activity, and shown tangible promoting growth of cell activity after adding in the culturing cell, this phenomenon explanation inclusion body has carried out activation recovering.Though the invention is not restricted to concrete theory,, its mechanism should be that PTD-hEGF has entered cell interior, has taken place again foldingly in cell, becomes the form that physiologically active is arranged by the form of non-activity.The concentration of hEGF is when 0.1-0.4ng/ml in cell cultures, its promoting growth of cell effect has tangible dose-effect relationship, but obviously the activity than standard hEGF is high again for the promoting growth of cell activity of PTD-hEGF, its reason may be that hEGF has entered in the nucleus under the traction of PTD, owing to have nuclear localization signal peptide (GRKKR) in the PTD sequence, mainly accumulate in cell behind its transmembrane transport, especially in the karyon, (Schwarze SR such as Schwarze, Ho A.Vocero-AkbamA, Dowdy SF.Science, 1999; 285 (5433): 1569-1572) exogenous protein of proof PTD transduction has nucleus accumulative characteristics.HEGF assembles in nuclear, direct regulation and control DNA, RNA and proteinic synthesizing.
(2) the short cell-proliferation activity of reorganization GST-hEGF fusion rotein
Detect with the biological activity of MTS method the GST-hEGF fusion rotein.Show that the GST-hEGF sample has similar ED to standard substance rhEGF
50(Effective dose 50: make cell yield reach the concentration that maximum is planted a half EGF) value.And reorganization GST-hEGF sample has higher biological activity (Figure 11) than standard substance hEGF.
The GST-hEGF fusion rotein can obviously promote the division and the growth of HEK-293 cell.Activity at 0.1-0.8ng/mL concentration range endonexin is higher than standard rhEGF, the mechanism of action of this peculiar phenomenon prompting hEGF except that with its cytolemma on receptors bind cause the series reaction, also may be brought into cell interior (the Namiki S that works by GST, Tomida T, Tanabe M, IinoM, Hirose K.Biochemical and Biophysical Research Communications, 2003,305:592 597.), perhaps arrive and link to each other with karyomit(e) in the nucleus, directly impel DNA, RNA and proteinic synthesizing.
The biological activity prompting that the GST-hEGF fusion rotein is shown: the utilization of hEGF can not need to remove the GST part of fusion rotein, brings great convenience for like this expression and purifying, can reduce production costs, and makes hEGF obtain utilizing more widely.
Sequence table
＜110〉Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences
＜120〉contain the fusion rotein of peptide carrier and Urogastron and nucleic acid and uses thereof
<130>IDC040097
<160>8
<170>PatentIn?version?3.2
<210>1
<211>168
<212>DNA
＜213〉artificial
<220>
