CN1202129C

CN1202129C - GDNF mutant and its medical application

Info

Publication number: CN1202129C
Application number: CN 00128679
Authority: CN
Inventors: 王嘉玺; 王金惠
Original assignee: Institute of Basic Medical Sciences of AMMS
Current assignee: Institute of Basic Medical Sciences of AMMS
Priority date: 2000-09-20
Filing date: 2000-09-20
Publication date: 2005-05-18
Anticipated expiration: 2020-09-20
Also published as: CN1343724A

Abstract

The present invention relates to a mutant of a glial cell line-derived neurotrophic factor (GDNF), which has the activity of being combined with a GDNF receptor but basically has no biological activity of GDNF. The present invention also relates to a polynucleotide molecule Containing a nucleotide sequence for coding the polypeptide of the present invention, a nucleic acid construction body, a carrier, a cell transforming the nucleic acid construction body or the carrier, and a method for the recombined production of the polypeptide of the present invention. Finally, the present invention relates to the use of the polypeptide of the present invention and a medical composition containing the polypeptide of the present invention.

Description

GDNF mutant and treatment thereof are used

Invention field

The present invention relates to a peptide species.More particularly, the present invention relates to the mutant of glial cell line-derived neurotrophic factor (GDNF).The invention still further relates to the nucleotide sequence that contains code book invention polypeptide polynucleotide molecule, nucleic acid construct, carrier, transformed the cell of above-mentioned nucleic acid construct or carrier and the method for recombinant production polypeptide of the present invention.The present invention relates to the purposes of polypeptide of the present invention at last, and the pharmaceutical composition that comprises polypeptide of the present invention.

Background technology

Glial cell line-derived neurotrophic factor is a kind of new neurotrophic factor (Lin LF etc., the Science.1993 May 21 of discovered in recent years; 260 (5111): 1130-2).As other neurotrophic factor, GDNF has neurotrophic effect comparatively widely to the multiple neurone of maincenter and periphery, it be found at present to Dopamine HCL (DA) serotonergic neuron, the most significant class neurotrophic factor of motor neuron effect (Henderson etc., Science.1994Nov 11; 266 (5187): 1062-4).

People GDNF mature peptide is formed (referring to SEQ ID NO:1) by 134 amino-acid residues, and 7 relative conserved cysteine residue in position are arranged in its molecular structure, forms the halfcystine ring structure of stumbling.Because these characteristics, it is belonged to the TGF-'beta ' family, but in the GDNF molecule homology of amino-acid residue therewith among the family member homology the highest also be no more than 20%, therefore, it is belonged to a new subfamily.The activity form of GDNF is the same subunit dimer that is formed by a pair of interchain disulfide bond.On evolving, GDNF presents suitable conservative property, and people and mouse GDNF molecule homology are up to 93%.Protein crystal (Eigenbrot C etc., Nat Struct Biol 1997 Jun of mouse GDNF molecule have been obtained at present; 4 (6): 435-8), and studied the three-dimensional structure of molecule with X-crystalline diffraction method, on GDNF X-crystalline diffraction collection of illustrative plates, perhaps the N terminal amino acid is the cause of flexible structure, and the structure of 1-37 amino acids residue fails to obtain to resolve.

GDNF has the effect that promotes neure growth and differentiation more widely, and is especially more single-minded to the dopaminergic neuron effect.In the isolated experiment, GDNF can promote the growth and the differentiation of tire mouse midbrain DA serotonergic neuron, and the neuronal cell perikaryon that is in vegetative period is increased, and kytoplasm is abundant, and aixs cylinder prolongs, and the dendron growth extensively.The effect of GDNF is that it can specificity strengthen the DA serotonergic neuron to the picked-up of DA high-affinity, and does not influence non-dopaminergic neuron simultaneously.GDNF not only to the nutritious and provide protection of DA energy neurocyte of isolated culture, has also obtained similar conclusion in the body experiment.GDNF also has very strong trophism to motor neuron, and in the growth course of motor neuron, GDNF has the inhibition apoptosis, promotes the sophisticated effect of cytodifferentiation (Houenou LJ etc., Cell Tissue Res 1996 Nov; 286 (2): 219-23).In addition it be up to now to be found to the most effective survival factors of the motor neuron that is in moribund condition.Show in the experiment in the mouse body, for the facial movement neurone of doing crosscut, ectogenic GDNF not only can stop these motor neurons dead because of damaged, and it also is the unique factor (Munson JB etc., Eur JNeurosci 1997 Jun that can stop the motor neuron atrophy of damaged; 9 (6): 1126-9).Therefore, GDNF can be used as disease such as the ALS and the amyotrophic physiology neurotrophic factor of ridge of treatment motor neuron afunction, degeneration and death and is used for clinical drug candidate.In recent years, a lot of live bodies and isolated experiment result show, GDNF for the treatment nerve carry out sexual involution disease such as Parkinson's disease (PD), lateral sclerosis of spinal cord diseases such as (ALS) has the application prospect of making us optimistic.

GDNF mainly is as a kind of neurotrophic factor, promotes neurone propagation, differentiation, survival.Behind neuronal damage, the signal path that relies on as Ret that the autocrine molecule mediated by GDNF in brain and schwann cell nerve pathway are rebuild, play an important role (Naveilhan P etc., Eur JNeurosci 1997 Jul; 9 (7): 1450-60).Equally, a large amount of studies confirm that, neuroglial cytoma can be secreted GDNF (Ma Duanduan etc., journal of Beijing Medical University, 1995,27 (4): 248) in a large number.The GDNF of this overexpression and autocrine may promote the propagation of self and keep survival, thereby prolong the survival time of oncocyte by the signal transduction pathway in the born of the same parents.Some researchs confirm that also self excretory GDNF can promote spongiocyte propagation equally, and therefore, the GDNF of excessive secretion may also play certain promoter action in the process of the generation of tumour, development.

The modern biology theory shows, the mechanism that GDNF brings into play its proliferation function is, the receptors bind on itself and target cell surface has activated a series of signal transduction path in the cell, comprising: Ras-MAPK path (Worby CA etc., J Biol Chem 1996 Sep 27; 271 (39): 23619-22), PI3K signal path (van Weering DH etc., Recent Results CancerRes 1998; 154:271-81), JNK path (Chiariello M etc., Oncogene 1998May 14; 16 (19): 2435-45) (16:2151-2163), thereby proliferation function is urged in performance for Borrello MG etc., MolCell Biol 1996 with PLC-γ signal path.

Therefore, a kind of competitive antagonist of exploitation GDNF suppressing combining of GDNF and its acceptor competitively, thereby is blocked the intracellular signal transduction that GDNF mediated, remove the function of its short propagation thus, this will be very significant in the treatment of some GDNF overexpression related diseases.

Summary of the invention

An object of the present invention is to provide a peptide species, it contains any aminoacid sequence that is selected from down group:

(a) compare identically substantially with glial cell line-derived neurotrophic factor (GDNF) mature polypeptide parent's aminoacid sequence, just wherein have the aminoacid sequence of one or more aminoacid replacement, disappearance and/or insertion at least; With

(b) with (a) in aminoacid sequence at least 80% homologous aminoacid sequence; And

Described polypeptide has the activity with the GDNF receptors bind, but the biologic activity of essentially no GDNF.

Another object of the present invention provides the polynucleotide molecule of the nucleotide sequence that contains code book invention polypeptide.

Another purpose of the present invention provides and comprises polynucleotide of the present invention, and the nucleic acid construct that can be operatively connected, can instruct one or more control sequence that polypeptide produces with it in suitable expressive host.

Another purpose of the present invention provides the carrier that comprises above-mentioned nucleic acid construct, the cell that has transformed above-mentioned nucleic acid construct or carrier, and the method for recombinant production aforementioned polypeptides.

