CN101775072A - Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides - Google Patents
Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides Download PDFInfo
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Abstract
The invention discloses an expression structure method for constructing Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides, the purification of an expressed protein product and the significance thereof, which is mainly characterized in that (1) GDNF parent is a human source mature type; (2) the N-tail end of the GDNF is fused with PEP-1 penetrating peptides comprising the 21 amino acid; and the expression product of the constructed penetrating peptides GDNF fusing the gene expression plasmid has the membrane penetrating function and biological function of GDNF and can be clinically applied in the treatment of the related diseases.
Description
Technical field
The present invention relates to expression plasmid structure and the expression and purification thereof of the human-derived neurotrophic factor GDNF (GDNF, down together) of fusing penetrating peptides, the technical field at its place is relevant with life science and biological medicine technology.
Background technology
Human brain neurogliocyte derived neurotrophic factor (GDNF, down together) is by the generation of the neurogliocyte in the cerebral tissue, a kind of neurocyte nutritional factor that important neural biological function is arranged of excretory.GDNF has the multiple neurocyte of maincenter and periphery the neurotrophic effect that promotes neure growth and differentiation is more widely arranged, be find at present to dopamine neuron and the most significant class neurotrophic factor of motor neuron effect, especially to the strongest (the Lin LT et al.Science 1993.260 (5111): 1130-2 of dopamine neuron effect; Henderson et al.Science 1994,266 (5183): 1062-4).
The function of GDNFF has been carried out extensive studies both at home and abroad, proved GDNF, obviously relaxed the concentrated and dna break of chromatin that 6-hydroxydopamine (6-OHDA) is brought out by the mediation of PI3K/Akt path; Start a series of anti-apoptotic signal approach and neuroprotective unit is considered to a kind of neuroprotective medicine likely.
Because the existence of hemato encephalic barrier, GDNF itself can't enter cerebral tissue performance smelting through peripheral blood and treat effect.In recent years abroad in of gene therapy or the conduit direct administration of animal or human's body, (Do Thi NA, Saillour P are studied in the effect of GDNF treatment central nervous system disease by virus vector, Ferrero L, DedieuJF, Mallet J, Paunio T.Gene Ther.2004 Jan 15; Patel NK et al, AnnNeurol.2005 Feb; 57 (2): 298-302) show GDNF, comprise that Parkinson's disease, amyotrophic lateral sclerosis and ischemic brain injury have better clinical treatment effect, be to treat one of parkinsonian popular research field now in the world to multiple nervous system disorders.There is the report that conducts a research at live body at domestic end still.
But, these approach had both increased painful and the treatment cost, also were helpless to GDNF widespread use clinically.Therefore, overcome the hemato encephalic barrier of brain, make GDNF pass through hemato encephalic barrier through the peripheral blood circulation system and enter cerebral tissue, bring into play its biological activity, the purpose that reaches treatment related neural systemic disease is the place of crux.
In recent years, find to like that scientist the TAT albumen of viral HIV has the function of permeates cell membranes and hemato encephalic barrier now, and proof fusion gene product protein this viral polypeptide, that have potential therapeutic action can pass through blood brain barrier too, bring into play (Ruzza P after its biological function, et al., J Pept Sci.2004 Jul; 10 (7): 423-6.), oneself confirms have several polypeptide also to have the function of similar HIV-TAT, these polypeptide have some amino acid polypeptide (Peng T, etal., Acta.Biochim.Biophys.Sin (Shanghai) the .2004 Sep in the last sequence of the N-of people's clock gene (clock gene); 36 (9): 629-36) and the amino acid polypeptide that constitutes by a plurality of Methionins (KKKKKKKKK) (Park, J, et al., Mol.Cells 2002, April 30; 13 (2): 202-8) etc., this class polypeptide be referred to as cell-penetrating peptides (penetrating peptideof cells, PPCs).。
Molecular chaperones HSP90 unfolds in high transduction rate and the dependent cells to obtain because the protein transduction of HIV-TAT mediation is subjected to pre-sex change, the influence of its biological effect of competence exertion and the more important thing is the load of the safety issue of HIV-TAT is disturbed, so this The Application of Technology is limited to some extent.
