CN101897953B - Non-invasive high-penetrability epidermal growth factor and application thereof - Google Patents
Non-invasive high-penetrability epidermal growth factor and application thereof Download PDFInfo
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- CN101897953B CN101897953B CN200910143772XA CN200910143772A CN101897953B CN 101897953 B CN101897953 B CN 101897953B CN 200910143772X A CN200910143772X A CN 200910143772XA CN 200910143772 A CN200910143772 A CN 200910143772A CN 101897953 B CN101897953 B CN 101897953B
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Abstract
The invention relates to a non-invasive high-penetrability epidermal growth factor (EGF) and application thereof, in particular to protein obtained by fusing protein transduction peptides with active fragments of human EGF and a nucleic acid molecule containing the nucleotide sequence coding the fragment. The invention relates to an expression vector containing the nucleic acid molecule. The EGF can treat virus infection, specifically kill tumor cells and treat neurodegenerative diseases, etc.
Description
Technical field
The present invention relates to fusion rotein and nucleic acid thereof that protein transduction domain merges the hEGF, comprise a series of technical matters such as construction expression purification.
Technical background
Epidermal growth factor is a kind of albumen that Cohen in 1962 and Montalcini professor find in mouse submandibular gland, and it has and impels that the newborn mice eyelid is early opened, the effect of tooth premature eruption.This active component is added in the substrate of cultivating epiderm skin, find that it can directly promote the growth of epiderm skin, therefore, with this active component called after " epidermal growth factor (epidermal growth factor, EGF).Epidermal growth factor (EGF) contains 3 intrachain disulfide bonds by the single chain polypeptide that 53 amino acid residues form.Isoelectric point, IP (PI) is 4.6, and molecular weight is 6045Da.
Gregory in 1975 extracts from Urina Hominis and obtains a kind of material, but its gastric acid secretion inhibiting and be called as urogastrone (urogastrone, UG).Proved afterwards that these two kinds of materials were very similar on structure and physiological function, so people regard EGF and this two peptide species of UG as the same class material, be referred to as " EGF-URO (EGF-UG) ".EGF content seldom (can only extract 1 gram) in the Urina Hominis in per 100,000 liters of urines, and has the probability of pollution and loss of activity.Utilize in recent years report and the Patents of the existing successful expression EGF of gene engineering method.
Experiment in vitro confirms EGF scalable neural precursor division growth.Adding EGF can make the neural precursor of BrdU labelling obviously increase.The retina precursor of mouse embryo can be divided into neuron and glial cell under the EGF effect.EGF also has proliferation to the neural precursor in the Mus olfactory bulb.EGF can stimulate the stem cells hyperplasia that separates with ventricles of the brain inferior segment from the Mus striatum.The reconstruction of early stage neuromechanism and function behind the EGF participation cerebral infarction.EGF is a kind of neurotrophic factor that cell in vitro is cultivated, promoted cell proliferation that is usually used in, and the potent effect of its short cell mitogen and the growth of enhancing neuron axon gains public acceptance.Behind the adults brain injury, although the NSC of subependymal region have propagation and migrate reaction, owing to the quantity that is divided into neuronal cell is difficult to improve function of nervous system very little.Craigig etc. inject EGF the tricorn of mouse, found that the cell number showed increased that ependyma district nestin is positive, showing very limited in the neuron regeneration ability of mammalian central nervous system is not to be owing to the enough neural stem cell of shortage, but owing to lacks the necessary neural factor of stimulation of neural stem cells differentiation.EGF is growing than working late period, and works in the propagation of neural stem cell and differentiation.The loss that excites endogenous NSC to breed to remedy neuronal cell might become the research direction for the treatment of from now on the neurodegenerative diseases such as AD.
But EGF is very unstable under the room temperature state, is easy to degraded and loses activity, and is difficult to cross the structures such as cell membrane, and makes drug effect loss, has limited application clinically.
Summary of the invention
Research is found, has the protein transduction of being called as district (proteintransduction domain at some protein, PTD) small fragment, be called again cell-penetrating peptide (cell-penetrating peptides, CPP) can effectively pass through biomembrane, enter various types of cells.Exogenous proteins, DNA or complex can enter cell and pass through blood brain barrier with after PTD is connected.Therefore, PTD is transporting protein, DNA or complex, and and then the treatment viral infection, there are huge potentiality the aspects such as specific killing tumor cell and neurodegenerative diseases.
HIV (human immunodeficiency virus)-1 (human immunodeficiency virus-1, HIV-1) antisense activated transcription (trans-activator transcription, TAT) albumen PTD (TAT-PTD) is a kind of source of present PTD.11 aminoacid (47-57 amino acids) among the TAT are the peptide sections with protein transduction, and its sequence is YGRKKRRQRRR (SEQ IDNO.1).TAT-PTD can transduce the polypeptide that is attached thereto and full-length proteins and enter cell in several minutes, and can be transported to cerebral tissue by blood circulation, entered in neuron or the glial cell thereby can cross over blood brain barrier.
The inventor is by having obtained the epidermal growth factor Isoforms with epidermal growth factor and PTD fusion.Experiment showed, that epidermal growth factor Isoforms of the present invention can pass through mucosa absorption, penetrates non-invasively mucosa and enters in the body, and then can be used for treating viral infection, specific killing tumor cell and neurodegenerative diseases etc.Therefore, epidermal growth factor Isoforms of the present invention is also referred to as non-invasive high-penetrability epidermal growth factor.
So one aspect of the present invention provides a kind of non-invasive high-penetrability epidermal growth factor, it comprises and is selected from following sequence:
A) aminoacid sequence shown in the SEQ ID NO.5; Or
B) aminoacid sequence in (a) through replaces, lack, insert or adds one or several aminoacid and keep to replace, disappearance, insert or add before activity by (a) derivative aminoacid sequence.