The present invention relates to polypeptide of the present invention at last and can be used for treating or prevent GDNF overexpression related disease or needs to reduce purposes in the medicine of the active situation of GDNF and the pharmaceutical composition that comprises polypeptide of the present invention and pharmaceutically acceptable carrier and/or vehicle in preparation.

The invention summary

The present invention relates to the mutant of glial cell line-derived neurotrophic factor, more particularly, the present invention relates to a peptide species, it contains any aminoacid sequence that is selected from down group:

The invention still further relates to the nucleotide sequence that contains code book invention polypeptide polynucleotide molecule, nucleic acid construct, carrier, transformed the cell of above-mentioned nucleic acid construct or carrier and the method for recombinant production polypeptide of the present invention.

Description of drawings

Fig. 1 shows 1.5% agarose gel electrophoresis figure of GDNF-28 pcr amplification product.Wherein the 1st swimming lane is the GDNF-28 amplified band, and the 2nd swimming lane is the nucleic acid molecular weight standard that pBR322 DNA cuts through Hinf I enzyme.

Fig. 2 is the design of graphics of the expression vector pBV-GDNF-28 of GDNF N end deletion mutant GDNF-28.

Fig. 3 is the SDS-PAGE and the western blotting thereof of expression product that carries the intestinal bacteria JM103 host cell of various carriers.Wherein the 1st swimming lane is a protein molecular weight standard, and the 2nd swimming lane is to carry the intestinal bacteria JM103 of empty carrier pBV-220 as negative control, and the 3rd swimming lane is the intestinal bacteria JM103 that carries expression vector pBV-GDNF-28; 4th, 5 roads are respectively that the 2nd, 3 swimming lanes use the western blotting at the polyclonal antibody (Santa Cruz company product) of GDNF.

Fig. 4 is the SDS-PAGE electrophorogram of the GDNF-28 of purifying.Wherein first swimming lane is a protein molecular weight standard; 2nd, 3 and 4 swimming lanes are the GDNF-28 behind the purifying.

Fig. 5 is the structure synoptic diagram of GDNF recombinant expression vector pBV-GDNF.

Fig. 6 shows the influence of recombinant expressed rhGDNF to the spongiocyte growth of vitro culture.Wherein light frame is the PBS contrast, and dark frame is rhGDNF.

Fig. 7 shows combining by the recombinant expressed rhGDNF of Flow cytometry and rhGDNF-28 and its acceptor.Wherein A is the negative control of KG-1a and PBS incubation; B is the result behind KG-1a and the fluorescently-labeled rhGDNF incubation; C is the result behind KG-1a and the fluorescently-labeled rhGDNF-28 incubation.

Fig. 8 shows the receptors bind competitive assay of rhGDNF and rhGDNF-28.Wherein A is the negative control of KG-1a and FITC-Avidine incubation; B is the result behind KG-1a and the fluorescently-labeled rhGDNF incubation; After C is KG-1a and fluorescently-labeled rhGDNF incubation, add the not result of fluorescently-labeled rhGDNF-28.

Fig. 9 shows the influence of GDNF-28 to 8 age in days dorsal root ganglion of chick embryo enations.Wherein the result of A for handling with recombinant expressed GDNF-28 shows the essentially no enation of Dorsal root ganglion; The contrast of B for handling with recombinant expressed GDNF shows that Dorsal root ganglion has obvious enation.

Figure 10 shows that GDNF-28 is to the influence of vitro culture motor neuron cell process growth under the various concentration.Wherein use PBS and vitro culture motor neuron cell incubation as negative control (black diamond is represented); RhGDNF and vitro culture motor neuron cell incubation are as positive control (square-shaped frame is represented).Show that protrusion cell number and negative control group behind GDNF-28 and the vitro culture motor neuron cell incubation (the triangle frame table shows) are suitable, and significantly be lower than the GDNF positive controls.

Figure 11 shows the influence of GDNF-28 to vitro culture motor neuron cell survival rate.Wherein use PBS and vitro culture motor neuron cell incubation as negative control (black diamond is represented); RhGDNF and vitro culture motor neuron cell incubation are as positive control (square-shaped frame is represented).Show that GDNF-28 and vitro culture motor neuron cell incubation (the triangle frame table shows) back cell survival rate is suitable with negative control group, and significantly be lower than the GDNF positive controls.

Preferred forms

In one embodiment, the present invention relates to the mutant of glial cell line-derived neurotrophic factor, more particularly, the present invention relates to a peptide species, it contains any aminoacid sequence that is selected from down group:

In one embodiment, the aminoacid sequence that polypeptide of the present invention has has one or more aminoacid replacement, disappearance and/or insertion at the N-end at least than its GDNF parent.Preferably, the aminoacid sequence that polypeptide of the present invention has has one or more disappearance than its GDNF parent at the N-end.

In a such scheme, polypeptide of the present invention is derived from the GDNF parent polypeptide in Mammals source.More preferably, the GDNF parent polypeptide in polypeptide derived from human of the present invention source.Most preferably, polypeptide of the present invention is derived from the GDNF parent polypeptide with aminoacid sequence shown in the SEQ ID NO:1.

Preferably, polypeptide of the present invention is compared with its GDNF parent polypeptide, N-end lacked be equivalent to the 1st amino acids Ser (1) among the SEQ ID NO:1 to the section of the 32nd amino acids Arg (32) at least about 11 amino-acid residues, better be at least about 15 amino-acid residues, be more preferably at least about 20 amino-acid residues, especially at least about 25 amino-acid residues, preferably at least about 28 amino-acid residues.

In another embodiment, polypeptide of the present invention has aminoacid sequence shown in the SEQ ID NO:1, N end Ser (1) about at least 11 amino-acid residues to the section of Arg (32) have just been lacked, about at least 15 amino-acid residues have preferably been lacked, about at least 20 amino-acid residues have more preferably been lacked, also more preferably lack about at least 25 amino-acid residues, especially lacked about at least 30 amino-acid residues.

In another embodiment, polypeptide of the present invention has the aminoacid sequence shown in the 11st, 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 or 32 amino acids residues to the 133 among the SEQ ID NO:1 or the 134 amino acids residues.Most preferably, polypeptide of the present invention has among the SEQ ID NO:1 Lys (29) to the aminoacid sequence shown in Cys (133) or the Ile (134).

In one embodiment, polypeptide of the present invention has aminoacid sequence with aforementioned polypeptides at least about 80% homology, preferably at least about 90% homology, more preferably at least about 95%, 96%, 97%, 98% or 99% homologous aminoacid sequence, this polypeptide has the activity with the GDNF receptors bind, but the biologic activity of essentially no GDNF.

In the present invention, the aminoacid sequence of " homology " refer to this aminoacid sequence with may be to insert with reference to the aminoacid sequence difference, add and/or lack 1 or a plurality of amino-acid residue and/or have 1 or a plurality of amino-acid residue replaced by the different aminoacids residue.Preferably, amino acid change is that character changes less variation, promptly be can the remarkably influenced Protein Folding and/or active conservative amino acid replace; Little amino or C-terminal extend, as the methionine residues of aminoterminal interpolation; The little connection peptides that reaches about 20-25 residue is arranged; Maybe can be by changing little extension such as poly Histidine fragment, epitope or the land that net charge or other function help purifying.

