CN1919868B - Chronic neuralgia related albumen and gene thereof - Google Patents
Chronic neuralgia related albumen and gene thereof Download PDFInfo
- Publication number
- CN1919868B CN1919868B CN2005100291135A CN200510029113A CN1919868B CN 1919868 B CN1919868 B CN 1919868B CN 2005100291135 A CN2005100291135 A CN 2005100291135A CN 200510029113 A CN200510029113 A CN 200510029113A CN 1919868 B CN1919868 B CN 1919868B
- Authority
- CN
- China
- Prior art keywords
- rsep1
- polypeptide
- sequence
- polynucleotide
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a new chronic neuralgia protein-RSEP1 protein, polynucleotide of RSEP1 encoding RSEP1 protein and method about generating RSEP1 protein through restructuring technology. The invention also discloses the usage to encode the polynucleotide of RSEP1 protein, which possesses a function of transmitting neuralgia. The invention also discloses the usage to encode the polynucleotide of RSEP1 protein. The RSEP1 protein possesses a function of transmitting neuralgia.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding chronic neuralgia related albumen RSEP1, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.
Background technology
Chronic neuralgia is the pain that periphery or central nervous system pathological change or damage cause, clinically to often not good [the Merskey H of the result of treatment of chronic neuralgia, Bogduk N.Classification of chronicpain.Classification of chronic pain.IASP Press, Seattle, 1994,394].
The clinical pain sensation and the paresthesia that mainly shows as injured nerve domination zone of chronic neuralgia is as hyperpathia (hyperalgesia strengthens normal pain irritant reaction); Touch bring out pain (allodynia, non-pain stimulation also can create a painful feeling feels), paresthesia (paresthesia), various burning sensation, the electric shock sample is felt and anesthesia or the like.
Had a lot of evidences to show, the dorsal horn of spinal cord participates in modulation process [IadarolaMJ, Berman, KF, Zeffiro TA, et al.Byas-Smith, M.G., Gracely, R.H., Max, the M.B.﹠amp of chronic neuralgia; Bennett, G.J.Neural activation during acute capsaicin-evoked painand allodynia assessed with PET.Brain, 1998,121:931-9471; Peyron R, Garcia-Larrea L, Gregoire MC, et al.Allodynia after lateral-medullary (Wallenberg) infarct.A PET study.Brain, 1998,121:345-356], and, there is a lot of genes to participate in process [the Boucher TJ that chronic neuralgia forms, Okuse K, Bennett DL, et al.Potent analgesic effects of GDNF in neuropathic pain states.Science, 2000,290:124-127; Malmberg AB, Chen C, Tonegawa S, et al.Preserved acute painand reduced neuropathic pain in mice lacking PKCgamma.Science, 1997,278:279-283].In addition, research also shows, neuromodulator such as tachykinin [Zieglgansberger W, BertheleA, Tolle TR.Understanding neuropathic pain.CNS.Spectr., 2005,10:298-308] and endogenic opioid peptides [Jones AK, Watabe H, Cunningham VJ et al.Cerebraldecreases in opioid receptor binding in patients with central neuropathicpain measured by[11C] diprenorphine binding and PET.Eur.J.Pain, 2004,8:479-485] also participated in the forming process of chronic neuralgia.Yet up to the present, the molecular mechanism of chronic neuralgia is not clear.
Therefore, this area presses for the new chronic neuralgia related albumen of exploitation.
Summary of the invention
The purpose of this invention is to provide a kind of new chronic neuralgia related albumen RSEP1 albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated chronic neuralgia related albumen-RSEP1 polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
Preferably, this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the neuralgic function of conduction by (a) polypeptides derived.
More preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned RSEP1 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or 3.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 4-429 position among the SEQ ID NO:1; (b) has the sequence of 1-917 position among the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of RSEP1 protein-active, this method comprises: (a) be fit to cultivate the above-mentioned host cell that is transformed or transduce under the proteic condition of expression RSEP1; (b) from culture, isolate polypeptide with RSEP1 protein-active.