Preferably, non-invasive high-penetrability epidermal growth factor of the present invention is:
A) aminoacid sequence shown in the SEQ ID NO.5; With
B) aminoacid sequence in (a) through replaces, lack, insert or adds one or several aminoacid and keep to replace, disappearance, insert or add before activity by (a) derivative aminoacid sequence.
Of the present inventionly in aminoacid sequence, replace, lack, insert or add 1 or several aminoacid, refer in sequence arbitrarily and the position in 1 or the several aminoacid sequence replaces, lack, insert or adds 1 or several amino acid residue (for example 2,3,4,5,6,7,8 or 9), and replace, disappearance, insert and interpolation in 2 kinds or two or more can the generation simultaneously.
Of the present invention in aminoacid sequence, replace, lack, insert or add 1 or several aminoacid can be by using the direct mutagenesis method acquisition of the records such as " Molecular Cloning 3 " (molecular cloning 3) and " Current Protocols in Molecular Biology " (modern molecular biology rule of operation).
For replacement of the present invention, preferably within following each group, carry out the mutual replacement of amino acid residue:
1: leucine, valine, alanine, methionine, serine, glycine;
2: aspartic acid, glutamic acid;
3: agedoite, glutamine;
4: lysine, arginine;
5: proline, hydroxyproline;
6: serine, threonine; With
7: phenylalanine, tyrosine.
For described conservative replacement herein, disappearance, insertion or active before adding, the protein after can being illustrated in replacements, disappearance, inserting or adding or the activity of aminoacid sequence are to replace, disappearance, insert or interpolation is front more than 10%, more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or 100% or higher.
The number of the amino acid residue of above-mentioned disappearance, replacement, insertion and/or interpolation, general preferred little number.And this proteinoid can also be protein, its have with the aminoacid sequence of serial number 5 have an appointment 60% or more than, approximately 70% or more than, 71% or more than, 72% or more than, 73% or more than, 74% or more than, 75% or more than, 76% or more than, 77% or more than, 78% or more than, 79% or more than, 80% or more than, 81% or more than, 82% or more than, 83% or more than, 84% or more than, 85% or more than, 86% or more than, 87% or more than, 88% or more than, 89% or more than, 90% or more than, 91% or more than, 92% or more than, 93% or more than, 94% or more than, 95% or more than, 96% or more than, 97% or more than, 98% or more than, 99% or more than, 99.1% or more than, 99.2% or more than, 99.3% or more than, 99.4% or more than, 99.5% or more than, 99.6% or more than, 99.7% or more than, 99.8% or more than, or 99.9% or the aminoacid sequence of above homogeneity, and has the non-invasive high-penetrability epidermal growth factor activity that serial number 5 has.The general preferred large numerical value of the numerical value of above-mentioned homology.
Preferably, above-mentioned replacement, disappearance, insertion or interpolation occur in the 12nd of the aminoacid sequence shown in the SEQ ID NO.5 and later on aminoacid deformity thereof.
Non-invasive high-penetrability epidermal growth factor of the present invention is except obtaining by gene engineering method (as described in following embodiment), also can pass through the chemical synthesis manufacturings such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (tertbutyloxycarbonyl method), or carry out chemosynthesis by peptide synthesizer.
Another aspect of the present invention also provides the nucleotide sequence of the non-invasive high-penetrability epidermal growth factor of the present invention of encoding.
Preferably, the encode nucleotide sequence of non-invasive high-penetrability epidermal growth factor of the present invention comprises:
A) nucleotide sequence shown in the SEQ ID NO.6;
B) under stringent condition with hybridized by the nucleotide sequence of serial number 6 and the polynucleotide of the aminoacid sequence with non-invasive high-penetrability epidermal growth factor activity of encoding; Or
C) above-mentioned a) or b) complementary series.
To have the activity that the non-invasive high-penetrability epidermal growth factor activity refers to a certain protein or aminoacid sequence herein, be more than 10% of SEQ ID NO.5, more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or 100% or higher.
Further preferably, the encode nucleotides sequence of non-invasive high-penetrability epidermal growth factor of the present invention is classified as:
A) nucleotide sequence shown in the SEQ ID NO.6;
B) under stringent condition with hybridized by the nucleotide sequence of serial number 6 and the polynucleotide of the aminoacid sequence with non-invasive high-penetrability epidermal growth factor activity of encoding;
C) above-mentioned a) or b) complementary series.
" stringent condition " as herein described can be in low stringent condition, middle stringent condition, the high stringent condition any, is preferably high stringent condition.Exemplarily, " low stringent condition " can be 30 ℃, the condition of 5 * SSC, 5 * Denhardt liquid, 0.5%SDS, 52% Methanamide; " middle stringent condition " can be 40 ℃, the condition of 5 * SSC, 5 * Denhardt liquid, 0.5%SDS, 52% Methanamide; " high stringent condition " can be 50 ℃, the condition of 5 * SSC, 5 * Denhardt liquid, 0.5%SDS, 52% Methanamide.Those skilled in the art are to be understood that temperature gets over the polynucleotide that Gao Yueneng obtains high homology.In addition, those skilled in the art can select to affect the synthesis result that a plurality of factors such as temperature, concentration and probe concentration, probe length, ionic strength, time, salinity of the stringency of hybridization form and realize corresponding stringency.