The example that conservative property replaces is the replacement of carrying out in basic aminoacids (arginine, Methionin and Histidine), acidic amino acid (L-glutamic acid and aspartic acid), polare Aminosaeren (glutamine and l-asparagine), hydrophobic amino acid (leucine, Isoleucine and Xie Ansuan), die aromatischen Aminosaeuren (phenylalanine, tryptophane and tyrosine) and p1 amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).The aminoacid replacement that can not change specific activity usually is known in the art, and by for example H.Neurath and R.L.Hill, 1979, at " protein " book, Academic Press described among the New York.Modal replacement is Ala/Seer, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly and the replacement that is reversed.

The polypeptide of the present invention's nucleic acid sequence encoding also is included in polypeptide or its segmental N-end or C-end and has merged the fusion polypeptide of another polypeptide or the fusion polypeptide of cleavable.The nucleotide sequence (or its part) of another polypeptide of coding just can be produced fusion polypeptide with nucleotide sequence of the present invention (or its part) fusion.The technology that produces fusion polypeptide is known in the art, comprise the encoding sequence that connects coded polypeptide, thereby make them in same frame, and the expression of fusion polypeptide is controlled by identical promotor and terminator.

Polynucleotide

The invention still further relates to the separation polynucleotide of the nucleotide sequence that contains code book invention aforementioned polypeptides.In one embodiment, the aminoacid sequence that the polypeptide of polynucleotide encoding of the present invention has is substantially the same with aminoacid sequence shown in the SEQ ID NO:1, just replaces, lacks and/or insert at least one amino-acid residue at the N-end.Preferably, the difference of aminoacid sequence shown in aminoacid sequence that the polypeptide of polynucleotide encoding of the present invention has and the SEQ ID NO:1 is that it has lacked N end Ser (1) about at least 11 amino-acid residues to the section of Arg (32), about at least 15 amino-acid residues have preferably been lacked, about at least 20 amino-acid residues have more preferably been lacked, about at least 25 amino-acid residues have also more preferably been lacked, especially about at least 30 amino-acid residues have been lacked, described polypeptide has the activity with the receptors bind of GDNF, but the biologic activity of essentially no GDNF.

In a preferred embodiment, polynucleotide of the present invention contain the subsequence of nucleotide sequence shown in the SEQ ID NO:2, its encoded polypeptides has the aminoacid sequence shown in the 11st, 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 or 32 amino acids residues to the 133 among the SEQ ID NO:1 or the 134 amino acids residues, this polypeptide has the activity with the receptors bind of GDNF, but the biologic activity of essentially no GDNF.The present invention also comprises the nucleotide sequence of the above-mentioned aminoacid sequence of encoding, and the subsequence of sequence is different because of the degeneracy of genetic code shown in it and the SEQ ID NO:1.Preferably, polynucleotide of the present invention contain among the SEQ ID NO:2 nucleotide sequence or its degeneracy nucleotide sequence shown in the 85th to 399 or 402 Nucleotide, and its encoded polypeptides has among the SEQ ID NO:1 Lys (29) to the aminoacid sequence shown in Cys (133) or the Ile (134).

In a preferred embodiment, these polynucleotide comprise the nucleotide sequence of SEQ ID NO:3.In another preferred embodiment, described polynucleotide comprise the nucleotide sequence that is had among the contained plasmid pBV-GDNF-28 in the intestinal bacteria CGMCC No.0480.2.The present invention comprises that also coding has among the SEQ ID NO:1 Lys (29) to the nucleotide sequence of aminoacid sequence shown in Cys (133) or the Ile (134), and sequence is different because of the degeneracy of genetic code shown in it and the SEQ ID NO:3.Nucleotide sequence of the present invention comprises genome sequence, and corresponding cDNA and RNA sequence.Term used herein " nucleotide sequence " is understood to include synthetic DNA in all interior these class mutation.

The invention still further relates to the separation polynucleotide that contain the mutant nucleic acid sequence that has a sudden change in the polypeptid coding area of SEQ ID NO:3 at least, wherein this mutant nucleic acid sequence encoding has among the SEQ ID NO:1 Lys (29) to the polypeptide of aminoacid sequence shown in the Ile (134).The invention still further relates to code book invention polypeptide, the nucleotide sequence of certain homology arranged with SEQ ID NO:3 polypeptid coding sequence, the homology degree is at least about 70%, preferred about 80%, more preferably from about 90%, also will be preferably about 95%, most preferably about 97% homology.With regard to the object of the invention, the homology degree between two nucleotide sequences is by Wilbur-Lipman method (Wilbur ﹠amp; Lipman, 1983, the journal 80:726-730 of NAS) uses LASERGENE ^TMMEGALIGN ^TMSoftware (DNASTAR, Inc., Madison, WI) and homogeny table and following multiple reduced parameter: breach point penalty and notch length point penalty are 10 and determine.Reduced parameter is Ktuple=3 in pairs, breach point penalty=3, window (windows)=20.

Nucleic acid construct

The invention still further relates to the nucleic acid construct of 1 or a plurality of regulating and controlling sequences that comprise nucleotide sequence of the present invention and can be operatively connected with it, described regulating and controlling sequence can instruct encoding sequence to express in proper host cell under its consistency condition.Expression be understood to include polypeptide produce in related any step, include, but are not limited to transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.

" nucleic acid construct " is defined as strand or double chain acid molecule in the text, and they separate from natural gene, and be perhaps modified and contain in the non-natural mode and make up and nucleic acid fragment arranged side by side.When nucleic acid construct comprises when expressing essential all regulating and controlling sequences of encoding sequence of the present invention term nucleic acid construct and expression cassette synonym.Term " encoding sequence " is defined as the part of directly determining the aminoacid sequence of its protein product in the nucleotide sequence in the text.The border of encoding sequence is normally determined by the ribosome bind site (for prokaryotic cell prokaryocyte) of next-door neighbour mRNA 5 ' end opening code-reading frame upstream and the transcription termination sequence in next-door neighbour mRNA 3 ' end opening code-reading frame downstream.Encoding sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

Can operate the isolated nucleic acid sequences of coding polypeptide of the present invention in many ways, make it express described variant.May expect or must before inserting carrier, process that this depends on expression vector to nucleotide sequence.The technology of using recombinant DNA method modification of nucleic acids sequence is known in the art.

Herein term " control sequence " be defined as comprise express polypeptide of the present invention institute must or favourable all components.Each regulating and controlling sequence can be natural that contain or external for the nucleic acid encoding sequence.These regulating and controlling sequences include, but not limited to leader sequence, polyadenylic acid sequence, propeptide sequence, promotor, signal sequence and transcription terminator.Bottom line, regulating and controlling sequence will comprise promotor and the termination signal of transcribing and translating.So that the coding region of regulating and controlling sequence with the nucleic acid encoding sequence is connected, can provide the regulating and controlling sequence of belt lacing in order to import specific restriction site.Term " can be operatively connected " and be defined as a kind of like this conformation in the text, and wherein regulating and controlling sequence is positioned at the appropriate location of the encoding sequence of relative dna sequence dna, so that regulating and controlling sequence instructs polypeptide expression.

Regulating and controlling sequence can be suitable promoter sequence, can be by the nucleotide sequence of the host cell of express nucleic acid sequence identification.Promoter sequence contains the transcription regulating nucleotide sequence that mediates expression of polypeptides.Promotor can be any nucleotide sequence that transcriptional activity is arranged in selected host cell, comprises sudden change, brachymemma and promotor heterozygosis, can get the gene of polypeptide in the outer or born of the same parents of own coding and host cell homology or allogenic born of the same parents.

Regulating and controlling sequence can also be suitable transcription termination sequence, thereby can be discerned one section sequence that termination is transcribed by host cell.Terminator sequence can be operatively connected 3 ' end in the nucleic acid encoding sequence.Any terminator that can bring into play function in selected host cell may be used to the present invention.

