CN101897953A

CN101897953A - Non-invasive high-penetrability epidermal growth factor and application thereof

Info

Publication number: CN101897953A
Application number: CN200910143772XA
Authority: CN
Inventors: 赵宝全; 李前; 孙曼霁
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2009-05-27
Filing date: 2009-05-27
Publication date: 2010-12-01
Anticipated expiration: 2029-05-27
Also published as: CN101897953B

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

Non-invasive high-penetrability epidermal growth factor and application thereof

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, finds that it can directly promote the growth of epiderm skin, therefore, with this active component called after " epidermal growth factor (and epidermal growth factor, EGF).The single chain polypeptide that epidermal growth factor (EGF) is made up of 53 amino acid residues contains 3 intrachain disulfide bonds.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 the secretion of its gastric acid inhibitory and be called as urogastrone (urogastrone, UG).Proved afterwards that these two kinds of materials were very similar on structure and physiological function,, be referred to as " EGF-URO (EGF-UG) " so people regard EGF and this two peptide species of UG as same class material.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 report and the relevant patent of the existing successful expression EGF of gene engineering method in recent years.

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 short proliferation function to the neural precursor in the Mus olfactory bulb.EGF can stimulate from Mus striatum and the isolating stem cells hyperplasia of ventricles of the brain inferior segment.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, though 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 proliferation of neural stem cells and differentiation.The loss that excites endogenous NSC to breed to remedy neuronal cell might become the research direction for the treatment of neurodegenerative diseases such as AD from now on.

But EGF is very unstable under the room temperature state, is easy to degraded and loses activity, and is difficult to cross structures such as cell membrane, and makes the drug effect loss, has limited application clinically.

Summary of the invention

Discover, (proteintransduction domain, small fragment PTD) are called cell-penetrating peptide (cell-penetrating peptides again to have the protein transduction of being called as district on some protein, CPP) can pass through biomembrane effectively, 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 is great potential aspects such as specific killing tumor cell and neurodegenerative diseases.

(human immunodeficiency virus-1, HIV-1) (trans-activator transcription, TAT) albumen PTD (TAT-PTD) is a kind of source of present PTD to the antisense activated transcription to HIV (human immunodeficiency virus)-1.11 aminoacid (47-57 amino acids) among the TAT are the peptide sections with protein transduction function, 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 epidermal growth factor allosteric body with epidermal growth factor and PTD fusion.Experiment showed, that epidermal growth factor allosteric body of the present invention can pass through mucosa absorption, penetrates mucosa non-invasively 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 allosteric body 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) is through replacing, lack, insert or add one or several aminoacid and reservation replacement, disappearance, insert or adding preceding active by (a) deutero-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

Of the present inventionly in aminoacid sequence, replace, lack, insert or add 1 or several aminoacid, be meant in sequence, to replace, lack, insert or add 1 or several amino acid residue (for example 2,3,4,5,6,7,8 or 9) arbitrarily and on the position in 1 or the several amino acid sequence, 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 the direct mutagenesis method acquisition of using " Molecular Cloning 3 " (molecular cloning 3) and " Current Protocols in Molecular Biology " (modern molecular biology rule of operation) etc. to put down in writing.

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 preceding 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, about 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 is had.The general preferred big numerical value of the numerical value of above-mentioned homology.

Preferably, above-mentioned replacement, disappearance, insertion or interpolation occur in the 12nd of aminoacid sequence shown in the SEQ ID NO.5 and aminoacid deformity later on thereof.

Non-invasive high-penetrability epidermal growth factor of the present invention is except that obtaining by gene engineering method (as described in following embodiment), also can pass through Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method chemosynthesis manufactured such as (tertbutyloxycarbonyl methods), 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, have the polynucleotide of the active aminoacid sequence of non-invasive high-penetrability epidermal growth factor with hybridizing and encode by the nucleotide sequence of serial number 6; Or

C) above-mentioned a) or b) complementary series.

To have the activity that the non-invasive high-penetrability epidermal growth factor activity is meant 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, have the polynucleotide of the active aminoacid sequence of non-invasive high-penetrability epidermal growth factor with hybridizing and encode by the nucleotide sequence of serial number 6;

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 influence the synthesis result that a plurality of factors such as temperature, concentration and probe concentration, probe length, ionic strength, time, salinity of the tight degree of hybridization form and realize corresponding tight degree.