In a fifth aspect of the present invention, provide and above-mentioned RSEP1 polypeptid specificity bonded antibody.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism RSEP1 polypeptide active is provided, and the compound that suppresses the RSEP1 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of RSEP1 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of RSEP1 in the test sample, it comprises: sample is contacted with the proteic specific antibody of RSEP1, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample RSEP1 albumen.
In a eighth aspect of the present invention, a kind of disease (as chronic neuralgia) relevant with RSEP1 polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes the RSEP1 polypeptide active, and perhaps screening suppresses the antagonist of RSEP1 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of RSEP1 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains RSEP1 polypeptide of the present invention or its agonist, antagonist (as antisense nucleic acid or antibody) and the pharmaceutically acceptable carrier of safe and effective amount (as 0.001-99.99wt%, preferably 0.01-90wt%).These pharmaceutical compositions can promote or suppress neuralgic conduction.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 has shown that the cDNA sequence of RSEP1 and the protein sequence of each species compare.Figure 1A wherein: rat RSEP1cDNA sequence and protein sequence, black matrix are translation initiation codons, the asterisk mark be terminator codon, the underscore mark be polyadenylation signal.Figure left side numerical markings is a Nucleotide, the numerical markings on the right side be amino acid.Inside the square frame is the sequence of design antisense nucleic acid and missense nucleic acid.Figure 1B: the comparison of the RSEP1 aminoacid sequence of rat, people, mouse, fruit bat and nematode.What short-term was represented is identical amino-acid residue, and the amino-acid residue of disappearance is represented with the space.
Fig. 2 has shown the Northern Blot analytical results of rat RSEP1 genetic expression.
Fig. 3 has shown the results of in situ hybridization of RSEP1mRNA at brain, cerebellum and spinal cord.Wherein use the RSEP1cDNA probe and the brain (A) of digoxigenin labeled, cerebellum (C) and spinal cord (E) carry out in situ hybridization.B, D, the enlarged image that F shows.Scale: A, C are 1000 μ m, and D is 500 μ m, and B, E are 250 μ m, and F is 100 μ m.
Fig. 4 has shown the Subcellular Localization result of EGFP-RSEP1 fusion rotein.Wherein, (A is C) with blank carrier EGFP-C2 transfection HEK293T cell with the EGFP-RSEP1 fusion protein expression vector.After the transfection 24 hours, with laser confocal microscope observe Expression of Fusion Protein (A, D).(B, E), C and F are synergetic images to dye nuclear with DAPI.What arrow was indicated is nucleus.Scale: 20 μ m.
Fig. 5 has shown that RSEP1 antisense oligonucleotide (a kind of RSEP1 antagonist) has alleviated the chronic neuralgia symptom of rat.X-coordinate is hind leg retraction waiting time (paw withdrawal latency) or hind leg retraction threshold value (g), ordinate zou for the operation back time (my god).
Embodiment
The inventor is through extensive and deep research, first by rat chronic neurodynia CCI (ChronicConstriction In jury, CCI) model, utilize the cDNA microarray technology to isolate a chronic neuralgia genes involved-RSEP1 (Rat Spinal-cord Expression Protein1 in conjunction with the method for in situ hybridization, RSEP1), experimental result shows that RSEP1 is a kind of chronic neuralgia related albumen.
Particularly, the inventor has studied the RSEP1 gene at the spinal cord of rat and the expression pattern of brain by hybridization in situ technique, found that in spinal cord, RSEP1 mainly is expressed in the I-II layer of cornu dorsale medullae spinalis, all has higher expression in the little Sensory neurone of V layer and the big motor neuron of cornu ventrale medullae spinalis.In pallium, RSEP1 mainly be expressed in cortex of frontal lobe (frontal cortex, Fr), radiation on the thalamus (superiorthalamic radiation, Str), and, in the CA1 district of hippocampus, (dentategyrus DG) has very strong expression for CA3 district and dentate gyrus.The expression of high level is also arranged in the granulosa cell of cerebellum in addition.The expression pattern prompting of RSEP1 in spinal cord, RSEP1 is a chronic neuralgia genes involved.