Regulating and controlling sequence can also be suitable leader sequence, promptly to the crucial mRNA non-translational region of the translation of host cell.Leader sequence can be operatively connected 5 ' end in the nucleic acid encoding sequence.Any leader sequence that can bring into play function in selected host cell all can be used for the present invention.

Regulating and controlling sequence can also be a signal peptide coding region, and this district's coding is connected in the N-terminal aminoacid sequence of polypeptide for one section, can guide coded polypeptide to enter the emiocytosis approach.5 ' the end in nucleic acid sequence encoding district may be natural contain the signal peptide coding region that the translation frame as one man is connected naturally with the coding region fragment of secrete polypeptide.Perhaps, can to contain encoding sequence be external signal peptide coding region to 5 ' of coding region end.When encoding sequence does not under normal circumstances contain signal peptide coding region, may need to add the extraneous signal peptide-coding region.Perhaps, can replace the natural signals peptide-coding region simply to strengthen the polypeptide secretion with external signal peptide coding region.But the signal peptide coding region that the polypeptide after any energy guiding is expressed enters the Secretory Pathway of used host cell may be used to the present invention.

Regulating and controlling sequence can also be peptide original encoding district, and this district's coding is positioned at the aminoterminal one section aminoacid sequence of polypeptide.The gained polypeptide is called as proenzyme or propolypeptide.Propolypeptide does not have activity usually, can be by catalysis or self-catalysis and be converted into sophisticated active polypeptide from propolypeptide cutting peptide is former.

When the existing signal peptide of the N-terminal of polypeptide has the former district of peptide again, the N-terminal of peptide former district next-door neighbour polypeptide, the signal peptide district then is close to the N-terminal in the former district of peptide.

The regulating and controlling sequence that interpolation can be regulated expression of polypeptides according to the growing state of host cell may also be needs.The example of regulator control system is that those can be reacted to chemistry or physical stimulation thing (being included under the situation of regulating compound), thereby opens or close the system of genetic expression.Other examples of regulating and controlling sequence are those regulating and controlling sequences that can make gene amplification.In these examples, nucleic acid encoding sequence and regulating and controlling sequence can should be operatively connected together.

Expression vector

The invention still further relates to and comprise nucleotide sequence of the present invention, promotor and transcribe and the recombinant expression vector of translation termination signal.Above-mentioned various nucleic acid and regulating and controlling sequence can be linked together prepares recombinant expression vector, and this carrier can comprise 1 or a plurality of restriction site easily, so that insert in these sites or replace the nucleic acid encoding sequence.Perhaps, can express nucleotide sequence of the present invention by nucleotide sequence or the nucleic acid construct that comprises this sequence are inserted suitable expression vector.Preparation is during expression vector, can make encoding sequence be arranged in carrier so that can be operatively connected with suitable expression regulation sequence.

Recombinant expression vector can be any carrier (for example plasmid or virus) of being convenient to carry out recombinant DNA operation and express nucleic acid sequence.The consistency of the host cell that carrier and it will import is depended in the selection of carrier usually.Carrier can be linearity or closed loop plasmid.

Carrier can be self-replicating type carrier (promptly be present in extrachromosomal complete structure, can be independent of karyomit(e) and duplicate), for example plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier can comprise any mechanism that guarantees self-replacation.Perhaps, carrier be one when importing host cell, with the carrier that is incorporated in the genome and duplicates with the karyomit(e) that is incorporated into.In addition, can use single carrier or plasmid, or totally comprise and will import the two or more carriers or the plasmid of all DNA of host cell gene group, or transposon.

Preferred carrier of the present invention contains 1 or a plurality of selective marker of being convenient to select transformant.Selective marker is such gene, and its product is given to the resistance of biocide or virus, to the resistance of heavy metal, or gives auxotroph prototroph etc.The dal gene of the example of bacterium selective marker such as subtilis or Bacillus licheniformis, the perhaps resistance marker of microbiotic such as penbritin, kantlex, paraxin or tsiklomitsin.

Preferred carrier of the present invention comprises can make the carrier stable integration in the host cell gene group, or guarantees carrier to be independent of cellular genome in cell and carry out the element of self-replicating.

With regard to the situation that is incorporated into the host cell gene group, carrier can depend on other elements of nucleic acid encoding sequence or carrier, so that carrier stably is incorporated in the genome by homology or non-homogeneous reorganization.Perhaps, carrier can contain and is useful on guiding and is incorporated into additional nucleotide sequence in the host cell gene group by homologous recombination.Additional nucleotide sequence can make vector integration accurate site on the karyomit(e) in the host cell gene group.In order to be increased in the possibility that accurate site is integrated, preferred integrated element should contain the nucleic acid of sufficient amount, as 100 to 1500 base pairs, preferred 400 to 1500 base pairs, 800 to 1500 base pairs most preferably, and with respective target sequence height homology, to increase the possibility of homologous recombination.Integrated element can be and hit any sequence of sequence homology of host cell gene group.In addition, integrated element can be non-coding or nucleic acid sequence encoding.On the other hand, carrier can be by non-homogeneous recombination and integration in the genome of host cell.

With regard to the situation of carrying out self-replicating, carrier can also comprise replication orgin, and carrier can independently be duplicated in target host cell.Replication orgin can have makes its sudden change that becomes responsive to temperature type in host cell (referring to for example, Ehrlich, 1978, the journal 75:1433 of NAS).

Can insert the nucleotide sequence of the present invention of copy more than 1 to improve the output of this gene product to host cell.The copy number increase of this nucleotide sequence can be by inserting 1 additional copies of this sequence in the host cell gene group at least, perhaps insert a selective marker that can increase with this nucleotide sequence, by culturing cell in the presence of the suitable selective reagents is being arranged, thereby pick out the cell that the selectable marker gene that containing the amplification copy contains the additional copies nucleotide sequence.

Be used to connect operation that above-mentioned each element makes up recombinant expression vector of the present invention and be well known to those skilled in the art (referring to for example, Sambrook etc., molecular cloning laboratory manual, second edition, press of cold spring harbor laboratory, cold spring port, New York, 1989).

Host cell

The invention still further relates to the recombinant host cell that comprises the nucleotide sequence of the present invention that can be used to the recombinant production polypeptide.The carrier that comprises the present invention's nucleotide sequence can be imported host cell, thereby this carrier is maintained with the outer carrier format of the karyomit(e) of above-mentioned chromosomal integration body or self-replacation.Term " host cell " is contained the sudden change that takes place between any because replicative phase and the offspring different with parental cell.Peptide coding gene and source thereof are depended in the selection of host cell to a great extent.

Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, and preferred prokaryotic cell prokaryocyte is as intestinal bacteria.Can carrier be imported host cell by technology well known to those skilled in the art.

The preparation method

The invention still further relates to recombinates prepares the method for polypeptide of the present invention, and this method comprises: (a) be suitable for producing under the condition of this polypeptide, cultivating the host cell that contains nucleic acid construct, this nucleic acid construct comprises the nucleotide sequence of this polypeptide of encoding; (b) reclaim this polypeptide.

In thorn Preparation Method of the present invention, with means known in the art culturing cell in the nutritional medium that is fit to the polypeptide generation.For example, can be in suitable medium, allowing under expression of polypeptides and/or the isolating condition, come culturing cell by shake-flask culture, laboratory or industrial fermentation jar middle and small scale or large scale fermentation (comprise continuously, in batches, batch charging or solid state fermentation).In the suitable medium that comprises carbon and nitrogenous source and inorganic salt, adopt step known in the art to cultivate.Suitable medium can be provided or can be prepared with reference to disclosed the composition (for example, described in the catalogue of American type culture collection) by supplier.If polypeptide is secreted in the substratum, then can directly from substratum, reclaim polypeptide.If polypeptide is not secreted, can from cell lysate, reclaim.