＜223〉according to the nucleic acid molecule of encoding human epidermal growth factor of the codon of plant-preference design
<220>
<221>CDS
<222>(1)..(168)
<400>1
atg?aat?agc?gat?tct?gaa?tgt?cca?ctt?tcc?cac?gac?gga?tac?tgc?ttg 48
Met?Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp?Gly?Tyr?Cys?Leu
1 5 10 15
cat?gat?ggt?gtt?tgc?atg?tac?att?gag?gct?ttg?gac?aag?tat?gca?tgc 96
His?Asp?Gly?Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys
20 25 30
aac?tgt?gtt?gtg?ggt?tac?atc?gga?gag?aga?tgt?caa?tac?cgt?gac?ctt 144
Asn?Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg?Cys?Gln?Tyr?Arg?Asp?Leu
35 40 45
aaa?tgg?tgg?gaa?ctt?cgt?taa?tag 168
Lys?Trp?Trp?Glu?Leu?Arg
50
<210>2
<211>54
<212>PRT
＜213〉artificial
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＜223〉composite structure
<400>2
Met?Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp?Gly?Tyr?Cys?Leu
1 5 10 15
His?Asp?Gly?Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys
20 25 30
Asn?Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg?Cys?Gln?Tyr?Arg?Asp?Leu
35 40 45
Lys?Trp?Trp?Glu?Leu?Arg
50
<210>3
<211>33
<212>DNA
＜213〉artificial
<220>
＜223〉the HIV-I TAT albumen PTD coding nucleotide sequence of synthetic
<220>
<221>CDS
<222>(1)..(33)
<400>3
tac?ggc?cgc?aag?aaa?cgc?cgc?cag?cgc?cgc?cgc 33
Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg
1 5 10
<210>4
<211>11
<212>PRT
＜213〉artificial
<220>
＜223〉composite structure
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Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg
1 5 10
<210>5
<211>678
<212>DNA
＜213〉artificial
<220>
＜223〉nucleotide sequence of coding GST
<220>
<221>CDS
<222>(1)..(678)
<400>5
atg?tcc?cct?ata?cta?ggt?tat?tgg?aaa?att?aag?ggc?ctt?gtg?caa?ccc 48
Met?Ser?Pro?Ile?Leu?Gly?Tyr?Trp?Lys?Ile?Lys?Gly?Leu?Val?Gln?Pro
1 5 10 15
act?cga?ctt?ctt?ttg?gaa?tat?ctt?gaa?gaa?aaa?tat?gaa?gag?cat?ttg 96
Thr?Arg?Leu?Leu?Leu?Glu?Tyr?Leu?Glu?Glu?Lys?Tyr?Glu?Glu?His?Leu
20 25 30
tat?gag?cgc?gat?gaa?ggt?gat?aaa?tgg?cga?aac?aaa?aag?ttt?gaa?ttg 144
Tyr?Glu?Arg?Asp?Glu?Gly?Asp?Lys?Trp?Arg?Asn?Lys?Lys?Phe?Glu?Leu
35 40 45
ggt?ttg?gag?ttt?ccc?aat?ctt?cct?tat?tat?att?gat?ggt?gat?gtt?aaa 192
Gly?Leu?Glu?Phe?Pro?Asn?Leu?Pro?Tyr?Tyr?Ile?Asp?Gly?Asp?Val?Lys
50 55 60
tta?aca?cag?tct?atg?gcc?atc?ata?cgt?tat?ata?gct?gac?aag?cac?aac 240
Leu?Thr?Gln?Ser?Met?Ala?Ile?Ile?Arg?Tyr?Ile?Ala?Asp?Lys?His?Asn
65 70 75 80
atg?ttg?ggt?ggt?tgt?cca?aaa?gag?cgt?gca?gag?att?tca?atg?ctt?gaa 288
Met?Leu?Gly?Gly?Cys?Pro?Lys?Glu?Arg?Ala?Glu?Ile?Ser?Met?Leu?Glu
85 90 95
gga?gcg?gtt?ttg?gat?att?aga?tac?ggt?gtt?tcg?aga?att?gca?tat?agt 336
Gly?Ala?Val?Leu?Asp?Ile?Arg?Tyr?Gly?Val?Ser?Arg?Ile?Ala?Tyr?Ser
100 105 110
aaa?gac?ttt?gaa?act?ctc?aaa?gtt?gat?ttt?ctt?agc?aag?cta?cct?gaa 384
Lys?Asp?Phe?Glu?Thr?Leu?Lys?Val?Asp?Phe?Leu?Ser?Lys?Leu?Pro?Glu
115 120 125
atg?ctg?aaa?atg?ttc?gaa?gat?cgt?tta?tgt?cat?aaa?aca?tat?tta?aat 432
Met?Leu?Lys?Met?Phe?Glu?Asp?Arg?Leu?Cys?His?Lys?Thr?Tyr?Leu?Asn