In addition, by suppress the expression of RSEP1 with antisense nucleic acid, found that the RSEP1 antisense oligonucleotide can alleviate the chronic neuralgia symptom of rat.This shows, a kind of really chronic neuralgia related albumen of RSEP1.
In the present invention, term " RSEP1 albumen ", " RSEP1 polypeptide " or " chronic neuralgia related albumen RSEP1 " are used interchangeably, all refer to the to have chronic neuralgia related albumen RSEP1 aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They comprise the chronic neuralgia related albumen RSEP1 that contains or do not contain initial methionine.
As used herein, term " RSEP1 albumen " also comprises the homologous protein from other animals, for example people's RSEP1 albumen.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating RSEP1 albumen or polypeptide " is meant that the RSEP1 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying RSEP1 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of RSEP1 polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of RSEP1, derivative and analogue.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps natural RSEP1 albumen of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " RSEP1 polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence of RSEP1 protein-active.This term also comprises having and variant form RSEP1 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of RSEP1 and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of RSEP1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-RSEP1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises RSEP1 polypeptide or its segmental fusion rotein (as merging formed fusion rotein with GST or 6His).Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of RSEP1 polypeptide.Usually, this fragment have the RSEP1 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of RSEP1 albumen or polypeptide.The difference of these analogues and natural RSEP1 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " RSEP1 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding RSEP1.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
RSEP1 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or RSEP1 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the RSEP1 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention RSEP1 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the RSEP1 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains RSEP1 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Experimental result of the present invention prompting, the low or forfeiture of RSEP1 protein function can cause the neurodynia level of conduction to descend, and the excited meeting of RSEP1 protein function causes the neurodynia level of conduction to rise.The RSEP1 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism RSEP1 protein function as pharmacological agent RSEP1 protein function.The peptide molecule that can suppress or stimulate the RSEP1 protein function that can be used for seeking therapeutic value with the reorganization RSEP1 protein screening peptide library of expressing.
On the other hand, the present invention also comprises RSEP1DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into RSEP1 gene product or fragment.Preferably, refer to that those can combine with RSEP1 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of RSEP1, comprise that also those do not influence the antibody of RSEP1 protein function.The present invention also comprise those can with modify or without the RSEP1 gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the RSEP1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing RSEP1 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the RSEP1 protein function and the antibody that does not influence the RSEP1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of RSEP1 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of RSEP1 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-RSEP1 can be used in the immunohistochemistry technology, detects the RSEP1 albumen in the biopsy specimen.
The disease that antibody among the present invention can be used for treating or prevention is relevant with RSEP1 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of RSEP1 or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of RSEP1 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of RSEP1 protein positive.
Available RSEP1 albumen of the production of polyclonal antibody or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with RSEP1 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Because the RSEP1 protein function is low or forfeiture can cause the neurodynia level of conduction to descend, and the excited meeting of RSEP1 protein function causes the neurodynia level of conduction to rise.Therefore, RSEP1 albumen and agonist can promote neuralgic conduction, and the inhibitor of RSEP1 (as antibody, antisense nucleic acid etc.) and antagonist can suppress neuralgic conduction.
Usually, these materials (albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor) can be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, preferably pH is about 6-8, although the pH value can change to some extent with being prepared Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
The inhibitor of polypeptide of the present invention or antagonist (as antisense nucleic acid) can be directly used in disease treatment, for example, are used for the treatment of chronic neuralgia aspect.When using RSEP1 albumen of the present invention, also can use the other treatment agent simultaneously.
The present invention also provides a kind of pharmaceutical composition, and it contains RSEP1 polypeptide of the present invention or its antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the RSEP1 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of RSEP1 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of RSEP1 of the proteic nothing expression of RSEP1 or unusual/non-activity.The RSEP1 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic RSEP1 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the RSEP1 transgenosis to cell.The method that structure carries the recombinant viral vector of RSEP1 gene is found in existing document (Sambrook, et al.).The RSEP1 gene of recombinating in addition can be packaged in the liposome, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of RSEP1mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of RSEP1 obtains.During screening, must carry out mark to the RSEP1 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization RSEP1 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The RSEP1 protein level that is detected in the test can be with laying down a definition the importance of RSEP1 albumen in various diseases and be used to the disease of diagnosing RSEP1 albumen to work.