In addition interfertile polynucleotide can also for, pass through FASTA, when the homology retrieval softwares such as BLAST calculate with the default parameters of default, with the polynucleotide of coded sequence numbers 6 have approximately 60% or more than, approximately 70% or more than, 71% or more than, 72% or more than, 73% or more than, 74% or more than, 75% or more than, 76% or more than, 77% or more than, 78% or more than, 79% or more than, 80% or more than, 81% or more than, 82% or more than, 83% or more than, 84% or more than, 85% or more than, 86% or more than, 87% or more than, 88% or more than, 89% or more than, 90% or more than, 91% or more than, 92% or more than, 93% or more than, 94% or more than, 95% or more than, 96% or more than, 97% or more than, 98% or more than, 99% or more than, 99.1 or more than, 99.2 or more than, 99.3% or more than, 99.4% or more than, 99.5% or more than, 99.6% or more than, 99.7% or more than, 99.8% or more than, or 99.9% or the polynucleotide of above homogeneity.
The homogeneity of aminoacid sequence, nucleotide sequence, algorithmic rule BLAST (Proc.Natl.Acad.Sci.USA 87:2264-2268,1990 that can use Karlin and Altschul; Proc.Natl.Acad.Sci.USA 90:5873,1993) determine.Program BLASTN, BLASTX based on the BLAST algorithmic rule is developed (AltschulSF, et al:J Mol Biol 215:403,1990).When using BLASTN to analyze base sequence, as to make parameter be score=100, wordlength=12; When using in addition the BLASTX analysis of amino acid sequence, as to make parameter be score=50, wordlength=3; When using BLAST and GappedBLAST program, adopt the system of each program can set default parameter value.
The present invention also provides the carrier that comprises nucleotide sequence of the present invention, for example plasmid vector.In addition, the present invention also provides the microorganism that imports carrier of the present invention, for example escherichia coli and yeast.
The present invention also provides the method for preparing non-invasive high-penetrability epidermal growth factor of the present invention, and it comprises cultivates microorganism of the present invention.
Again one side of the present invention provides a kind of pharmaceutical composition or pharmaceutical preparation, and it comprises non-invasive high-penetrability epidermal growth factor of the present invention or its nucleotide sequence.For pharmaceutical composition of the present invention or pharmaceutical preparation, it can be used for treating neurodegenerative diseases, such as alzheimer disease, parkinson and black substance degeneration etc.
Another aspect of the present invention provides non-invasive high-penetrability epidermal growth factor of the present invention and nucleotides sequence thereof to be listed in for the preparation of in the medicine of neurodegenerative diseases (such as alzheimer disease, parkinson, black substance degeneration) or be used for the purposes of the value-added medicine of neural stem cell.
For pharmaceutical composition of the present invention, it can be used by various ways such as injection, sublingual administration, lung suction, nasal mucosa and anus bolt or skin absorption, eye drops.
Description of drawings
Fig. 1: NcoI and EcoR I double digestion carrier pUC57,2% low melting point glue reclaim purpose fragment OmpA-PTD-hEGF.Wherein:
Swimming lane M, DNA Marker; Swimming lane 1, pUC57-OmpA-PTD-hEGF.
Fig. 2: NcoI and EcoR I double digestion carrier pET-28a (+), the carrier segments of recovery 5400bp.Swimming lane M, DNA Marker; Swimming lane 1, pET-28a (+).
Fig. 3: bacterium colony PCR identifies figure.Wherein:
Swimming lane M, DNA Marker; Swimming lane 1-5,5 single bacterium colonies.
Fig. 4: PTD-hEGF secreting, expressing figure.Wherein:
Swimming lane M, peptide Marker; Swimming lane 1, PTD-h EGF; Swimming lane 2 is not induced.
Fig. 5: the Western blotting of secreting, expressing PTD-hEGF fusion rotein is identified figure, wherein:
1, PTD-hEGF fusion rotein; 2, negative control.
Fig. 6: Nco I and BamH I double digestion carrier pET-28a (+), the carrier segments of recovery 5400bp.Wherein:
Swimming lane M, DNA Marker; Swimming lane 1, pET-28a (+).
Fig. 7: PCR method is angled and is got the PTD-hEGF gene, fragment NcoI and BamHI double digestion, and reclaim test kit with low melting-point agarose gel DNA and reclaim carrier and genes of interest fragment.Wherein:
Swimming lane M, DNA Marker; Swimming lane 1, the PTD-hEGF gene.
Fig. 8: inclusion body is expressed PTD-h EGF figure.Wherein:
1,2,3 swimming lanes induce respectively 1,3,5h; M, peptide Marker; Swimming lane 4 is not induced.
Fig. 9: inclusion body is expressed the Western blotting of PTD-h EGF and is identified figure.Wherein:
1, PTD-hEGF fusion rotein; 2, negative control.
Figure 10: fusion rotein blood brain barrier breakthrough experiment, wherein: 1, PTD-hEGF; 2, hEGF; 3, negative control.
Figure 11: water maze laboratory, wherein: A, AD model mouse intravenously administrable; B, natural aging Mus intravenously administrable; C, AD model mouse rectally; D, natural aging Mus rectally.
Figure 12: the shuttle box experiment, wherein: A, AD model mouse intravenously administrable; B, natural aging Mus intravenously administrable; C, AD model mouse rectally; D, natural aging Mus rectally.
Figure 13: the neural stem cell experiment, hippocampus of mice nestin (Nestin) is expressed figure, wherein: A, AD model mice; B, PTD-hEGF treats mice; C, the sham-operation mice.
Figure 14: the neural stem cell experiment, the acid albumen of hippocampus of mice nerve fiber (GFAP) is expressed figure, wherein: A, AD model mice; B, PTD-hEGF treats mice; C, the sham-operation mice.
Figure 15: the neural stem cell experiment, hippocampus of mice bromination Brdurd (Brdu) mixes spirogram, wherein: A, AD model mice; B, PTD-hEGF treats mice; C, the sham-operation mice.