130 135 140
ggt?gat?cat?gta?acc?cat?cct?gac?ttc?atg?ttg?tat?gac?gct?ctt?gat 480
Gly?Asp?His?Val?Thr?His?Pro?Asp?Phe?Met?Leu?Tyr?Asp?Ala?Leu?Asp
145 150 155 160
gtt?gtt?tta?tac?atg?gac?cca?atg?tgc?ctg?gat?gcg?ttc?cca?aaa?tta 528
Val?Val?Leu?Tyr?Met?Asp?Pro?Met?Cys?Leu?Asp?Ala?Phe?Pro?Lys?Leu
165 170 175
gtt?tgt?ttt?aaa?aaa?cgt?att?gaa?gct?atc?cca?caa?att?gat?aag?tac 576
Val?Cys?Phe?Lys?Lys?Arg?Ile?Glu?Ala?Ile?Pro?Gln?Ile?Asp?Lys?Tyr
180 185 190
ttg?aaa?tcc?agc?aag?tat?ata?gca?tgg?cct?ttg?cag?ggc?tgg?caa?gcc 624
Leu?Lys?Ser?Ser?Lys?Tyr?Ile?Ala?Trp?Pro?Leu?Gln?Gly?Trp?Gln?Ala
195 200 205
acg?ttt?ggt?ggt?ggc?gac?cat?cct?cca?aaa?tcg?gat?ctg?gtt?ccg?cgt 672
Thr?Phe?Gly?Gly?Gly?Asp?His?Pro?Pro?Lys?Ser?Asp?Leu?Val?Pro?Arg
210 215 220
gga?tcc 678
Gly?Ser
225
<210>6
<211>226
<212>PRT
＜213〉artificial
<220>
＜223〉composite structure
<400>6
Met?Ser?Pro?Ile?Leu?Gly?Tyr?Trp?Lys?Ile?Lys?Gly?Leu?Val?Gln?Pro
1 5 10 15
Thr?Arg?Leu?Leu?Leu?Glu?Tyr?Leu?Glu?Glu?Lys?Tyr?Glu?Glu?His?Leu
20 25 30
Tyr?Glu?Arg?Asp?Glu?Gly?Asp?Lys?Trp?Arg?Asn?Lys?Lys?Phe?Glu?Leu
35 40 45
Gly?Leu?Glu?Phe?Pro?Asn?Leu?Pro?Tyr?Tyr?Ile?Asp?Gly?Asp?Val?Lys
50 55 60
Leu?Thr?Gln?Ser?Met?Ala?Ile?Ile?Arg?Tyr?Ile?Ala?Asp?Lys?His?Asn
65 70 75 80
Met?Leu?Gly?Gly?Cys?Pro?Lys?Glu?Arg?Ala?Glu?Ile?Ser?Met?Leu?Glu
85 90 95
Gly?Ala?Val?Leu?Asp?Ile?Arg?Tyr?Gly?Val?Ser?Arg?Ile?Ala?Tyr?Ser
100 105 110
Lys?Asp?Phe?Glu?Thr?Leu?Lys?Val?Asp?Phe?Leu?Ser?Lys?Leu?Pro?Glu
115 120 125
Met?Leu?Lys?Met?Phe?Glu?Asp?Arg?Leu?Cys?His?Lys?Thr?Tyr?Leu?Asn
130 135 140
Gly?Asp?His?Val?Thr?His?Pro?Asp?Phe?Met?Leu?Tyr?Asp?Ala?Leu?Asp
145 150 155 160
Val?Val?Leu?Tyr?Met?Asp?Pro?Met?Cys?Leu?Asp?Ala?Phe?Pro?Lys?Leu
165 170 175
Val?Cys?Phe?Lys?Lys?Arg?Ile?Glu?Ala?Ile?Pro?Gln?Ile?Asp?Lys?Tyr
180 185 190
Leu?Lys?Ser?Ser?Lys?Tyr?Ile?Ala?Trp?Pro?Leu?Gln?Gly?Trp?Gln?Ala
195 200 205
Thr?Phe?Gly?Gly?Gly?Asp?His?Pro?Pro?Lys?Ser?Asp?Leu?Val?Pro?Arg
210 215 220
Gly?Ser
225
<210>7
<211>372
<212>DNA
＜213〉artificial
<220>
＜223〉proteic nucleotide sequence of exemplary fused
<220>
<221>CDS
<222>(1)..(372)
<400>7
cat?cat?cat?cat?cat?cat?ggt?atg?gct?agc?atg?act?ggt?gga?cag?caa 48
His?His?His?His?His?His?Gly?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln
1 5 10 15
atg?ggt?cgg?gat?ctg?tac?gac?gat?gac?gat?aag?gat?cga?tgg?gga?tcc 96
Met?Gly?Arg?Asp?Leu?Tyr?Asp?Asp?Asp?Asp?Lys?Asp?Arg?Trp?Gly?Ser
20 25 30
aag?ctt?ggc?tac?ggc?cgc?aag?aaa?cgc?cgc?cag?cgc?cgc?cgc?ggt?gga 144
Lys?Leu?Gly?Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Gly?Gly
35 40 45
tcc?acc?atg?tcc?ggc?tat?cca?tat?gac?gtc?cca?gac?tat?gct?ggc?tcc 192
Ser?Thr?Met?Ser?Gly?Tyr?Pro?Tyr?Asp?Val?Pro?Asp?Tyr?Ala?Gly?Ser
50 55 60
atg?gcc?ggt?acc?atg?aat?agc?gat?tct?gaa?tgt?cca?ctt?tcc?cac?gac 240
Met?Ala?Gly?Thr?Met?Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp
65 70 75 80
gga?tac?tgc?ttg?cat?gat?ggt?gtt?tgc?atg?tac?att?gag?gct?ttg?gac 288