Recently, domestic and international many 21 amino acid polypeptide PEP-1 that studies have shown that Morris according to natural fine after birth penetrating peptide characteristics design, have and directly to carry bioactive protein and enter cell and pass through hemato encephalic barrier and enter cerebral tissue, and the function of performance biological effect, have transduction efficiency height,, stability and advantages such as nontoxicity that height in physiological buffer arranged insensitive again simultaneously to serum, thereby be considered to be more suitable for carrier instrument (Morris MC. in protein therapeutic, et al, J.Nat.Biotech.2001.19 (12): 1173-6; Dong Xiao etc., Nanfang Medical Univ's journal 2006,26 (8): 1114;
Choi HS, Free Radic Biol Med.2006 Oct 1; 41 (7): 1058-68;
Choi SH, et a; .,
Mol Cells.2005 Dec 31; 20 (3): 401-8;
Choi HS, et al.,
Free Radic Biol Med.2006 Oct1; 41 (7): 1058-68.Epub 2006 Jun 15).
Therefore, based on above research, make up penetrating peptide/neurotrophic factor GDNF fusion gene, development can intravascular input, pass through hemato encephalic barrier and enter the GDNF fusion rotein that cranial nerve tissue is brought into play its biological function, it will be possible becoming a kind of biotherapeutics that some nervous system disorders is had a better therapeutic action.
Summary of the invention
One of purpose of the present invention provides a kind of neurocyte nutritional factor GDNF expression structure of fusing penetrating peptides and the method for marking protein purifying thereof.
Two of purpose of the present invention provides the application of above-mentioned fusing penetrating peptides recombinant human gdnf protein matter.
One of purpose of the present invention realizes by following technical measures:
1, from human brain tissue information extraction Yeast Nucleic Acid (mRNA), and utilize ThermoScript II with the synthetic complementary DNA (cDNA) (cDNA) of mRNA, then, use round pcr, clone gdnf gene, and the coding deoxynucleotide of penetrating peptide PEP-1 is fused to the amino-terminal end (N-end) of people source neurocyte nutritional factor gdnf gene;
2, above-mentioned GDNF fusion gene is inserted the protokaryon protein carrier, be built into the GDNF fusion gene expression plasmid;
3, above-mentioned GDNF fusion gene expression plasmid transformed competence colibacillus is expressed bacterium, form the transformant that contains the GDNF fusion gene expression plasmid;
4, cultivate transformant under proper condition and express bacterium, and with the transcribing of the expression plasmid GDNF fusion gene in the inductor abduction delivering bacterium, and synthesize the protein of this genes encoding;
5, determine the biological function activity of GDNF fusion rotein by purified technology of protein, Western Blot analysis and cell research.
Penetrating peptide in the people GDNF fusion gene of the present invention is a kind of in some amino acid polypeptide in the sequence of penetrating peptide TAT, PEP-1, people's clock gene N-end and the amino acid polypeptide that is made of a plurality of Methionins (KKKKKKKKK).
Expression vector of the present invention is a procaryotic cell expression carrier, also can be carrier for expression of eukaryon and virus expression carrier.
Two of purpose of the present invention provide described people GDNF fusion gene marking protein as preparation treatment as the application in some the nervous system disease medicine described in this specification sheets, its effective active composition behaviour gdnf protein matter.