In addition interfertile polynucleotide can also for, pass through FASTA, when homology retrieval softwares such as BLAST calculate with the default parameters of default, with the polynucleotide of coded sequence numbers 6 have about 60% or more than, about 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 the BLASTX analysis of amino acid sequence in addition, 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.

One side more 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, 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 the purposes that preparation is used for the medicine of neurodegenerative diseases (as alzheimer disease, parkinson, black substance degeneration) or is used for the value-added medicine of neural stem cell.

For pharmaceutical composition of the present invention, it can be used by injection, sublingual administration, lung suction, nasal mucosa and anus bolt or multiple mode such as skin absorbs, eye drop.

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 E G F; Swimming lane 2 is not induced.

Fig. 5: the Western blotting of secreting, expressing PTD-hEGF fusion rotein is identified figure, wherein:

1, the PTD-hEGF fusion rotein; 2, negative control.

Fig. 6: NcoI and BamHI 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 target gene fragment.Wherein:

Swimming lane M, DNA Marker; Swimming lane 1, the PTD-hEGF gene.

Fig. 8: inclusion body is expressed PTD-h E G F figure.Wherein:

1,2,3 swimming lanes induce 1,3 respectively, 5h; M, peptide Marker; Swimming lane 4 is not induced.

Fig. 9: inclusion body is expressed the Western blotting of PTD-h E G F and is identified figure.Wherein: 1, the 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, mice Hippocampus 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, mice alveus acidic protein (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, mice Hippocampus 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.

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

Under the constant situation of aminoacid sequence, adjust nucleotide sequence according to hEGF's sequence, directly merge PTD sequence (centre does not add any base) at EGF sequence N end, before PTD, add OMPA sequence (SEQ ID NO.7) again, in order to be connected on pET-28a (+) prokaryotic expression carrier, before the OMPA sequence, add NcoI restriction enzyme site (CCATGG) again, utilize the ATG start codon in the NcoI restriction enzyme site, for frameshit not adds bases G C behind CAATGG, methionine and glycine just 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 target gene fragment with NcoI and EcoR I double digestion.Wherein, described recovery is to use following reaction system:

NcoI 1μl

EcoR?I 1μl

The general buffer 2 μ l of NcoI and EcoR I

BSA 0.2μl

Puc?57(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 (see figure 1).PET-28a (+) carrier segments reclaims the genetic fragment about 5400bp with 1.2% low melting-point agarose gel electrophoresis (see figure 2), determines to downcut the gel of this position with blade in the segmental position of purpose under uviol lamp.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 NcoI and EcoR I, and reclaim the purpose carrier segments.For target gene fragment being connected on pET-28a (+) carrier, carry out under following reaction system, reacting.

Reaction system is as follows:

DNA (target gene fragment) 5 μ l

DNA (pET-28a (+) carrier segments) 2 μ l

T4 ligase 1 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 promptly connecting) transformed into escherichia coli DH5 α, and it will be cultivated.Picking list bacterium colony then, 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 to transform 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, and the T4 ligase is available from Promega company, and DH5 α and BL21 (DE3) strain is a 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;

Preparation 5～30% glucoses were sterilized 20 minutes, and were added among the above-mentioned LB for 8 pounds;

Kanamycin adopts 50～60 μ g/ml working concentrations;

Derivant IPTG mother solution 1M/L.

The plasmid of recombination to construct among the embodiment 1 is transformed BL21 (DE3) escherichia coli 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 following 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.Add IPTG then to final concentration 0.1-1mM/L, simultaneously the kanamycin that restock is once new is to 50-60 μ g/ml, and in 2～10 hours (see figure 4)s of 25～35 ℃ of following abduction deliverings.Fig. 4 is the SDS-Page electrophoretogram of expressing protein, and as can be seen, the size of expressing protein is consistent with theoretical value from the figure.

The proteic purification of embodiment 3 secreting, expressings

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 following 8000g are centrifugal, remove supernatant, precipitation MgSO ₄Solution suspends, ice bath, vibration gently;

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 pH7.0～9.0) balance phenyl hydrophobic medium, the supernatant (being the thick product of albumen) that previous step obtains is gone up sample, reuse 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 last 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 the long-pending solution (20～70mMTris-HCl pH7～9.0) of monoploid and go up sample, reuse solution (20～70mMTris-HCl pH7.0～9.0) balance, use solution (20～70mMTris-HCl pH7.0～9.0 then, 0.5～2MNaCl) gradient elution, collect eluting peak, be the destination protein sample.Purifying protein is identified by Western blotting, shows it is correct (see figure 5).