Gly?Tyr?Cys?Leu?His?Asp?Gly?Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp
85 90 95
aag?tat?gca?tgc?aac?tgt?gtt?gtg?ggt?tac?atc?gga?gag?aga?tgt?caa 336
Lys?Tyr?Ala?Cys?Asn?Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg?Cys?Gln
100 105 110
tac?cgt?gac?ctt?aaa?tgg?tgg?gaa?ctt?cgt?taa?tga 372
Tyr?Arg?Asp?Leu?Lys?Trp?Trp?Glu?Leu?Arg
115 120
<210>8
<211>122
<212>PRT
＜213〉artificial
<220>
＜223〉composite structure
<400>8
His?His?His?His?His?His?Gly?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln
1 5 10 15
Met?Gly?Arg?Asp?Leu?Tyr?Asp?Asp?Asp?Asp?Lys?Asp?Arg?Trp?Gly?Ser
20 25 30
Lys?Leu?Gly?Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Gly?Gly
35 40 45
Ser?Thr?Met?Ser?Gly?Tyr?Pro?Tyr?Asp?Val?Pro?Asp?Tyr?Ala?Gly?Ser
50 55 60
Met?Ala?Gly?Thr?Met?Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp
65 70 75 80
Gly?Tyr?Cys?Leu?His?Asp?Gly?Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp
85 90 95
Lys?Tyr?Ala?Cys?Asn?Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg?Cys?Gln
100 105 110
Tyr?Arg?Asp?Leu?Lys?Trp?Trp?Glu?Leu?Arg
115 120
Claims (10)
1. the fusion rotein that comprises peptide carrier sequence (preferred protein transduction territory (PTD) or glutathione-S-transferase (GST)) and Urogastron (EGF);
Preferably, wherein peptide carrier sequence is positioned at the N-end of EGF, preferably is close to EGF, also can link to each other with EGF by intervening sequence;
Preferably, wherein the peptide carrier sequence homology abnormal shape that is selected from fruit bat is transcribed the PTD or the glutathione-S-transferase (GST) of the transcription activating protein (TAT) of the structural protein VP22 of albumin A ntp, hsv or human I type immunodeficiency virus; The PTD of the transcription activating protein (TAT) of preferred human I type immunodeficiency virus, especially, have the sequence shown in the SEQ ID NO:4,
Preferably, wherein EGF is people EGF, preferred recombinant human EGF;
Preferably, wherein said fusion rotein also contains the appended sequence that helps purifying, for example holds the label that contains 6 Histidines at N end or C;
Preferably, described fusion rotein has the sequence shown in the SEQ ID NO:8.
2. nucleic acid molecule, it has the sequence that is selected from following nucleotide sequence: each the nucleotide sequence of fusion rotein of (1) coding claim 1-6; (2) with the nucleotide sequence complementary nucleotide sequence of (1);
Preferably, described nucleic acid molecule comprises according to plant for example nucleotide sequence or its complementary sequence (sequence shown in SEQ ID NO:1) of the coding EGF of the codon design of tomato preference, perhaps according to prokaryotic organism for example nucleotide sequence or its complementary sequence of the coding EGF of the codon design of intestinal bacteria preference.