Recombinant human GDNF fusion gene expression structure provided by the invention has following feature:
1). people source GDNF is adult form people source GDNF, and promptly its N-end lacks 58 amino acid from the first to the 58 than wild-type people source GDNF, and its C-terminal sequence remains unchanged;
2). the N-of adult form people GDNF is terminal to be merged by the PEP-1 penetrating peptide of being made up of 23 amino acid polypeptides (KETWWETWWTEWSQPKKKRKV);
3). recombinant human GDNF fusion gene is made up of 157 amino acid, comprises transmembrane polypeptide and 134 adult form GDNF that amino acid constitutes that 23 amino acid are formed.
4). recombinant human GDNF fusion gene is connected in procaryotic cell expression carrier pET-45b (+) with the Nco1 of N-end and the Hind111 of C-end, be positioned at the downstream of T7 promotor, build up the expression plasmid of recombinant human GDNF fusion gene, and the competence that transforms expression bacterium is the expression bacterium that is suitable for procaryotic cell expression carrier pET-45b (+).
Therefore, described expression plasmid pET-45b (+)-PEP-1/GDNF has the effect of expressing human GDNF fusion rotein, and the GDNF fusion rotein of purifying has penetrating peptide and the two function of GDNF.
Description of drawings
Accompanying drawing 1: people source neurocyte nutritional factor gdnf gene cDNA fragment (RT-PCR) product: first swimming lane is deoxynucleotide (DNA, a down together) molecular weight standard; The second and the 3rd swimming lane is reverse transcriptase-polymerase chain reaction (RT-PCR) product of this gene.The arrow indication is the position of this gene cDNA fragment on agarose electrophoresis.
The fusion of accompanying drawing 2:PEP-1 and people source gdnf gene: adopt PCR method, with the gdnf gene is template, the nucleotide primer of using 5 '-not terminal amino acid of coding PEP-1 polypeptide and GDNF carries out the PCR reaction, finishes the fusion of PEP-1 and human-derived neurotrophic factor GDNF.First road is that dna molecular amount standard, the second and the 3rd road are PCR product (arrow indication).
Accompanying drawing 3:pET-45b (+)-PEP-1/GDNF expression plasmid structure: use dna ligase the PEP-1/GDNF fusion gene fragment of Not1～BamH1 is inserted the procaryotic cell expression carrier pET-45b (+) that digests with restriction endonuclease Not1 and BamH1, conclusive evidence is pET-45b (+)-PEP-1/GDNF expression plasmid after transformed competence colibacillus bacterium, cultivation amplification and enzyme are cut screening.
The expression of accompanying drawing 4:pET-45b (+)-PEP-1/GDNF: this figure is the inductive expression product in the result of SDS-PAGE glue after the bright blue dyeing of Ka Mashi, rises from left to right, and first swimming lane is a standard protein molecular weight marker thing; Second swimming lane is inductive bacterial product not; Three, the 4th swimming lane is respectively the product of different induction times.Arrow is depicted as expression product.
The Western Blot of accompanying drawing 5:pET-45b (+)-PEP-1/GDNF expression product analyzes: first swimming lane is a standard protein molecular weight marker thing; Second swimming lane is that bacterial product is induced at the end; Three, the 4th be respectively the product of different induction times with the 5th swimming lane.Show that expression product is the gdnf gene product that meets expection.
Accompanying drawing 6:PEP-1/GDNF expression product enters the eukaryotic Western Blot of Hela and analyzes: first road is a standard protein molecular weight marker thing; Second swimming lane is a control group; Third and fourth road, the 6th and the 7th road be respectively hatched Hela cell result altogether for the PEP-1/GDNF expression product in 2 hours and 4 hours, the 5th swimming lane is the PEP-1/GDNF expression product.
The separation and purification of accompanying drawing 7:PEP-1/GDNF expressed fusion protein matter: adopt non-sex change to gather bright acrylamide gel isolated protein.First road is a standard protein molecular weight marker thing; Second swimming lane is that control group is a sample solution; Three, the 4th and the 5th road is the collection liquid of dextran G50 molecular sieve; Six, the 7th and the 8th road is the protein liquid that Zeo-karb is leant on separation and purification.