The structure of embodiment 4 inclusion body expression vector pET PTD-hEGF

On the basis of the secreting, expressing plasmid that in the foregoing description 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,, just before PTD, added two aminoacid (methionine and glycine) for frameshit not adds bases G C.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:

P1 5，-CCCATGGGCTACGGTCGTAAGAAACG-3’(SEQ?ID?NO.8)

P2 5’-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 pre-degeneration 7min

72℃ 7min

4 ℃ are extended ∞

PET-28a (+) carrier and PCR fragment are carried out double digestion with NcoI and BamHI respectively, reclaim test kit (is Time Inc. available from the sky) with low melting-point agarose gel DNA and reclaim carrier and target gene 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 target gene fragment.

Reaction system:

PET-28a (+) carrier segments 1 μ l

Target gene fragment PTD-hEGF 1 μ l

T4 ligase 1 μ l

Ligase buffer 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 promptly connecting) transformed into escherichia coli DH5 α, and it will be cultivated.Picking list bacterium colony then, and it is carried out PCR identify and determined dna sequence.From the correct escherichia coli of checking order, extract plasmid to be used to transform 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 in match Parkson company, pET-28a (+) carrier is purchased the company in Novagen, and T4 ligase purchases the company in Promega, and it is Time Inc. that DH5 α and BL21 (DE3) strain is purchased in the sky.

One, the fermentation of engineering bacteria

1. planting daughter bacteria cultivates

The strain that glycerol is preserved inserts LB culture medium (containing 50 μ g/ml kanamycin) by 1% inoculum concentration, cultivated one day down with 200rpm in 25～37 ℃, insert LB culture medium (containing 50 μ g/ml kanamycin) by 0.5% inoculum concentration again, overnight incubation under 25～37 ℃ and 200rpm.

2. ferment and abduction delivering

To go up the step inoculum and insert the LB culture medium, and under 37 ℃ and 200rpm, begin to cultivate by 1～10%.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 fermentation afterwards, centrifugal collection thalline is also weighed.The proteic expression of SDS-PAGE testing goal.

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 following 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 urea liquid (2M carbamide, 50mMTris-HCl, the pH7.0～9.0) precipitation that fully suspends, and in 12000rpm and 4 ℃ centrifugal 30min down, 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 is obtained), add 1M DTT again to final concentration 20mM, stirring at room is to all dissolvings, in 12000rpm and 4 ℃ centrifugal 30min down, collects supernatant then.

Two, dilution refolding

1. make renaturation solution (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. go up and drip the abundant mixing of renaturation solution in the step inclusion body supernatant, stop to drip, 4 ℃ of placements at least 20 hours until just precipitation occurring.

Three, phenyl hydrophobic chromatography

1. will go up step solution at 12000rpm and 4 ℃ centrifugal 30min down, adding ammonium sulfate to final concentration then in supernatant is 2M, again in 12000rpm and 4 ℃ centrifugal 30min down, 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, sample on the supernatant that previous step is obtained, reuse 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 pH7.0～9.0) eluting, collects eluting peak.

3. dialysis

The eluting peak collected of the last 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, last sample.Reuse 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 to use solution then.Purifying protein is identified by Western blotting the result is right-on (sees Fig. 9, the 1st, destination protein, the 2nd, negative control).

Embodiment 5 blood brain barrier penetrate activity

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 PTD-hEGF and hEGF respectively, wherein in contrast with the mice of injecting normal saline.Injection executions of craning one behind the 4h is got brain and is put into formalin immediately 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 irritated 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 computer earlier, open miscellaneous equipment again, begin experiment.Every mouse is trained 2 times every day.The mouse place of entry is at I and IV quadrant.Computer write down automatically animal from entry to finding the platform required time, as incubation period.After mouse is climbed up platform, allow the mouse 30s that on platform, 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 ability for quantitative criterion.Be normal mice less than 120s incubation period.