Especially, described nucleic acid molecule is DNA, for example has the sequence shown in the SEQ ID NO:7.
3. the carrier that contains the nucleic acid molecule of claim 2; Preferably, it contains be suitable for controlling element that described fusion rotein is expressed, that be operably connected with described nucleic acid molecule in the purpose host cell.
4. the host cell that contains the carrier of claim 9 or 10, for example eukaryotic host cell such as zooblast, vegetable cell, insect cell or fungal cell, perhaps prokaryotic host cell such as Bacillus coli cells.
5. composition, it contains each fusion rotein or the nucleic acid molecule of claim 7 or 8 or carrier or its one or more any combination of claim 9 or 10 of claim 1-6; Randomly, described composition also contains vehicle, thinner or auxiliary substance; Preferably, described composition is pharmaceutical composition (being used for prevention or treatment EGF relative disease), Halth-care composition, perhaps make-up composition.
Peptide carrier sequence (preferred protein transduction territory (PTD)) preparation claim 1-6 each fusion rotein or the purposes in the nucleic acid molecule of claim 7 or 8.
Claim 1-6 each fusion rotein or the purposes of carrier in preparation medicine, healthcare products or makeup of the nucleic acid molecule of claim 7 or 8 or claim 9 or 10.
8. nucleic acid molecule, it has: according to the plant nucleotide sequence of the coding EGF of the codon design of tomato preference for example, or its complementary sequence, the sequence shown in the SEQ ID NO:1 for example is perhaps according to the prokaryotic organism nucleotide sequence of the coding EGF of the codon design of intestinal bacteria preference for example.
9. nucleic acid molecule, it has following nucleotide sequence: described nucleotide sequence and SEQ IDNO:1 or 3 or 5 or 7 have at least 80% identity, more preferably at least 90% identity, most preferably at least 95% even 98%, 99% identity; Preferably, described nucleic acid molecule has the sequence of SEQ ID NO:I or 3 or 5 or 7.
10. polypeptide, it has following aminoacid sequence: described aminoacid sequence and SEQ IDNO:2 or 4 or 6 or 8 have at least 80% identity, more preferably at least 90% identity, most preferably at least 95% even 98%, 99% identity; Preferably, described polypeptide has the aminoacid sequence of SEQ ID NO:2 or 4 or 6 or 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100901593A CN100528899C (en) | 2004-10-29 | 2004-10-29 | Fusion protein and nucleic acid containing peptide carrier and epidermal growth factor and its uses |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100901593A CN100528899C (en) | 2004-10-29 | 2004-10-29 | Fusion protein and nucleic acid containing peptide carrier and epidermal growth factor and its uses |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008043241A1 (en) * | 2006-09-25 | 2008-04-17 | Southern Medical University Zhujing Hospital | A fusion protein carrying neurotrophin across the blood-brain barrier, encoding gene and uese thereof |
| CN101921314A (en) * | 2010-06-28 | 2010-12-22 | 华中科技大学 | Brain-targeting neurotrophic factor fusion polypeptide for preventing and treating Alzheimer's disease |
| CN101081870B (en) * | 2006-05-31 | 2012-07-11 | 华中科技大学 | Antineoplastic biological medicament PTD4-GFP-Apoptin fusion protein and preparation method thereof |
| CN101711874B (en) * | 2008-10-08 | 2012-07-25 | 广州暨南大学医药生物技术研究开发中心 | Application of cell penetrating peptide Tat-mediated growth factor in transdermal transfer |
| CN101897953B (en) * | 2009-05-27 | 2013-03-27 | 中国人民解放军军事医学科学院毒物药物研究所 | Non-invasive high-penetrability epidermal growth factor and application thereof |