Whether having the proteic method of RSEP1 in a kind of detection test sample is to utilize the proteic specific antibody of RSEP1 to detect, and it comprises: sample is contacted with the RSEP1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample RSEP1 albumen.
The proteic polynucleotide of RSEP1 can be used for the diagnosis and the treatment of RSEP1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of RSEP1 can be used for detecting the proteic expression of RSEP1 RSEP1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of RSEP1 as the RSEP1DNA sequence.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of RSEP1 albumen and also can detect the proteic transcription product of RSEP1.
The sudden change that detects the RSEP1 gene also can be used for the disease of diagnosing RSEP1 albumen relevant.The form of RSEP1 protein mutation comprises that the point mutation compared with normal wild type RSEP1DNA sequence, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of RSEP1 prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from the rat cdna library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 917 bases, and its open reading frame is positioned at the 4-429 position, and the coding total length is 509 amino acid whose RSEP1 albumen (SEQ ID NO:2).Albumen of the present invention provides the novel targets of treatment chronic neuralgia, thereby has great application prospect.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Material
Oligotex Direct mRNA Mid﹠amp; Max Kit is available from Qiagen company
The hybridization nylon membrane is available from Schleicher﹠amp; Schuell company
Taq polymerase is available from New England Biolabs company
TRIzol is an Invitrogen company product
DNTP, DTT are Promega company product
DNA labeling kit is a TAKARA company product
Random primer is an Invitrogen company product
Sephadex G-50 is a Pharmacia company product
α-32P-dATP isotropic substance is available from Amersham Pharmacia Biotech company
Sprague-Dawley adult rat (200-280g) is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
SP6/T7Transcription Kit: available from Roche company
Universal method:
(a) the CCI rat model is made:
The making method of CCI rat model is as follows: with vetanarcol anesthetized rat (40mg/kg, i.p.), make otch at big midleg, separate muscle with blunt to both sides, expose sciatic nerve, do the slight ligation in four roads with 4.0 trumpeter's art suture lines, being advisable should appear with the leg of rat once twitching in the dynamics of ligation, between the suture line of ligation every distance, suture muscles and the skin of about 1mm.The control group profit exposure sciatic nerve that uses the same method passes below sciatic nerve with operating sutures, but not ligation, suture muscles and skin.Every rat before carrying out the study of behaviour detection, recovered 24 hours after operation at least.
The clone of embodiment 1:RSEP1 albumen cDNA
The inventor is by rat chronic neurodynia CCI model, expands total RNA of spinal cord of portion with TRIzol reagent extracting rat waist, and the reverse transcription label probe comes the cDNA microarray of the cDNA library making of differential screening rat brain.And, a series of difference expression genes have been obtained by the contained dna sequence dna of each differential expression clone is carried out two-way mensuration.One of them cDNA clone's dna sequence dna is new full-length cDNA.Computer Analysis shows that this full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as RSEP1 albumen, its encoding gene called after RSEP1 protein gene.
By order-checking, Blast finds that relatively it is a new gene for RSEP1, and has comprised ORF total length (Fig. 1 and SEQID NO:1).The cDNA total length of RSEP1 is 917bp, the long 426bp of its ORF, and 142 the amino acid whose eggs (SEQ ID NO:2) of encoding are white.At poly (A)
+There is a polyadenylation signal (ATTAAA) at 19 base places, the upstream of tail.