Figure 16: PET brain imaging experiment, wherein: A, AD rat model; B, the AD rat model of hEGF treatment; C, the AD rat model of PTD-hEGF treatment; D, the sham-operation rat.
The below will be by illustrating in greater detail the present invention by following examples.Following examples only are illustrative, should be understood that the present invention is not subjected to the restriction of following embodiment.
The structure of embodiment 1 secreted expression carrier OmpA-PTD-hEGF
According to hEGF's sequence in the situation that the constant nucleotide sequence of adjusting of aminoacid sequence, directly merge PTD sequence (centre does not add any base) at EGF sequence N end, before PTD, add again OMPA sequence (SEQ ID NO.7), in order to be connected on pET-28a (+) prokaryotic expression carrier, before the OMPA sequence, add again NcoI restriction enzyme site (CCATGG), utilize the ATG start codon in the NcoI restriction enzyme site, for frameshit not adds bases G C behind CAATGG, methionine and glycine namely before OMPA, have been added.The C-terminal of hEGF has added TAA TAA (termination codon subsequence), and the termination codon back connects EcoRI restriction enzyme site (GAATTC).Whole gene order is CAATGGGC+OMPA sequence (SEQ ID NO.7)+PTD (SEQ IDNO.3)+hEGF sequence (SEQ ID NO.4)+TAA TAA+GAATTC (this integral body gene order is synthetic by Shanghai bio-engineering corporation), and is implemented on the pUC57 carrier.Transform DH5 α escherichia coli, picking list bacterium colony shakes bacterium upgrading grain.The pUC57 plasmid vector reclaims the genes of interest fragment with NcoI and EcoR I double digestion.Wherein, described recovery is to use following reaction system:
NcoI 1μl
EcoRI 1μl
NcoI and EcoR I universal buffering liquid 2 μ l
BSA 0.2μl
Puc57(pET-28a(+)) 8μl
H
2O 7.8μl
Total reaction system is 20 μ l, bathes 3 hours in 37 ℃ of temperature.
Enzyme action product OmpA-PTD-hEGF reclaims the genetic fragment about the 220bp in the swimming lane 1 through 2% low melting-point agarose gel electrophoresis (seeing Fig. 1).PET-28a (+) carrier segments reclaims the genetic fragment about 5400bp with 1.2% low melting-point agarose gel electrophoresis (seeing Fig. 2), determines the position of purpose fragment under uviol lamp, downcuts the gel of this position with blade.Blob of viscose is put in the 1.5ml centrifuge tube, reclaims test kit with low melting-point agarose gel DNA and reclaim the purpose fragment.
PET-28a (+) carrier is also carried out double digestion with Nco I and EcoR I, and reclaim the purpose carrier segments.For the genes of interest fragment is connected on pET-28a (+) carrier, carry out under following reaction system, reacting.
Reaction system is as follows:
DNA (genes of interest fragment) 5 μ l
DNA (pET-28a (+) carrier segments) 2 μ l
10 * ligase buffer, 1 μ l
H
2O 1μl
Total reaction system is 10 μ l, spends the night in 4 ℃.
To connect product (pET-28a (+) carrier after namely connecting) and transform bacillus coli DH 5 alpha, and it will be cultivated.Then picking list bacterium colony, and it is carried out PCR identify, picking 5 single bacterium colonies, Fig. 3 is qualification result (all entirely true).PCR is identified that correct gene carries out determined dna sequence.From the correct escherichia coli of checking order, extract plasmid to be used for transforming BL21 (DE3) escherichia coli.
Used various enzymes and relevant strain in the present embodiment, its source-information is as follows: NcoI and EcoR I enzyme are available from match Parkson company, pET-28a (+) carrier is available from Novagen company, the T4 ligase is available from Promega company, and DH5 α and BL21 (DE3) strain is Time Inc. available from the sky.
The abduction delivering of embodiment 2 secreted expression carriers
Fluid medium (LB prescription)
2～15g tryptone, 2～10g yeast extract, 2～8g NaCl, 10gNaH
2PO
412H
2O, 0.33g Na
2HPO
42H
2O is dissolved in the 1000ml distilled water, sterilizes 25 minutes for 15 pounds;
Kanamycin adopts 50～60 μ g/ml working concentrations;
Derivant IPTG mother solution 1M/L.
With Plasmid Transformation BL21 (DE 3) escherichia coli of recombination to construct among the embodiment 1 and cultivate.Select single bacterium colony and be seeded in the LB culture medium (glucose 0.2%+ kanamycin 50-60 μ g/ml), and cultivated 5 to 15 hours in 25～35 ℃ of lower shaking tables.Get seed liquor and add to liquid LB culture medium (adding glucose to final concentration 0.2%, kanamycin 50-60 μ g/ml), and under 25～37 ℃, be cultured to OD
600Be about 0.5～1.0.Then add IPTG to final concentration 0.1-1mM/L, replenish once more simultaneously new kanamycin to 50-60 μ g/ml, and in 25～35 ℃ of lower abduction deliverings 2～10 hours (seeing Fig. 4).Fig. 4 is the SDS-Page electrophoretogram of expressing protein, can find out on scheming, and the size of expressing protein is consistent with theoretical value.