Accompanying drawing 8: the Western Blotting check and analysis of purifying PEP-1/GDNF fusion rotein: first road is a standard protein molecular weight marker thing; Second swimming lane is that control group is a sample solution; The the 3rd to the 5th road is that dextran G50 molecular sieve is collected liquid; The the 6th to the 8th road is that cationic exchange is leant on separation and purification collection liquid.
Accompanying drawing 9: the PEP-1/GDNF albumen of purifying is to the influence of nerve growth: first road is not for containing the situation of the cell culture fluid of PEP-1/GDNF fusion rotein to the SH-SY5Y nerve growth; Second observes the influence of SH-SY5Y nerve growth with to be the cell culture fluid that contains equivalent PEP-1/GDNF fusion rotein ask when different the cultivation in the 3rd road.
Embodiment
Embodiment one: people's gdnf gene clone, gene fusion and expression plasmid thereof make up:
1). people's gdnf gene clone and fusion gene thereof merge:
With the human brain tissue information ribonucleotide (mRNA) for preparing is template, with Poly (T) primer under the reverse transcriptase effect, the complementary DNA Nucleotide (cDNA) of synthetic human brain tissue; Then with human brain tissue cDNA as template, reverse primer with the P-1-1 that contains coding penetrating peptide PEP-1 Nucleotide and people's gdnf gene N-end sequence Nucleotide and P-1-2 forward primer and people source gdnf gene C-terminal nucleotide sequence carries out the secondary PCR amplification, human cloning source gdnf gene, and finish the reconstruction that its N does not hold.
The a:PEP-1 design of primers is as follows:
P-1-1：
5’-tcgagctcaggaggaaacgccatggagaaagaaacctggtgggaaacctggtggaccg-3’
P-1-2：
5’-cctggtggaccgaatggtctcagccgaaaaaaaaacgtaaagtgtcaccagataaacaaatgg-3’
B. the design of people source gdnf gene reverse primer is as follows:
5’-tcaagcttcagatacatccacaccttt-3’
For the second time pcr amplification product reacts through tailing, add VITAMIN B4 (T) after, be connected to the T-easy carrier, behind the transform bacteria, amplification, extracting, purifying are determined through dna sequencing.
2). contain the structure of people GDNF fusion gene pronucleus cell expressing plasmid:
With suitable restriction endonuclease, promptly Not1 and BamH1 digest the above-mentioned clone's of containing segmental T-easy carrier of people PEP-1-GDNF fusion gene and procaryotic cell expression carrier pET-45b (+) DNA respectively, separate with agarose electrophoresis, and carry out purifying; People PEP-1/GDNF fusion gene fragment and procaryotic cell expression carrier pET-45b (+) DNA behind the purifying splice under the ligase enzyme effect; Behind the transform bacteria, amplification, extracting, purifying contain people pET-45b (+)-PEP-1/GDNF fusion gene pronucleus expression plasmid through what the restriction endonuclease dna digestion was determined successfully to make up.
Embodiment two: the expression of expression plasmid pET-45b (+)-PEP-1/GDNF in former pyrenomycetes:
1). competence is expressed bacterium and is transformed: get 1～2 microgram pET-45b (+)-PEP-1/GDNF prokaryotic expression plasmid DNA and be added in the competence of thawing on ice and express bacterium BL21 (DE3) PlysS, mix, place hatch 30 minutes on ice after; Then, 42 ℃ of 42 seconds of water-bath, placed 1 minute on ice; Add 900 microlitre LB inoculums, cultivated 1 hour in 37 ℃ of shaking table concussions; Get an amount of nutrient solution and coat and contain on the suitable antibiotic culture plate, overnight incubation in 37 ℃ of incubators is until strain growth is arranged.