Can detect polypeptide with the special method at described polypeptide known in the art.These detection methods comprise the use of specific antibody, active test experience etc.

Can reclaim the polypeptide that is produced with means known in the art.For example, can from substratum, reclaim polypeptide by routine operation (including, but are not limited to centrifugal, filtration, extracting, spraying drying, evaporation or precipitation).

Can come purifying variant polypeptide of the present invention by various operations known in the art, these operations comprise, but (for example be not limited to chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size exclusion chromatography), electrophoresis (for example, the isoelectric focusing of preparation property), differential solubleness (for example ammonium sulfate precipitation), SDS-PAGE or extracting is (referring to for example, protein purification, J.C.Janson and Lars Ryden compile, VCH Publishers, NewYork, 1989).

Polypeptide of the present invention has the activity with the receptors bind of GDNF, and the biologic activity of essentially no GDNF.Therefore, the GDNF variant can be used for combining the GDNF acceptor with the GDNF competition, as the antagonist of GDNF.

The invention still further relates to the pharmaceutical composition that contains polypeptide of the present invention and pharmaceutical acceptable carrier and/or vehicle.Described pharmaceutical composition can be used for the GDNF overexpression related disease or needs reduce GDNF active situation, especially tumour, in particularly gliomatous treatment or the prevention.In one embodiment, the pharmaceutical composition that contains the polypeptide of the present invention for the treatment of significant quantity is beneficial to medicinal mode and prepares and administration, and need consider individual patient clinical condition, transport site, medication, administration schedule and the known other factors of doctor.Therefore be used for " significant quantity " of this paper purpose consideration decision by these aspects.

The pharmaceutical composition that contains the polypeptide of the present invention for the treatment of significant quantity can be oral, administration etc. in the rectal administration, parenterai administration, brain pond." pharmaceutical acceptable carrier " refers to the prescription subsidiary of nontoxic solid, semisolid or liquid filler material, diluent, capsule material or any kind.The administering mode of term used herein " parenteral " expression comprises intravenously, intramuscular, intraperitoneal, breastbone is interior, subcutaneous and intra-articular injection and infusion.Polypeptide of the present invention also can pass through slow-released system administration rightly.

In another embodiment, the invention provides a kind of method that suppresses the neurogliocyte growth, comprise described cell is contacted with polypeptide of the present invention.

Further describe the present invention by following examples, but it should be interpreted as limitation of the scope of the invention.

Embodiment

The clone of embodiment 1:GDNF cDNA

Adding in 10% calf serum and the conventional IMDM substratum (available from GIBCO company) 37 ℃, 5%CO with antibiotic (penicillin 100U/ml, Streptomycin sulphate 50 μ g/ml) ₂Cultivator neuroastrocytoma cell BT-325 (available from institute of neurosurgery, Capital University of Medical Sciences Tiantan Hospital) under the condition.Gather in the crops well-grown BT-325 cell, according to the sour phenol-SDS single stage method of improvement extract cell total rna (Zhao Yongfang compiles, Measurement for Biochemistry principle and application thereof, press of Wuhan University, 1988:152).

GDNF cDNA sequences Design according to report such as Lin (seeing above) is synthesized following primer:

Upstream primer: 5 '-AAATGTCACCAGATAAACAA-3 '

Downstream primer: 5 '-CG GGATCCTTAGATACATCCACACCT-3 ' (the BamHI restriction enzyme site of underscore) for introducing

Adopt the RT-PCR test kit of Promega company, in the reaction system of 50 μ l, carry out RT-PCR.In brief, in the 0.5ml centrifuge tube, mix following component: 25mM MgSO ₄2 μ l, 10x damping fluid 5 μ l, 10mM dNTP 1 μ l, RNA 1 μ g, upstream primer 0.5 μ l, downstream primer 0.5 μ l, AMV reversed transcriptive enzyme 0.5 μ l, TF1 0.5 μ l reaches in right amount through the DEPC treated water.Adopt following reaction cycle parameter: 48 ℃ were carried out reverse transcription reaction in 45 minutes; 94 ℃ of sex change are 1 minute then, 48 ℃ of annealing 1 minute, and 68 ℃ were extended 1 minute, carried out 30 circulations altogether, extended 5 minutes at 68 ℃ at last, thereby carried out PCR reaction amplification.Detect with 1.5% agarose gel electrophoresis, the result shows the amplified fragments that obtains about 400bp, identical size with experimental design.Through the automatic dna sequencer analysis, the result shows, the dna sequence dna of PCR reaction gained conform to fully with experimental design (referring to Lin etc., the source is the same).

The clone of embodiment 2:GDNF-28 modification D NA sequence

For obtaining the GDNF variant of N-end 1-28 amino acids residue disappearance, design following primer with reference to (source are the same) such as Lin:

Upstream primer: 5 '-CG GAATTCAAATGAAAGGTCGGAGAGGC-3 ' (the EcoR I restriction endonuclease sites of underscore) for introducing;

Downstream primer: 5 '-CG GGATCCTTAGATACATCCACACCT-3 ' (the BamHI restriction endonuclease sites of underscore) for introducing,

The wild-type GDNF cDNA that obtains with embodiment 1 utilizes designed primer to carry out PCR reaction amplification as template, obtains the GDNF-28 dna sequence dna.In brief, in the 0.5ml centrifuge tube, mix following component: 25mM MgSO ₄2 μ l, 10xPCR damping fluid 5 μ l, 10mM dNTP1 μ l, DNA 1 μ g, upstream primer 0.5 μ l, downstream primer 0.5 μ l, Taq archaeal dna polymerase (available from Promega company) 2.5U and suitable quantity of water are to final volume 50 μ l.The PCR reaction conditions is: 94 ℃ of sex change 1 minute, and 50 ℃ of annealing, 40 seconds, 72 ℃ of extensions, 1 minute, carry out 30 circulations altogether, extended 5 minutes at 72 ℃ at last.Detect pcr amplification product with 1.5% agarose gel electrophoresis, the result shows the amplified fragments that obtains about 300bp, and identical size with experimental design is as Fig. 1.According to the test kit explanation, this fragment is inserted in the pGEM-T Easy carrier (available from Promega company), obtain pGEM-Teasy-GDNF-28, referring to Fig. 2.Through the automatic dna sequencer analysis, the result shows that the dna sequence dna of PCR reaction gained conforms to fully with experimental design.

The structure of embodiment 2:GDNF N end deletion mutant GDNF-28 expression vector

Select pBV220 expression vector (Zhang Zhiqing etc., viral journal, 1990; 6 (2): 111) carry out the recombinant expressed of GDNF-28.The pBV220 expression vector is about 3.66kb, contains two placed in-line promotors of PLPR, and very strong rnn terminator sequence is arranged.Utilize EcoRI and BamHI that purpose fragment GDNF-28 is downcut from carrier pGEM-Teasy-GDNF-28, handle expression vector pBV200 with identical double digestion, utilize glass milk to reclaim test kit (Huamei Bio-Engrg Co.,'s product) and the recovery dna fragmentation is described according to the manufacturer, in 10 μ l linked systems, add 100ng carrier DNA fragment and 50ng purpose small segment, the T4 dna ligase (available from Promega company) that adds 10x connection damping fluid 1 μ l and 1-3weiss, 16 ℃ of connections of spending the night.With reference to (molecular cloning experiment guides such as Sambrook, the 2nd edition, 1989, press of cold spring harbor laboratory, the cold spring port, New York) preparation competence e.colidh5, and will connect product transformed into escherichia coli DH5 α, obtain positive recombinant DH5 α/pBV-GDNF-28 by extracting the plasmid evaluation.Structure synoptic diagram such as Fig. 2 of expression vector pBV-GDNF-28.From positive recombinant DH5 α/pBV-GDNF-28, extract plasmid pBV-GDNF-28 according to a conventional method.