The purification of embodiment 3 secreting, expressing albumen
1), from embodiment 2 in the culture medium of abduction delivering by centrifugal collection thalline, with solution (10～30mM Tris-HCl, 10～30% sucrose, 0.5～1mM EDTA pH7.0～9.0, by 50～100 milliliters of calculating of every gram thalline) suspend, ice bath vibrated 10 minutes gently;
2), 4 ℃ of lower 8000g are centrifugal, remove supernatant, precipitation MgSO
4Solution suspends, ice bath, gently vibration;
3), centrifugal 15 minutes of 12000g, get supernatant, supernatant is the thick product of albumen;
4), phenyl hydrophobic chromatography
The phenyl hydrophobic medium that swelling is the good chromatographic column of packing into, with solution (1～5M ammonium sulfate, 20～70mMTris-HCl pH 7.0～9.0) balance phenyl hydrophobic medium, with supernatant obtained in the previous step (being the thick product of albumen) loading, use again solution (0.5～5M ammonium sulfate, 20～70mMTris-HCl pH7.0～9.0) balance phenyl hydrophobic medium is stable to electric conductance, with solution (50mMTris-HCl pH pH 7.0～9.0) eluting, collects eluting peak;
5), dialysis
The eluting peak collected of the upper step bag filter of packing into is put into dialysis solution (20～70mMTris-HClpH7.0～9.0) dialysed overnight;
6), DEAE anion-exchange chromatography
At first use solution (20～70mMTris-HCl pH7.0～9.0) balance DEAE anion medium; Then in dialysis solution, add monoploid long-pending solution (20～70mMTris-HCl pH7～9.0) and loading, use again solution (20～70mMTris-HCl pH7.0～9.0) balance, then use solution (20～70mMTris-HCl pH7.0～9.0,0.5～2MNaCl) gradient elution, collect eluting peak, be the destination protein sample.Purifying protein is identified by Western blotting, shows it is correct (seeing Fig. 5).
The structure of embodiment 4 inclusion body expression vector pET PTD-hEGF
On the basis of the secreting, expressing plasmid that in above-described embodiment 1, makes up, utilize PCR method to angle and get the PTD-hEGF genetic fragment.Add NcoI restriction enzyme site (CCATGG) and protectiveness base thereof at its front end, utilize the ATG start codon in the NcoI site, for frameshit not adds bases G C, namely before PTD, added two aminoacid (methionine and glycine).Add BamHI restriction enzyme site and protection base sequence thereof after behind hEGF, adding two termination codoies of TAA TAA.
Design of primers is as follows:
P15’-CCCATGGGCTACGGTCGTAAGAAACG-3’(SEQ ID NO.8)
P25’-CGGGATCCTTATTAACGCAGTTCCCAC-3’(SEQ ID NO.9)
The PCR reaction system:
P1 1μl
P2 1μl
Template (OmpA-PTD-hEGF) 0.5 μ l (derive from the connection described in the embodiment 1 after pET-28a (+) carrier)
2×DNAMix 10μl
H2O 7.5μl
Total system 20 μ l
Reaction condition:
95 ℃ of denaturation 7min
72℃ 7min
4 ℃ are extended ∞
PET-28a (+) carrier and PCR fragment are carried out double digestion with NcoI and BamHI respectively, receive test kit (be Time Inc. available from the sky) with low melting-point agarose gel DNA and reclaim carrier and genes of interest fragment.Fig. 6 is pET-28a (+) carrier NcoI and BamHI double digestion figure, reclaims 5400bp left and right sides genetic fragment.Fig. 7 is PCR fragment NcoI and BamHI double digestion figure, reclaims the genetic fragment about 220bp.
Under following reaction system, carrier segments is connected with the genes of interest fragment.
Reaction system:
PET-28a (+) carrier segments 1 μ l
Genes of interest fragment PTD-hEGF 1 μ l
H
2O 6μl
Total reaction system is 10 μ l, spends the night in 4 ℃ of connections.
To connect product (pET-28a (+) carrier after namely connecting) and transform bacillus coli DH 5 alpha, and it will be cultivated.Then picking list bacterium colony, and it is carried out PCR identify and determined dna sequence.From the correct escherichia coli of checking order, extract plasmid to be used for transforming BL21 (DE3) escherichia coli.
Used various enzymes and relevant strain in the present embodiment, its source-information is as follows: NcoI and BamHI enzyme are purchased from match Parkson company, pET-28a (+) carrier is purchased from Novagen company, T4 ligase is purchased from Promega company, and DH5 α and BL21 (DE3) strain is purchased from the sky and is Time Inc..
One, the fermentation of engineering bacteria
1. planting daughter bacteria cultivates
The strain that glycerol is preserved accesses LB culture medium (containing 50 μ g/ml kanamycin) by 1% inoculum concentration, under 25～37 ℃ and 200rpm, cultivated one day, again by 0.5% inoculum concentration access LB culture medium (containing 50 μ g/ml kanamycin), overnight incubation under 25～37 ℃ and 200rpm.
2. ferment and abduction delivering
Inoculum of upper step by 1～10% access LB culture medium, and is begun to cultivate under 37 ℃ and 200r pm.Work as OD
600Be about 1.0, the adding final concentration is that the IPTG of 0.1～1.0mM induces, and continues fermentation 3-5 hour.Stop afterwards fermentation, centrifugal collection thalline is also weighed.SDS-PAGE testing goal protein expression situation.
3. the collection of inclusion body, washing and degeneration
Broken thalline adds an amount of PMSF and TritonX-100 with thalline with TE (Tris-HCl, 1mMEDTA, pH7.0～9.0) dissolving, fully carrying out ultrasonic bacteria breaking (ultrasound intensity is 70% * 500 hertz, ultrasonic time 30min) behind the mixing.At 12000rpm and 4 ℃ of lower centrifugal 30min, collecting precipitation (Fig. 8 has shown different time points inclusion body expression, and 1,2,3 swimming lanes are respectively the inclusion bodys of expressing 1,3,5 hours).With fully suspension precipitation of urea liquid (2M carbamide, 50mMTris-HCl, pH7.0～9.0), and in 12000r pm and 4 ℃ of lower centrifugal 30min, thereby remove other foreign protein, to obtain purer destination protein precipitation.With denaturing soln (8～10M carbamide, 50mMTris-HCl, 2mM EDTA pH7.0～9.0) dissolution precipitation inclusion body (being the precipitation that obtains), add again 1M DTT to final concentration 20mM, stirring at room is to all dissolvings, then in 12000rpm and 4 ℃ of lower centrifugal 30min, collect supernatant.