2). the expression of people GDNF fusion gene in prokaryotic cell prokaryocyte BL21 (DE3) PlysS: select single bacterial strain from growth plate, grow overnight in containing suitable antibiotic 5 milliliters of LB nutrient solutions; In getting the LB nutrient solution of 500 microlitre inoculums to 50 milliliter the morning on the secondth, cultivated about 4 hours in 37 ℃ of shaking tables concussions; When the O.D/550 value reaches 0.5, add an amount of inductor IPTG, induce the expression of people PEP-1/GDNF fusion gene; After 3-4 hour, centrifugal thalline, thalline are collected supernatant liquor after ultrasonication, use 12%SDS-PAGE (poly-propionic acid amide glue) isolated protein; After the separation, a glue dyes, observe target protein expression, another piece glue with albumen through electrotransfer to cellulose acetate film, carry out Western blot immune response, determine it is the product of expection.
Embodiment three: the functional study of people PEP-1/GDNF expression product permeates cell membranes:
1) preparation of .PEP-1/GDNF fusion gene expression product: express bacterium behind abduction delivering, centrifugal expression bacterium abandons supernatant liquor, adds in thalline and does not contain the serum eukaryotic cell culture medium, fully suspends; After ultrasonication inclusion body, renaturation, get solubility expression product supernatant liquor, through the membrane filtration of 0.2 ц, and the serum of adding 10% in the filtrate and an amount of microbiotic, standby;
2) the .PEP-1/GDNF fusion rotein is worn the film functional study:
A. wear film test: eukaryotic cell Hela overnight incubation added above-mentioned PEP-1/GDNF fusion rotein filtrate with behind the PBS damping fluid washing Hela cell three times, cultivated altogether respectively 2 hours and 4 hours in second day; Then, remove above-mentioned filtrate, wash the Hela cell three times with capacity PBS damping fluid;
The b.PEP-1/GDNF fusion rotein is worn the film validity check: the above-mentioned Hela cell of centrifugal collection, and through ultrasonication, centrifugal, the collecting cell supernatant is molten, carries out electrophoretic separation, transfer; Then, carry out Western blot and analyze, the result shows that PEP-1/GDNF fusion gene expression product has the film of wearing function.
Embodiment four: the separation of PEP-1/GDNF expressed fusion protein, purifying and detection thereof:
1) separation of .PEP-1/GDNF expressed fusion protein, purifying: 37 ℃ of the engineering bacterias of expressing the PEP-1/GDNF fusion gene after 4 hours, separate inclusion body through abduction delivering; Inclusion body is through the dissolving of thorough washing and 8M urea, renaturation, centrifugal after, it is standby to get its supernatant liquor.Use the on-column refolding method, successively use dextran G50 molecular sieve to lean on to separate, the purifying expressed proteins with Zeo-karb;
2). the PEP-1/GDNF detection of fusion proteins of purifying: use the poly-propionic acid amide gel electrophoresis PEP-1-GDNF expressing protein of 15% non-sex change subsequently, and electrotransfer is to film, experiment condition routinely, carry out Western blotting with mouse GDNF antibody and detect, what the result showed purifying is people GDNF fusion rotein.
Embodiment five: the influence of PEP-1/GDNF fusion rotein confrontation SH-SY5Y nerve growth: get about 500,000 passage cells for every group and cultivate, treat that adherent growth is after 12 hours, in fresh medium, add equivalent PEP-1/GDNF albumen, cultivated respectively 12 and 24 hours, the collecting cell counting, and with in fresh medium, do not add the proteic cellular control unit of PEP-1/GDNF counting relatively, analyze the influence of PEP-1/GDNF albumen to nerve growth, the result shows that PEP-1/GDNF albumen has the effect that promotes the SH-SY5Y nerve growth.