Expression and the Western blot of embodiment 3:GDNF N end deletion mutant GDNF-28 in intestinal bacteria

Expression plasmid pBV-GDNF-28 transformed into escherichia coli competent cell JM103 with constructed gathers in the crops bacterium with reference to (seeing above) described methods such as Sambrook abduction delivering in 5ml LB substratum, with the proteic expression of SDS-PAGE analysis purposes after 4 hours.The result shows, the intestinal bacteria JM103 (the 3rd swimming lane) that carries expression vector pBV-GDNF-28 compares with the same intestinal bacteria JM103 negative control of handling (the 2nd swimming lane) that carries empty carrier pBV-220 of process, molecular weight is less than the protein expression showed increased of 14kDa, therefore as shown in Figure 3, may express the target protein rhGDNF-28 that molecular weight is about 11.6kDa.Through the thin layer scanning analysis, the expression level of rhGDNF-28 in intestinal bacteria JM103 can reach more than 25% of full bacterium total protein.According to (seeing above) described methods such as Sambrook, adopt ECL Westernboltting analysis system (Amersham company product) to utilize GDNF polyclonal antibody (Santa Cruz company product) to carry out western blot analysis to the foreign protein of expressing.The result shows that people GDNF-28 protein (rhGDNF-28) recombinant expressed in intestinal bacteria JM103 can combine with the GDNF polyclonal antibody specifically.Shown in Fig. 3 the 5th road.

The purifying of embodiment 4:rhGDNF-28 and renaturation

The JM103 cell that transformed pBV-GDNF-28 is carried out abduction delivering according to embodiment 3 is described in 2000ml LB, and carry out protein purification and renaturation according to (source are the same) such as Sambrook.Briefly, be about to the fermentation using bacteria culture behind 4 ℃, the centrifugal 30min of 4000rpm, bacterial precipitation is resuspended among the 50ml TE, add N,O-Diacetylmuramidase, behind the ultrasonic disruption (6 * 30 seconds), cellular lysate thing centrifugal 10 minutes through 10000rpm, precipitation is after 2%Triton X-100 and the washing of 3mol/L urea, and the purity of inclusion body can reach 78%.Behind 8mol/L urea, select solubilization of inclusion bodies for use Sephacryl-200 (S-200) post, carry out further purifying through gel filtration method.After elution samples was collected with automatic collector, the component with SDS-PAGE identifies each elution peak behind coomassie brilliant blue staining, showed the expection band of the about 11.6kD of molecular weight, and does not have other assorted band, as Fig. 4 on the running gel.Identify that through thin layer scanning the purity of target protein reaches more than 90%.

In each fraction behind the S-200 purifying, add renaturation buffer (100mmol/L Na ₂HPO ₄, 2mmol/L Cys, the 0.2mmol/L sodium tetrathionate pH8.3), is diluted to 0.1mg/ml with protein concentration, and 5 ℃ were advanced following renaturation 72 hours.Protein after the renaturation is concentrated through conventional method, freeze-drying.

The preparation of embodiment 5:GDNF recombinant protein

The GDNF cDNA of clone among the embodiment 1 is inserted in the pBV220 carrier (referring to Fig. 6), make up the recombinant expression vector pBV-GDNF of GDNF, obtain positive recombinant DH5 α/pBV-GDNF.With embodiment 2-4 described preparation and purifying GDNF recombinant protein rhGDNF similarly.

Embodiment 6:GDNF is to the effect of the spongiocyte of vitro culture

This experiment as experiment material, has tentatively been inquired into rhGDNF influence to rat spongiocyte growth with mtt assay with 2 days rat cerebral cortex spongiocyte of new life.Get newborn 2 days Wistar rat (available from Military Medical Science Institute's Experimental Animal Center), cut off brain skin under aseptic condition, cut the soft or hard meninx open, cut pallium, tissue block is shredded, the pancreatin with 0.25% is at 37 ℃ of digestion 30min; The collecting cell suspension is by 1 * 10 ⁵In the culture dish that scribbles poly-lysine, culture condition is for containing 10%FCS with cell inoculation for cell/ml, the 2mmol/L L-glutaminate, and the DMEM of 0.6% glucose (available from GIBCO company), in 36 ℃, 10%CO ₂Cultivate under the condition, changed liquid once in per 3 days.The spongiocyte that goes down to posterity after 2 times is bred in a large number, adopts mtt assay volumes such as (, cytokine research methodology, People's Health Publisher,, 65 pages in 1999) Sun Weimin to analyze the influence of rhGDNF to the spongiocyte growth.

Collect cell, and the adjustment cell count is 1.2 * 10 ⁵Cell/ml, with serum free medium (2mmol/LL-glutamine, the DMEM of 0.6% glucose) inoculating cell, add different concns rhGDNF (concentration is respectively 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml) in the time of inoculating cell, in 36 ℃, 10%CO ₂After cultivating 48h under the condition, the diluent (5mg MTT/ml is in the PBS of pH7.4) that adds 20ulMTT continues to cultivate 4h; Add 10%SDS-0.01mol/L NH ₄Cl (100ul/ hole), 36 ℃ of incubated overnight, enzyme connection detector is measured OD ₅₇₀Control group adds PBS.Found that rhGDNF all can promote the propagation of spongiocyte, compare that cell number has tangible increase, show that GDNF can obviously promote the propagation of spongiocyte with control group.As Fig. 6.

The receptor-binding activity of embodiment 7:rhGDNF-28

According to (Blood such as Gattei V., 1997, April 15,89 (8): 2925-2937) report, human myeloid leukemia cell KG-1a expresses two the component GDNFR-α and the RET of the receptor complex of GDNF, therefore utilizes the receptor-binding activity of KG-a cell research rhGDNF-28.Containing 10% calf serum, penicillin 100U/ml, in the PRMI1640 substratum (available from GIBCO company) of Streptomycin sulphate 50 μ g/ml, in 37 ℃, 5%CO ₂Cultivate KG-1a cell (doctor Liu Hezhong of fundamental research institute of Military Medical Science Institute is so kind as to give) under the condition.Utilize biotin-avidin system (all available from Sigma company), write rhGDNF and the rhGDNF-28 of (immunoassay technology (third edition), 1991, Science Press) described method after with reference to Xu Yiwei and carry out mark and detection purification renaturation.4 ℃ of incubations 90 minutes, after finishing, reaction utilize cells were tested by flow cytometry fluorescencepositive cell number KG-1a cell and fluorescently-labeled rhGDNF and rhGDNF-28 to determine rhGDNF and rhGDNF-28 and to be present in KG-1a surface receptor GDNFR-α bonded situation.Adopt KG-1a cell and FITC-Avidin incubation as negative control.The result shows, rhGDNF and rhGDNF-28 are under following concentration 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml, compare with the fluorescence curve (Fig. 7 A) of negative control, GDNF-28 (Fig. 7 C) obviously moves to right with the fluorescence curve of wild-type GDNF molecule (Fig. 7 B), and the degree that moves to right is similar, shows that binding ability is similar.As shown in Figure 7.Wherein the fluorescencepositive cell of rhGDNF counting is 27.8%, and the fluorescencepositive cell counting of rhGDNF-28 is 22.3%, and the fluorescencepositive cell of negative control counting only is 0.2%.