Two, dilution refolding
1. renaturation solution processed (0.5～2.5M carbamide, 20～70mMTris-HCl pH7.0～9.0,1～10% glycerol, 0.1～5% glycine, 1.5～2.5mM oxidized form of glutathione, 1～5mM reduced glutathion).
2. drip the abundant mixing of renaturation solution in the upper step inclusion body supernatant, stop to drip until precipitation just will occur, 4 ℃ of placements at least 20 hours.
Three, phenyl hydrophobic chromatography
With solution of upper step at the lower centrifugal 30min of 12000rpm and 4 ℃, then adding ammonium sulfate to final concentration in supernatant is 2M, again in 12000rpm and 4 ℃ of lower centrifugal 30min, to collect supernatant.
2. the phenyl hydrophobic medium that swelling is the good chromatographic column of packing into, with solution (1～5M ammonium sulfate, 20～70mMTris-HCl pH7.0～9.0) balance phenyl hydrophobic medium, with supernatant loading obtained in the previous step, use again solution (0.5～5M ammonium sulfate, 20～70mMTris-HCl pH7.0～9.0) balance phenyl hydrophobic medium is stable to electric conductance, with solution (50mMTris-HCl pH 7.0～9.0) eluting, collects eluting peak.
3. dialysis
The eluting peak collected of the upper step bag filter of packing into is put into dialysis solution (20～70mMTris-HClpH7.0～9.0) dialysed overnight.
4.DEAE anion-exchange chromatography
4.1. with solution (20～70mMTris-HCl pH7.0～9.0) balance DEAE anion medium.
4.2. add the long-pending solution (20～70mMTris-HCl pH7～9.0) of monoploid in the dialysis solution, loading.Use solution (20～70mMTris-HCl pH7.0～9.0) balance, (20～70mMTris-HCl pH7.0～9.0,0.5～2MNaCl) gradient elutions collects eluting peak, is the destination protein sample then to use solution again.Purifying protein is identified by Western blotting, and the result is right-on (sees Fig. 9, the 1st, destination protein, the 2nd, negative control).
With embodiment 3 and 4 purification obtain PTD-hEGF and hEGF (available from Sigma, following examples are identical therewith) by waiting molal quantity to be mixed with solution.Mouse tail vein is injected respectively PTD-hEGF and hEGF, wherein with the mice of injecting normal saline in contrast.Injection executions of craning one behind the 4h is got brain and is put into immediately formalin and fix.Tissue slice, washing and dehydration, transparent, waxdip, embedding, section and bonding die, dewaxing, immunohistochemical analysis are all carried out according to a conventional method, the results are shown in accompanying drawing 10.
The group experiment of the mouse brain of tail vein injection PTD-hEGF section shows very dark painted, and that hEGF mice and matched group are cut into slices is painted shallow.Show that PTD-hEGF can pass through blood brain barrier and enter in the mouse brain, and hEGF fails to enter in the brain.
The therapeutical effect of 6 pairs of AD model mouses of embodiment and natural aging Mus
(1) water maze laboratory
The water maze that this experiment is used is the Morris water maze that institute of Materia Medica,Chinese Academy of Medical Sciences is produced.
Before the experiment tank is filled with clear water to predetermined pond height.Laboratory temperature remains on 23 ℃～25 ℃, adds entry (temperature transfers to 23 ± 1 ℃) in the Morris tank to exceeding security platform 1cm, and water temperature remains on 23 ± 1 ℃.Platform is placed on the II quadrant predetermined position in pond, as the target of searching for after the mouse entry.Mounted camera head is connected with computer, monitor and printer.Open first computer, open again miscellaneous equipment, begin experiment.Every mouse is trained 2 times every day.The mouse place of entry is at I and IV quadrant.Computer automatically record animal from entry to finding the platform required time, as incubation period.After mouse is climbed up platform, allow mouse at the platform 30s that stands.If mouse with the interior platform of failing to find in the pond, can be positioned over mouse rest 30s on the platform at entry 120s, train again afterwards next time.Trained altogether 5 days.With mouse from entry to the time of finding platform (being incubation period) weigh its spatial memory capacity for quantitative criterion.Be normal mice less than 120s incubation period.
The result shows, to (hEGF net content 4～40 μ g/ pcs/days of the PTD-hEGF of AD model mice and natural aging mouse mainline embodiment 3 and 4 purified acquisitions, continuous 5～10 days) can significantly shorten its mice and find the time of platform (incubation period), and with AD model group and natural aging non-administered group comparing difference very significantly (p＜0.01); And hEGF group fails to improve the incubation period of AD model mice and natural aging mice, with AD model group and administration natural aging Mus comparison there was no significant difference (p＞0.05) (Figure 11 A and B) not.
Simultaneously, per rectum gives (hEGF net content 40～400 μ g/ pcs/days of the PTD-hEGF of embodiment 3 and 4 purified acquisitions, continuous 5～10 days) also can significantly shorten incubation period of AD model mice and natural aging mice, and compare difference extremely significantly (p＜0.01) with administration natural aging Mus not with the AD model group, and the hEGF group fails to improve the incubation period of AD model mice and natural aging mice, with AD model group and administration natural aging Mus there was no significant difference (p＞0.05) relatively not, (Figure 11 C and D).
(2) shuttle box experiment
The principle of shuttle box experiment gives the buzz signal when being the animals received stimulation, and by repetitious stimulation, animal produces the sound trained reflex, hears buzz again, and initiatively escaping stimulates, and has increased the reaction hub process than water maze laboratory.