| CN103705395A (en) * | 2014-01-03 | 2014-04-09 | 广州瑾洋化妆品有限公司 | Whey protein composition and application thereof to face beautifying |
| CN109563173A (en) * | 2016-07-14 | 2019-04-02 | 生化制剂有限公司 | Chimeric protein, polynucleotides, genetic structure, the preparation (type) for activating body, regenerating bone or cartilage |
| WO2019240430A1 (en) * | 2018-06-14 | 2019-12-19 | (주) 에빅스젠 | Fusion protein bound to cell-permeable peptide, and composition comprising fusion protein or cell-permeable peptide and epithelial cell growth factor as active ingredients |
| CN112625089A (en) * | 2020-12-14 | 2021-04-09 | 四川省恩乐生物工程有限公司 | Active protein peptide gene with excellent repairing effect and coded protein peptide |
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| CN107365784A (en) * | 2017-08-02 | 2017-11-21 | 滨州医学院 | A kind of fusion protein and synthetic method for delivery to central nervous system |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1279288A (en) * | 2000-07-19 | 2001-01-10 | 深圳市华生元基因工程发展有限公司 | Recombination human epidermal growth factor gene, its expression product and its application |
| US20020142299A1 (en) * | 2001-01-09 | 2002-10-03 | Davidson Beverly L. | PTD-modified proteins |
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- 2004-10-29 CN CNB2004100901593A patent/CN100528899C/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101081870B (en) * | 2006-05-31 | 2012-07-11 | 华中科技大学 | Antineoplastic biological medicament PTD4-GFP-Apoptin fusion protein and preparation method thereof |
| WO2008043241A1 (en) * | 2006-09-25 | 2008-04-17 | Southern Medical University Zhujing Hospital | A fusion protein carrying neurotrophin across the blood-brain barrier, encoding gene and uese thereof |
| CN101711874B (en) * | 2008-10-08 | 2012-07-25 | 广州暨南大学医药生物技术研究开发中心 | Application of cell penetrating peptide Tat-mediated growth factor in transdermal transfer |
| CN101897953B (en) * | 2009-05-27 | 2013-03-27 | 中国人民解放军军事医学科学院毒物药物研究所 | Non-invasive high-penetrability epidermal growth factor and application thereof |
| CN101921314A (en) * | 2010-06-28 | 2010-12-22 | 华中科技大学 | Brain-targeting neurotrophic factor fusion polypeptide for preventing and treating Alzheimer's disease |
| CN101921314B (en) * | 2010-06-28 | 2012-11-21 | 华中科技大学 | Brain-targeting neurotrophic factor fusion polypeptide for preventing and treating Alzheimer's disease and preparation method |
| CN103705395A (en) * | 2014-01-03 | 2014-04-09 | 广州瑾洋化妆品有限公司 | Whey protein composition and application thereof to face beautifying |
| CN103705395B (en) * | 2014-01-03 | 2015-11-18 | 广州瑾洋化妆品有限公司 | Lactalbumin composition and the purposes in beauty treatment thereof |
| CN109563173A (en) * | 2016-07-14 | 2019-04-02 | 生化制剂有限公司 | Chimeric protein, polynucleotides, genetic structure, the preparation (type) for activating body, regenerating bone or cartilage |
| WO2019240430A1 (en) * | 2018-06-14 | 2019-12-19 | (주) 에빅스젠 | Fusion protein bound to cell-permeable peptide, and composition comprising fusion protein or cell-permeable peptide and epithelial cell growth factor as active ingredients |
| US11547649B2 (en) | 2018-06-14 | 2023-01-10 | Avixgen Inc. | Fusion protein bound to cell-permeable peptide, and composition comprising fusion protein or cell-permeable peptide and epithelial cell growth factor as active ingredients |
| CN112625089A (en) * | 2020-12-14 | 2021-04-09 | 四川省恩乐生物工程有限公司 | Active protein peptide gene with excellent repairing effect and coded protein peptide |
| CN112625089B (en) * | 2020-12-14 | 2023-01-24 | 四川省恩乐生物工程有限公司 | Active protein peptide gene with excellent repairing effect and coded protein peptide |
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| CN100528899C (en) | 2009-08-19 |
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