Also carried out the combine competitive assay of GDNF-28 with GDNF.In this experiment, at first combine with the KG-1a cell receptor in order to the biotin labeled wild-type GDNF of 200ng/ml, measure fluorescencepositive cell number (Fig. 8 B), and then in this reaction system, add the biotin labeled GDNF-28 that do not use of same amount, measure fluorescencepositive cell number (Fig. 8 C) once more, equally with KG-1a cell and FITC-Avidin incubation as negative control (Fig. 8 A).The result shows, biotin labeled wild-type GDNF combines with the KG-1a cell receptor and adds unlabelled GDNF-28 again, unlabelled GDNF-28 compares before with adding, its fluorescent absorption obviously moves to left, the fluorescencepositive cell number reduces to 15% by 33%, illustrates that GDNF-28 can combine with its acceptor GDNFR-α with wild-type GDNF molecule is competitive.

The biologic activity analysis of embodiment 9:GDNF N end deletion mutant rhGDNF-28

The most frequently used neurotrophic factor measuring method for activity be with E8-E10 dorsal root ganglion of chick embryo neurone as material, observe neuronal cell enation state after cultivating 24h.From 8 days instar chicken embryo (available from herding institute of the Chinese Academy of Agricultural Sciences) separate dorsal root ganglion, plant in scribbling poly-lysine, and use 24 good porocyte culture plates of Hank ' s liquid balance or culturing bottle cultivation, every bottle kind 1-2 joint; 37 ℃, 5%CO ₂Following adherent 1h, add serum free medium DMEM (available from GIBCO company) then, the recombinant factor rhGDNF and the rhGDNF-28 that in the dorsal root ganglion of chick embryo nutrient solution, add purification renaturation respectively with 50ng/ml, 100ng/ml, three kinds of concentration of 200ng/ml, the result shows, rhGDNF all can promote the whole Dorsal root ganglion of E8 chicken embryo to grow a large amount of projections under three kinds of concentration, and rhGDNF-28 group only has a spot of inoblast to move out on every side at Dorsal root ganglion tissue, shows that this mutant lost biologic activity.As Fig. 9.

Embodiment 10 rhGDNF-28 are to the effect of vitro culture tire mouse anterior horn motor neurons enation

The Wistar rat (available from Beijing Medical University's Experimental Animal Center) that sacrificed by decapitation is pregnant 14 days, under gnotobasis, open the abdominal cavity, cut the tire mouse, under stereoscope, cut off skin from dorsal part, take out the whole piece spinal cord, scalper cuts the veutro part, and tissue block is shredded, and digests 30min with 0.125% pancreatin (Sigma company product) at 37 ℃; The collecting cell suspension is by 10 ⁵Cell/ml cell count with cell inoculation in the culture dish that scribbles poly-lysine (Sigma company product) (motor neuron culture condition: 1%B27 additive, 1%N2 additive, 98% neurocyte serum free medium, all available from GIBCO company), two groups of experimental group add recombinant factor rhGDNF and the rhGDNF-28 of 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml and 500ng/ml respectively, negative control group adds the PBS of equal volume, 36 ℃, 10%CO ₂Cultivate 16h under the condition, counting is respectively organized the protrusion cell number under the inverted microscope.Experimental result shows that rhGDNF all can obviously promote the growth of motor neuron cell process under six kinds of concentration that experiment is given, but the rhGDNF-28 group is compared no significant difference with control group.As Figure 10.

Embodiment 11 rhGDNF-28 are to cultivating the influence of E14 rat spinal cord anterior angle motor neuron survival rate

Separate E14 rat spinal cord anterior angle motor neuron cell with method described in the embodiment 10, by 10 ⁵Cell/ml cell count in the culture dish that scribbles poly-lysine, adds 10ng/ml rhGDNF and rhGDNF-28 with cell inoculation respectively in the time of two groups of experimental group inoculating cells, negative control group adds the PBS of equal volume.Cell culture condition is with embodiment 10, in cell cultivation process, change liquid weekly twice, add corresponding nutritional factor rhGDNF and rhGDNF-28 when changing liquid, change liquid measure at every turn and be half of stoste, 5-7 days (deciding on the spongiocyte upgrowth situation) cultivating at neurone add cytosine arabinoside one time with 3 μ g/ml final concentrations, suppress the growth of spongiocyte.Motion neuron survival rate when calculate cultivating 3 days, 7 days, 14 days and 21 days respectively.Experimental result shows that rhGDNF can significantly improve vitro culture 3 days, 7 days, 14 days and the survival rate of 21 days motion neuronal cells, but the rhGDNF-28 group is compared no significant difference with control group.As Figure 11.

The above results explanation, this GDNF N end deletion mutant GDNF-28 can competitive inhibition GDNF and the combining of its acceptor, thereby as the antagonist of GDNF, promotion neuronal survival and the proliferation function of inhibition GDNF.

Following biomaterial has been followed the regulation of Budapest treaty, is deposited in the Zhong Guan-cun, BeiJing, China, China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is as follows:

Preservation thing preserving number preservation date

DH5 α/pBV-GDNF-28 GMCC No.0480.2 on August 17th, 2000

DH5 α/pBV-GDNF GMCC No.0480.1 on August 17th, 2000

Described herein and claimed invention is not limited in the scope of disclosed specific embodiments, because these embodiments are intended to illustrate several aspect of the present invention.The embodiment of any equivalence includes within the scope of the invention.In fact, except shown in this paper and described, after the description of reference front, be conspicuous to those skilled in that art for modification of the present invention.These modifications are also contained in the scope of appended claims.When conflict is arranged,, comprise that definition is as the criterion with this paper disclosure.

This paper has quoted a lot of documents, and they all are incorporated herein by reference in full.

Sequence table

＜110〉applicant: fundamental research institute of Academy of Military Medicine, PLA

＜120〉denomination of invention: the treatment of GDNF N end deletion mutant is used