Sequence table
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＜120〉Title: a kind of neurocyte nutritional factor GDNF of fusing penetrating peptides
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Sequence
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<210>1
<211>504
<212>DNA
＜213〉GDNF artificial sequence
<400>1
<210>2
<211>157
<212>PRT
＜213〉GDNF artificial sequence
<400>2
Claims (9)
1. the neurocyte nutritional factor GDNF of a fusing penetrating peptides, its principal character is:
A) .GDNFN is adult form GDNF, and its N-terminal deletion is from 58 amino acid of the first to the 58, and its C-terminal sequence is constant;
B). the terminal fusing penetrating polypeptide of the N-of adult form GDNF;
C). described GDNF parental source is in the people;
2. a kind of GDNF fusion gene of root a tree name claim 1, contain following amino acid sequences:
A). ripe parent's amino acid sequence of polypeptide of people GDNF;
B). merge GDNF and form, comprise the adult form GDNF that penetrate polypeptide and 134 amino acid formations that 23 amino acid are formed by 157 amino acid;
3. root a tree name claim 1, the terminal fusing penetrating peptides of the N-of described GDNF, penetrating peptide be by 21 amino acid, i.e. the PEP-1 penetrating peptide of being made up of KETWWETWWTEWSQPKKKRKV amino acid.
4. root a tree name claim 1, described penetrating peptide is not limited to above-mentioned penetrating peptide, also can be a kind of in the penetrating peptide that constitutes of the TAT penetrating peptide of people's clock gene (clockgene), penetrating peptide (VKRVSRNKSEKKRRG), virus of AIDS HIV-1 or a plurality of Methionin (KKKKKKKKK).
5. root a tree name claim 1, the construction process that people's gdnf gene N-does not hold, be template promptly with people's gdnf gene, use synthetic, coding penetrating peptide PEP-1 Nucleotide and the forward primer P-1-1 of people's gdnf gene N-end sequence Nucleotide and the reverse primer of P-1-2 and people's gdnf gene C-terminal nucleotide sequence, carry out the pcr amplification of two step method, finish the reconstruction that its N does not hold, i.e. the fusion of GDNF and PEP-1 penetrating peptide;
The a.PEP-1 design of primers is as follows:
P-1-1：
5’-tcgagctcaggaggaaacgccatggagaaagaaacctggtgggaaacctggtggaccg-3’
P-1-2：
5’-cctggtggaccgaatggtctcagccgaaaaaaaaacgtaaagtgtcaccagataaacaaatgg-3’
B. the design of people's gdnf gene reverse primer thing is as follows:
5’-tcaagcttcagatacatccacaccttt-3’
Pcr amplification product reacts through tailing, add VITAMIN B4 after, be connected to the T-easy carrier, behind the transform bacteria, amplification, extracting, purifying are determined through dna sequencing.
6. root a tree name claim 1, the prokaryotic expression plasmid pET45b-PEP-1-GDNF transformed competence colibacillus that contains above-mentioned expression structure is expressed the engineering bacteria that bacterium BL21 (DE3) plysS forms, but be not limited to expression vector pET45b of the present invention and competence and express bacterial classification, comprise that also all pET express serial carrier and other prokaryotic expression carrier series and BL21 (DE3) serial competence expression bacterium and other prokaryotic cell prokaryocyte competence and express bacterium.
7. root a tree name right 1, right 2 and right 3, the expressed sequence structure of penetrating peptide GDNF fusion gene is at prokaryotic cell prokaryocyte and/or eukaryotic protein expressioning product.
8. root a tree name right 1, right 2 and right 3, penetrating peptide GDNF fusion gene expression structure is in the application of adenovirus (Adenoviruse), adeno-associated virus (AAVs) or other virus carrier system.
9. a tree name right 1, described penetrating peptide GDNF fusion gene expressing protein product are treated relative disease clinically, as ischemic brain injury, and the application in the related neural systemic diseases such as amyotrophic lateral sclerosis and Parkinson's disease.
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| CN101008012A (en) * | 2006-10-27 | 2007-08-01 | 王尚武 | Novel human-derived neurotrophic factor BDNF expression structure and its expression |
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