10 circulations have been adopted in this experiment, estimate animal learning by electric shock time or active escape time and remember and respond.The electric shock time is short, and the active escape time is long, proves that medicine is effective, otherwise invalid.The result shows, no matter PTD-hEGF prepared among the embodiment 3 and 4 is intravenous injection or rectal administration, also no matter AD model mice or natural aging mice all there is obvious therapeutical effect, and compare the equal highly significant of difference (P＜0.01) with the natural aging mice of the AD model mice of not administration or not administration, the results are shown in accompanying drawing 12.
The experiment of water maze laboratory and shuttle box shows, PTD-hEGF can improve alzheimer disease (Alzheimer ' sdisease, AD) model mouse and natural aging Mus learning and memory ability, and AD is had therapeutical effect.Other carrying out property degenerative disease of central nervous system also had therapeutical effect.
The experiment of embodiment 7 neural stem cell
Nestin (Nestin) belongs to fibroin in the middle of the VI class, and mainly by one crossing the property expression in period of embryo and the neural precursor of growing up, under the various damage conditions of central nervous system, reactive neuron reproduces again its expression in the brain.Nestin is the characteristic albumen of neural stem cell.Experiment finds that the Expression of Nestin amount (Figure 13 A) in the AD model mice hippocampal dentate is starkly lower than normal mouse (sham-operation intracerebroventricular normal saline) (Figure 13 C), and can cause that the Nestin expression increases (accompanying drawing 13B) in the AD model mice hippocampal dentate after PTD-hEGF (among the embodiment 3 or 4 prepared destination protein sample) treatment.
The acid albumen of nerve fiber (GFAP) is the characteristic protein of astrocyte.Experiment finds that GFAP expression (Figure 14 A) is higher than normal mouse (Figure 14 C) in the AD model mice hippocampal dentate, after PTD-hEGF (among the embodiment 3 or 4 prepared destination protein sample) treatment, the expression of GFAP reduces (accompanying drawing 14B) in the AD mouse model hippocampal dentate.
Bromination Brdurd (Brdu) can be incorporated among the new synthetic DNA as the thymidine analog, and the cell by detecting the Brdu labelling can be qualitative and the vegetative state of sxemiquantitative ground reflection cell.The amount of participating in that experiment shows BrdU in the AD model mice hippocampal dentate reduces (accompanying drawing 15A) than normal mouse, and PTD-hEGF (among the embodiment 3 or 4 prepared destination protein sample) treatment can increase the amount of participating in (accompanying drawing 15B) of AD model mice hippocampal dentate BrdU.
The imaging of embodiment 8PET brain
Normal cerebral tissue will keep its function, consumes a large amount of glucoses.The deoxyglucose of F18 labelling is similar to glucose molecule, also can be utilized by cerebral tissue, and the fluorescence of its emission can show at the PET instrument, therefore can be used as labelled compound as consumption sugar amount.The normal brain activity fluorescence intensity is large, and AD brain fluorescence intensity is little, treats when effective, and intensity can increase again.AD rat model tail vein injection PTD-hEGF (among the embodiment 3 or 4 prepared destination protein sample) injected for two weeks continuously.The water maze method detects therapeutic effect after drug withdrawal 4-7 days.Choose the successful Mus for the treatment of and negative control (the AD model is the administration rat not) and carry out the PET detection.The imaging of PET brain shows, the clear zone of PTD-hEGF treatment rat brain near (Figure 16 D), shows that the carbohydrate metabolism reaction enlivens than untreated AD model mouse distribution area large (Figure 16 C) and Sham-operated control group.And the AD rat model (Figure 16 B) that hEGF processes approaches with AD rat model (Figure 16 A), and is kind without significantly changing.
In addition, the present inventor by the aminoacid sequence shown in the SEQ ID NO.5 is replaced, lacks, inserts or add behind one or several aminoacid the aminoacid sequence that obtains or activity with corresponding homogeneity aminoacid sequence also carried out experiment such as embodiment 5 and 6, the result has confirmed that all it is active accordingly.At this, the present inventor is exemplary from wherein having enumerated the mutant of following SEQ ID NO.5: SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14.Its corresponding nucleotide sequence is respectively: SEQ ID NO.11, SEQ IDNO.13 and SEQ ID NO.15, therefore, also be equivalent to exemplify under the described stringent condition herein and hybridize with the nucleotide sequence of serial number 6 or have polynucleotide that corresponding homogeneity and coding have the aminoacid sequence of non-invasive high-penetrability epidermal growth factor activity.
Although described the present invention with above-mentioned embodiment, should be understood that, under the prerequisite that does not deviate from spirit of the present invention, the present invention can further modify and change, and these modifications and the change all belong within protection scope of the present invention.