MAAAAEGVPA?TRREEPPRDD?AAVETAEEAK?EPAEADINEL?CRDMFSKMAT?YLTGELTATS60
EDYKLLENMN?KLTSLKYLEM?KDIAINISRN?LKDLNQKYAG?LQPYLDQINV?IEEQVAALEQ120
AAYKLDAYSK?KLEAKYKKLE?KR142
(SEQ?ID?NO：2)
By BLASTP, the inventor compares the sequence in the aminoacid sequence of this supposition and the Protein Data Bank, found that the homologous sequence of four different plant species.Sequential analysis shows that this albumen is an albumen of striding the high conservative of species, and homology similarity aminoacid sequence of rat and people, mouse, fruit bat and nematode is respectively 100%, 98%, 39% and 31% (Figure 1B).Because the homologous protein of other four species and the proteic functional study of the RSEP1 of rat be report not also, thus the inventor this unnamed gene be RSEP1 (
RAt
SPinal-cord
EXpression
PRotein1, RSEP1).By http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html, find that the RSEP1 gene is positioned at the zone of rat karyomit(e) 1q54.
Embodiment 2: with the proteic encoding sequence of RT-PCR method clone RSEP1
Extract total RNA of the spinal cord of the L4-5 be in rat with Trizol (Gibco company), get total RNA and 0.5 μ g Oligo-dT
12-
18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ lddH after finishing
2O dilutes.The used primer of pcr amplification is as follows: adopted primer is arranged: 5 '-ggc atg gcg gct gctgcg gag ggc-3 ' (SEQ ID NO:3), antisense primer 5 '-tgagacacac gaacatgatt ctca-3 ' (SEQ ID NO:4).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mMdNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.The dna sequence analysis result shows DNA sequences encoding and the coding region shown in the SEQ ID NO:1 identical (the PCR product is corresponding to the 1-453 position among the SEQ ID NO:1) of this PCR product.
Recombinant expressed and the purifying of embodiment 3RSEP1 albumen
In this embodiment, be template with the pcr amplification product among the embodiment 2,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains RSEP1DNA as inserting fragment.
5 ' the end Oligonucleolide primers sequence of using in the PCR reaction is:
5′-CAGGTACCGGTCCGGAATTC-3′(SEQ?ID?NO：7)
This primer contains the restriction enzyme site of EcoRI restriction enzyme, is the part encoding sequence that is begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5′-GGGGTACCTCAGCCTCAGGATTTAGTGG-3′(SEQ?ID?NO：8)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the RSEP1 of KpnI restriction enzyme.
RSEP1 albumen cDNA PCR product purification after EcoR I/KpnI enzyme cut and recombinate according to a conventional method with plasmid pUC18 again and be converted into the competence bacillus coli DH 5 alpha, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The RSEP1 albumen cDNA EcoR I endonuclease bamhi of correct sequence is cloned into commercially available expression vector pGEX-2T, forms carrier pGEX-2T-RSEP1, transform people DH5 α then.Positive colony is cut the evaluation direction of insertion through enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete RSEP1 encoding sequence.
Choosing the positive DH5 α clone who expresses RSEP1 is inoculated in 100ml2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mMIPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,000g4 ℃ of centrifugal 10min removes supernatant, puts and uses 50ml1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na
2HPO
4, 1.8mM KH
2PO
4PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,000g4 ℃ of centrifugal 10min, supernatant with 0.8 μ m membrane filtration after, cross 1ml50% glutathione S epharose4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10mM gsh, 50mM Tris-HCl, pH8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeat wash-out 2-3 time, obtain RSEP1 albumen, molecular weight conforms to predictor.
The generation of embodiment 4 anti-RSEP1 protein antibodies
The reorganization RSEP1 albumen that obtains among the embodiment 3 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation RSEP1 protein gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
The tissue distribution of embodiment 5RSEP1mRNA:
Study the expression and distribution situation of RSEP1 gene in each tissue of rat by the method for Northern Blot.Method is as follows: total RNA of about 20 micrograms of extracting from the spinal cord of 8 all adult rats, behind the formaldehyde gel electrophoresis, change film to the nylon membrane of positively charged, and use α then
-32The probe hybridization of P mark.The size of RSEP1mRNA is about 1.8kb, and 18S rRNA contrasts as last sample.