The result shows, to the PTD-hEGF of AD model mice and natural aging

mouse mainline embodiment

3 and 4 purified acquisitions (hEGF net content 4～40 μ g/ only/day, continuous 5～10 days) can significantly shorten its mice and find the time of platform (incubation period), and with AD model group and not administration group of natural aging 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 give

embodiment

3 and 4 purified acquisitions PTD-hEGF (hEGF net content 40～400 μ g/ only/day, 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 shuttle box experiment principle 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 the obvious treatment 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 ' s disease, AD) model mouse and natural aging Mus learning and memory ability have therapeutical effect to AD.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 the period of embryo and the neural precursor of growing up, under the various damage conditions of central nervous system, reactive neuron reproduces its expression again in the brain.Nestin is the characteristic albumen of neural stem cell.Experiment finds that nest expressing quantity (Figure 13 A) is starkly lower than normal mouse (sham-operation intracerebroventricular normal saline) (Figure 13 C) in the AD model mice hippocampal dentate, 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 decreased of GFAP (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 its emitted fluorescence can show on the PET instrument, so can be used as labelled compound as consumption sugar amount.The normal brain activity fluorescence intensity is big, 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 successful Mus of treatment 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 big (Figure 16 C) and Sham-operated control group.And the AD rat model (Figure 16 B) that hEGF handles is approaching with AD rat model (Figure 16 A), and it is kind not have significantly change.

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 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 sequences is respectively: SEQ ID NO.11, SEQ IDNO.13 and SEQ ID NO.15, therefore, also be equivalent to exemplify under the described herein stringent condition and hybridize with the nucleotide sequence of serial number 6 or have polynucleotide that corresponding homogeneity and coding have the active aminoacid sequence of non-invasive high-penetrability epidermal growth factor.

Though 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

1. pharmaceutical composition or pharmaceutical preparation, it comprises non-invasive high-penetrability epidermal growth factor, or the nucleotide sequence of the described non-invasive high-penetrability epidermal growth factor of encoding, and wherein said non-invasive high-penetrability epidermal growth factor comprises following sequence:

A) aminoacid sequence shown in the SEQ ID NO.5; Or

B) aminoacid sequence in (a) is through replacing, lack, insert or add one or several aminoacid and reservation replacement, disappearance, insert or adding preceding active by (a) deutero-aminoacid sequence;

Wherein said nucleotide sequence comprises:

A) nucleotide sequence shown in the SEQ ID NO.6;

C) above-mentioned a) or b) complementary series.

2. pharmaceutical composition according to claim 1 or pharmaceutical preparation, wherein said non-invasive high-penetrability epidermal growth factor is:

A) aminoacid sequence shown in the SEQ ID NO.5; Or

3. pharmaceutical composition according to claim 1 and 2 or pharmaceutical preparation, wherein said nucleotides sequence is classified as:

A) nucleotide sequence shown in the SEQ ID NO.6;

C) above-mentioned a) or b) complementary series.

4. pharmaceutical composition or the pharmaceutical preparation described in each among the claim 1-3, its be suitable for injecting, dosage form that sublingual administration, lung suction, nasal mucosa approach, anus bolt or forms such as skin absorbs, eye drop are used.

5. pharmaceutical composition or the pharmaceutical preparation described in each among the claim 1-3, it further contains and is selected from following adjuvant: binding agent, disintegrating agent, diluent, lubricant, fluidizer, emulsifying-solubilizing agent, sweeting agent, coating material and antibiotic antiseptic, for example sulphur butyl-beta-schardinger dextrin-and Polyethylene Glycol.

6. pharmaceutical composition described in the claim 4 or pharmaceutical preparation, its application dosage are 30mg non-invasive high-penetrability epidermal growth factor/kg body weight-35mg non-invasive high-penetrability epidermal growth factor/kg body weight.

Among the claim 1-3 in each defined non-invasive high-penetrability epidermal growth factor or its corresponding nucleotides sequence be listed in preparation and be used for the treatment of or prevent purposes in the medicine of neurodegenerative diseases.

8. the described purposes of claim 7, wherein said neurodegenerative diseases is alzheimer disease, parkinson or black substance degeneration.

Among the claim 1-3 in each defined non-invasive high-penetrability epidermal growth factor or its corresponding nucleotides sequence be listed in the purposes that preparation is used for the value-added medicine of neural stem cell.