Sequence table
＜110〉Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences
＜120〉non-invasive high-penetrability epidermal growth factor and application thereof
<130>IDC080126
<160>15
<170>PatentIn version 3.5
<210>1
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉PTD aminoacid sequence
<400>1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210>2
<211>53
<212>PRT
＜213〉artificial sequence
<220>
＜223〉hEGF's aminoacid sequence
<400>2
Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His
1 5 10 15
Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn
20 25 30
Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys
35 40 45
Trp Trp Glu Leu Arg
50
<210>3
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉PTD nucleotide sequence
<400>3
tacggtcgta agaaacgtcg ccagcgtcgc cgt 33
<210>4
<211>159
<212>DNA
＜213〉artificial sequence
<220>
＜223〉human epidermis factor nucleic sequence
<400>4
aattccgact ctgaatgccc gctgtctcac gacggttact gcctacacga tggtgtttgc 60
atgtatatcg aagctctgga caaatacgcg tgcaactgtg ttgttggtta catcggtgaa 120
cgttgccagt accgtgacct gaaatggtgg gaactgcgt 159
<210>5
<211>64
<212>PRT
<213>E.Coli
<400>5
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Asn Ser Asp Ser Glu
1 5 10 15
Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met
20 25 30
Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr
35 40 45
Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg
50 55 60
<210>6
<211>192
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the mutant of epidermal growth factor
<400>6
tacggtcgta agaaacgtcg ccagcgtcgc cgtaattccg actctgaatg cccgctgtct 60
cacgacggtt actgcctaca cgatggtgtt tgcatgtata tcgaagctct ggacaaatac 120
gcgtgcaact gtgttgttgg ttacatcggt gaacgttgcc agtaccgtga cctgaaatgg 180
tgggaactgc gt 192
<210>7
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉OMPA sequence
<400>7
aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt agcgcaggcc 60
<210>8
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer 1
<400>8
cccatgggct acggtcgtaa gaaacg 26
<210>9
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer 2
<400>9
cgggatcctt attaacgcag ttcccac 27
<210>10
<211>53
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>10
Asn Ser Asp Ser Glu Ser Pro Leu Ser His Asp Gly Tyr Ser Leu His
1 5 10 15
Asp Gly Val Ser Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Ser Asn
20 25 30
Ser Val Val Gly Tyr Ile Gly Glu Arg Ser Gln Tyr Arg Asp Leu Lys
35 40 45
Trp Trp Glu Leu Arg
50
<210>11
<211>159
<212>DNA
＜213〉artificial sequence
<220>
＜223〉mutant
<400>11
aattccgact ctgaatcgcc gctgtctcac gacggttact cgctacacga tggtgtttcg 60
atgtatatcg aagctctgga caaatacgcg tcgaactcgg ttgttggtta catcggtgaa 120
cgttcgcagt accgtgacct gaaatggtgg gaactgcgt 159
<210>12
<211>53
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>12
Asn Ser Asp Ser Glu Ala Pro Leu Ser His Asp Gly Tyr Ala Leu His
1 5 10 15
Asp Gly Val Ala Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Ala Asn
20 25 30
Ala Val Val Gly Tyr Ile Gly Glu Arg Ala Gln Tyr Arg Asp Leu Lys
35 40 45
Trp Trp Glu Leu Arg
50
<210>13
<211>159
<212>DNA
＜213〉artificial sequence
<220>
＜223〉mutant
<400>13
aattccgact ctgaagctcc gctgtctcac gacggttacg ctctacacga tggtgttgct 60
atgtatatcg aagctctgga caaatacgcg gctaacgctg ttgttggtta catcggtgaa 120
cgtgctcagt accgtgacct gaaatggtgg gaactgcgt 159
<210>14
<211>47
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>14
Asn Ser Asp Ser Glu Pro Leu Ser His Asp Gly Tyr Leu His Asp Gly
1 5 10 15
Val Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Asn Val Val Gly Tyr
20 25 30
Ile Gly Glu Arg Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg
35 40 45
<210>15
<211>141
<212>DNA
＜213〉artificial sequence
<220>
＜223〉mutant
<400>15
aattccgact ctgaaccgct gtctcacgac ggttacctac acgatggtgt tatgtatatc 60
gaagctctgg acaaatacgc gaacgttgtt ggttacatcg gtgaacgtca gtaccgtgac 120
ctgaaatggt gggaactgcg t 141
Claims (5)
1. the nucleotide of coding non-invasive high-penetrability epidermal growth factor, it is: the hEGF sequence that the PTD+ of the OMPA sequence that CAATGGGC+ is comprised of SEQ ID NO.7+be comprised of SEQ ID NO.3 is comprised of SEQID NO.4+TAA TAA+GAATTC.
2. carrier, it comprises nucleotide claimed in claim 1.
3. according to claim 2 carrier, it is plasmid vector.
4. microorganism, it comprises the carrier of claim 2 or 3.
5. according to claim 4 microorganism, it is escherichia coli or yeast.
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| KR101705603B1 (en) * | 2015-03-19 | 2017-02-13 | 을지대학교 산학협력단 | cell-penetrating protein from enterobacteriacea and use thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0454026A1 (en) * | 1990-04-26 | 1991-10-30 | ZAMBON GROUP S.p.A. | Pharmaceutical compositions containing epidermal growth factor for the treatment of neurodegenerative disorders |
| CN1765929A (en) * | 2004-10-29 | 2006-05-03 | 中国人民解放军军事医学科学院毒物药物研究所 | Contain the fusion rotein of peptide carrier and Urogastron and nucleic acid and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0454026A1 (en) * | 1990-04-26 | 1991-10-30 | ZAMBON GROUP S.p.A. | Pharmaceutical compositions containing epidermal growth factor for the treatment of neurodegenerative disorders |
| CN1765929A (en) * | 2004-10-29 | 2006-05-03 | 中国人民解放军军事医学科学院毒物药物研究所 | Contain the fusion rotein of peptide carrier and Urogastron and nucleic acid and uses thereof |
Non-Patent Citations (3)
| Title |
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| B. Connor等.the role of neuronal groth factors in neurodegenerative disorders of the human brain.《brain research reviews》.1998,第27卷1-39页. * |
| 付爱玲.蛋白质转导的研究进展.《国外医学药学分册》.2004,第31卷(第5期),260-263页. * |
| 赵宝全.人源表皮生长因子变构体对实验性阿尔采默病的治疗作用及其他应用性研究.《中国博士学位论文全文数据库医药卫生科技辑》.2007,(第04期),摘要、第21-26页实验结果、第33页最后一段、第35页第1-9行、第47页最后一段. * |
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