The result as shown in Figure 2, RSEP1mRNA has only detected the band of an about 1.8kb in each tissue, in most brain district, except cerebellum, very high expression is arranged all; At spinal cord, RSEP1mRNA also has higher expression; In addition, in neural kidney of periphery and ovary, express also higherly, and other tissue expression level is lower.
The expression pattern of embodiment 6RSEP1 gene in CNS:
Present embodiment utilizes the method for in situ hybridization to study the expression pattern of RSEP1 at central nervous system in rat.Method is as follows: with the RSEP1cDNA probe and the brain (A) of digoxigenin labeled, cerebellum (C) and spinal cord (E) carry out in situ hybridization.
The result as shown in Figure 3.At pallium, RSEP1mRNA mainly is expressed on prefrontal lobe and the thalamus and radiates, in the CA1 district of hippocampus, the granulosa cell in CA3 district and DG district also have the expression of high level (Fig. 3 A, B).At spinal cord, RSEP1mRNA mainly be expressed in the I-II layer of dorsal horn and the big motor neuron of V-VI confluent monolayer cells and ventral horn (Fig. 3 E, F).At cerebellum, mainly be expressed in granular cell layer (Fig. 3 C, D).
The Subcellular Localization of embodiment 7RSEP1 fusion rotein:
(a) structure of EGFP-RSEP1 fusion protein expression plasmid carrier:
According to the sequence before the RSEP1 gene ORF,, RSEP1 gene ORF (4-432bp) is inserted in the commercially available pEGFP-C2 expression vector by the method for PCR.The design primer is as follows:
Forward primer: 5 '-CAGGTACCGGTCCGGAATTC-3 ' (SEQ ID NO:7)
Reverse primer: 5 '-GGGGTACCTCAGCCTCAGGATTTAGTGG-3 ' (SEQ ID NO:8)
The PCR product is cut with EcoRI and KpnI enzyme, and same, the pEGFP-C2 carrier is also cut with EcoRI and KpnI enzyme, reclaims, and connects then, makes up fusion vector.
(b) Subcellular Localization
Because RSEP1 is the cDNA sequence of a new rat, it may be helpful to the function meeting of this gene of research RSEP1 so understand the proteic Subcellular Localization of RSEP1 without any the known sequences motif.EGFP-RSEP1 fusion protein expression vector transfection human embryo kidney 293T cell with building scans cell with laser confocal microscope.
The result as shown in Figure 4.The EGFP-RSEP1 fusion rotein is positioned in the tenuigenin fully, in nucleus, do not have the EGFP-RSEP1 Expression of Fusion Protein (Fig. 4 A, 4C); On the contrary, blank carrier EGFP-C2 has expression (Fig. 4 D, 4F), these results shows that RSEP1 albumen is a cytoplasm protein in tenuigenin and nucleus.
Embodiment 8RSEP1 antisense oligonucleotide alleviates rat chronic neurodynia:
(a) the synthetic and perfusion of antisense oligodeoxyribonucleotide:
In order to explore the function that the RSEP1 gene has in rat chronic neurodynia forming process, the inventor is according to the sequences Design of RSEP1 gene and synthesized that antisense nucleic acid 5 '-(SEQ ID NO:5 and missense nucleic acid 5 '-ctt acc gca gca cct ctg ct-3 ' (SEQ ID NO:6), the sequence of RSEP1 gene is seen Fig. 1 to tct cca ccg cag cat cgt ct-3 '.Under the situation of vetanarcol anesthesia (40mg/kg), by the 4th, the intervertebral foramen of fifth lumbar vertebra is inserted into the L4-L5 lumbar vertebrae to PE-10 sheath inner catheter and expands the subarachnoid space of portion, inject aseptic physiological saline (about 4 μ l) in the conduit, expose external end stopper jam-pack.The rat feeding that inserts conduit in one cage, was recovered 4 days.Synthetic antisense nucleic acid and missense be dissolved in 0.9% the physiological saline, the L4-L5 lumbar vertebrae that is then injected into experimental rat expands the subarachnoid space of portion (4 μ l perrat, oligonucleotide at 0.5 μ g/ μ l), the normal saline flushing of using 5 μ l again once.Injection in per 24 hours was once injected 4 days altogether.Survey pain, analyze its ethological variation, study its function.