＜150〉application number: undetermined

＜151〉applying date: undetermined

＜160〉sequence number: 3

<210>1

<211>134

＜212〉protein

＜213〉people

<400>1

TCA?CCA?GAT?AAA?CAA?ATG?GCA?GTG?CTT?CCT 30

Ser?Pro?Asp?Lys?Gln?Met?Ala?Val?Leu?Pro

1 6

AGA?AGA?GAG?CGG?AAT?CGG?CAG?GCT?GCA?GCT 60

Arg?Arg?Glu?Arg?Asn?Arg?Gln?Ala?Ala?Ala

11 16

GCC?AAC?CCA?GAG?AAT?TCC?AGA?GGA?AAA?GGT 90

Ala?Asn?Pro?Glu?Asn?Ser?Arg?Gly?Lys?Gly

21 26

CGG?AGA?GGC?CAG?AGG?GGC?AAA?AAC?CGG?GGT 120

Arg?Arg?Gly?Gln?Arg?Gly?Lys?Asn?Arg?Gly

31 36

TGT?GTC?TTA?ACT?GCA?ATA?CAT?TTA?AAT?GTC 150

Cys?Val?Leu?Thr?Aal?Ile?His?Leu?Asn?Val

41 46

ACT?GAC?TTG?GGT?CTG?GGC?TAT?GAA?ACC?AAG 180

Thr?Asp?Leu?Gly?Leu?Gly?Yyr?Glu?Thr?Lys

51 56

GAG?GAA?CTG?ATT?TTT?AGG?TAC?TGC?AGC?GGC 210

Glu?Glu?Leu?Ile?Phe?Arg?Tyr?Cys?Ser?Gly

61 66

TCT?TGC?AGT?GCA?GCT?GAG?ACA?ACG?TAC?GAC 240

Ser?Cys?Asp?Ala?Ala?Glu?Thr?Thr?Tyr?Asp

71 76

AAA?ATA?TTG?AAA?AAC?TTA?TCC?AGA?AAT?AGA 270

Lys?Ile?Leu?Lys?Asn?Leu?Ser?Arg?Asn?Arg

81 86

AGG?CTG?GTG?AGT?GAC?AAA?GTA?GGG?CAG?GCA 300

Arg?Leu?Val?Ser?Asp?Lys?Val?Gly?Glu?Ala

91 96

TGT?TGC?AGA?CCC?ATC?GCC?TTT?GAT?GAT?GAC 330

Cys?Cys?Arg?Pro?Ile?Ala?Phe?Asp?Asp?Asp

101 106

CTG?TCG?TTT?TTA?GAT?GAT?AAC?CTG?GTT?TAC 360

Leu?Ser?Phe?Leu?Asp?Asp?Asn?Leu?Val?Tyr

111 116

CAT?ATT?CTA?AGA?AAG?CAT?TCC?GCT?AAA?AGG 390

His?Ile?Leu?Arg?Lys?His?Ser?Aal?Lys?Arg

121 126

TGT?GGA?TGT?ATC?TGA 412

Cys?Gly?Cys?Ile

134

<210>2

<211>412

<212>DNA

＜213〉people

<400>2

TCA?CCA?GAT?AAA?CAA?ATG?GCA?GTG?CTT?CCT 30

AGA?AGA?GAG?CGG?AAT?CGG?CAG?GCT?GCA?GCT 60

GCC?AAC?CCA?GAG?AAT?TCC?AGA?GGA?AAA?GGT 90

CGG?AGA?GGC?CAG?AGG?GGC?AAA?AAC?CGG?GGT 120

TGT?GTC?TTA?ACT?GCA?ATa?CAT?TTA?AAT?GTC 150

ACT?GAC?TTG?GGT?CTG?GGC?TAT?GAA?ACC?AAG 180

GAG?GAA?CTG?ATT?TTT?AGG?TAC?TGC?AGC?GGC 210

TCT?TGC?AGT?GCA?GCT?GAG?ACA?ACG?TAC?GAC 240

AAA?ATA?TTG?AAA?AAC?TTA?TCC?AGA?AAT?AGA 270

AGG?CTG?GTG?AGT?GAC?AAA?GTA?GGG?CAG?GCA 300

TGT?TGC?AGA?CCC?ATC?GCC?TTT?GAT?GAT?GAC 330

CTG?TCG?TTT?TTA?GAT?GAT?AAC?CTG?GTT?TAC 360

CAT?ATT?CTA?AGA?AAG?CAT?TCC?GCT?AAA?AGG 390

TGT?GGA?TGT?ATC?TGA 412

<210>3

<211>324

<212>DNA

＜213〉people

<400>3

AAA?GGT?CGG?AGA?GGC?CAG?AGG?GGC?AAA?AAC 30

Lys Gly Arg Arg?Gly?Gln?Arg?Gly?Lys?Asn

1 6

CGG?GGT?TGT?GTC?TTA?ACT?GCA?ATA?CAT?TTA 60

Arg?Gly?Cys?Val?Leu?Thr?Aal?Ile?His?Leu

16

AAT?GTC?ACT?GAC?TTG?GGT?CTG?GGC?TAT?GAA 90

Asn?Val?Thr?Asp?Leu?Gly?Leu?Gly?Tyr?Glu

21 26

ACC?AAG?GAG?GAA?CTG?ATT?TTT?AGG?TAC?TGC 120

Thr?Lys?Glu?Glu?Leu?Ile?Phe?Arg?Tyr?Cys

31 36

AGC?GGC?TCT?TGC?AGT?GCA?GCT?GAG?ACA?ACG 150

Ser?Gly?Ser?Cys?Asp?Ala?Ala?Glu?Thr?Thr

41 46

TAC?GAC?AAA?ATA?TTG?AAA?AAC?TTA?TCC?AGA 180

Tyr?Asp?Lys?Ile?Len?Lys?Asn?Leu?Ser?Arg

51 56

AAT?AGA?AGG?CTG?GTG?AGT?GAC?AAA?GTA?GGG 210

Asn?Arg?Arg?Leu?Val?Ser?Asp?Lys?Val?Gly

61 66

CAG?GCA?TGT?TGC?AGA?CCC?ATC?GCC?TTT?GAT 240

Glu?Ala?Cys?Cys?Arg?Pro?Ile?Ala?Phe?Asp

71 76

GAT?GAC?CTG?TCG?TTT?TTA?GAT?GAT?AAC?CTG 270

Asp?Asp?Leu?Ser?Phe?Leu?Asp?Asp?Asn?Leu

81 86

GTT?TAC?CAT?ATT?CTA?AGA?AAG?CAT?TCC?GCT 300

Val?Tyr?His?Ile?Leu?Arg?Lys?His?Ser?Aal

91 96

AAA?AGG?TGT?GGA?TGT?ATC?TGA 324

Lys?Arg?Cys?Gly?Cys?Ile

101 106

Claims

1. a peptide species, described polypeptide has the activity with the GDNF receptors bind, but does not have the biologic activity of GDNF, and contains aminoacid sequence shown in the 29-133 position residue of SEQ ID NO:1 or the 29-134 position residue.

2. according to the polypeptide of claim 1, wherein said polypeptide has among the SEQ ID NO:1 Lys (29) to the aminoacid sequence shown in the Ile (134).

3. a fusion rotein wherein comprises claim 1 or 2 described polypeptide.

4. the polynucleotide that comprise the nucleotide sequence of coding claim 1 or 2 described polypeptide or the described fusion rotein of claim 3.

5. according to the polynucleotide of claim 4, wherein said polynucleotide contain nucleotide sequence shown in the SEQ IDNO:3 or its degeneracy sequence.

6. a nucleic acid construct wherein comprises the polynucleotide of claim 4 or 5, and one or more control sequence that can be operatively connected, can instruct polypeptide to produce in suitable expressive host with it.

7. according to the nucleic acid construct of claim 6, wherein said control sequence comprises and is selected from λ P _L, λ P _R, the T7 promotor promotor.

8. recombinant expression vector wherein comprises the nucleic acid construct of claim 6 or 7.

9. recombinant expression vector according to Claim 8, it is pBV-GDNF-28.

10. a cell has wherein transformed the nucleic acid construct of claim 6 or 7 or the recombinant expression vector of claim 8 or 9.

11. according to the cell of claim 10, it is a Bacillus coli cells.

12. according to the cell of claim 11, it is CGMCC No:0480.2.

13. method of producing claim 1 or 2 described polypeptide or the described fusion rotein of claim 3, be included in the cell of cultivating among the claim 10-12 any one under the condition that is suitable for producing described polypeptide, and from this cell or its substratum, reclaim this polypeptide.

14. the described fusion rotein of the polypeptide of claim 1 or 2 or claim 3 can be used for treating and/or preventing purposes in the medicine that GDNF overexpression related disease or needs reduce the active situation of GDNF in preparation.

15. the described fusion rotein of the polypeptide of claim 1 or 2 or claim 3 can be used for treating and/or preventing purposes in the gliomatous medicine in preparation.

16. the described fusion rotein of the polypeptide of claim 1 or 2 or claim 3 is as the purposes of the antagonist of GDNF.

17. the method in the growth of vitro inhibition neurogliocyte comprises described cell is contacted with the claim 1 of significant quantity or 2 polypeptide or the described fusion rotein of claim 3.

18. comprise the polypeptide of claim 1 or 2 or the pharmaceutical composition of the described fusion rotein of claim 3 and pharmaceutically acceptable carrier and/or vehicle.

19. according to the pharmaceutical composition of claim 18, it is used for the treatment of and/or prevents GDNF overexpression related disease or needs to reduce the active situation of GDNF.

20. according to the pharmaceutical composition of claim 18, it is used for the treatment of and/or prevents neurospongioma.