(b) study of behaviour detection method
1, the heat pain is quick:
The research method that adopts Hargreaves etc. [Hargreaves etc., 1988] to introduce is measured rat and is come to harm after the stimulation hot in nature, the contraction latent period of its hind leg.Method is as follows: be put in the resin glass cage rat is single, a glass platform of raising is wherein arranged, the thermal source of a diversity is arranged below platform.After rat is known well the environment of the cage of detection, begin hind leg with the thermal source irradiation in rats of diversity, when the claw of rat lifts, turn off thermal source, note rat from the thermal stimulus of coming to harm property to time that its hind leg shrinks, this time is defined as the latent period that hind leg shrinks, thereby it is quick to measure its heat pain.
Thermal source keeps a constant intensity, has produced the contraction latent period of more stable 8-10 second like this in the not side of operation of CCI rat model.If rat will be turned off thermal source to not reaction of thermal source when shining 20 seconds, in order to avoid tissue is burnt.Rat is divided into 4 every group detects, like this, the left hind (side of contrast) of rat is detected once, after 5 minutes, detect right hind (CCI one side) more at interval.Every group of 4 rats repeat to survey twice again, and are average this result of three times of every side at last.
2, the machinery pain is quick:
Measure the contraction thresholding of rat hindlimb from the von Frey metal needle of 0.6-18 gram with the scope of series of standards.Single rat is put into the bottom to be had in the cage of metal grill, makes it to environmental adaptation 30 minutes.Come the middle section of stimulation in rats hind leg sole, each 6 seconds according to the order that progressively raises (0.6,0.9,1.3,2.2,4.8,6,7.2,9,13 and 18 gram) with von Frey metal needle.Having only when the four limbs of rat all land just stimulates with von Frey metal needle.After the stimulation, have only and at the bottom of hind leg has left the cage of metal grill completely, just think once to shrink reflection.Again this hind leg is detected, after shrinking reflection foundation, stimulate with next von Frey metal needle again, up to not reaction generation.Come the hind leg 5 times of stimulation in rats with a series of von Frey metal needle, in 5 times stimulation, if minimum stimulation can cause 3 times contraction reflection is arranged each 15 seconds at interval, the stimulation of this minimum is exactly to shrink thresholding.In case the thresholding of left side (side of contrast) has been determined, after 5 minutes, detect right side (side of CCI) again with the similar detection program, determine that it shrinks thresholding.
Statistical study:
Data are represented with mean number ± standard error, analyze by the dual factors multiple covariance and calculate its statistical significance.P＜0.05 has been considered to statistical significance.
The result:
Present embodiment utilizes chronic neuralgia model-CCI model [the Bennett GJ of the nerve injury-induced of rat, Xie YK.A peripheral mononeuropathy in rat that produces disorders of painsensationlike those seen in man.Pain, 1988,33:87-107] function of probing into the RSEP1 gene.As before report, performed the operation back 3 days at one-sided CCI, rat demonstrate intensive heat pain quick and mechanical quick bitterly (Fig. 5, A, B).After the CCI operation, as far back as the quick reaction of just appearance pain in 2 days.These neurodynia behaviors can be kept one month behind the surgical injury hind leg, and offside blank one side pain do not show in the quick reaction very big variation (Fig. 5, A, B).
The rear flank limb sciatic nerve of rat is accepted the CCI operation, and opposite side is as blank.Heat pain quick (A) and machinery pain quick (B).Side in damage shows heat pain quick (A) and machinery pain quick (B), and that blank one side does not show is bitterly quick.Cavitas subarachnoidealis spinalis intrathecal injection physiological saline does not weaken the behavior of CCI damage inductive neurodynia.By having weakened CCI damage inductive heat pain quick (C) and machinery quick (D) bitterly at intrathecal injection RSEP1 antisense oligonucleotide (inject once every day, injected 4 days), and that injecting normal saline or RSEP1 missense oligonucleotide can not weaken this pain is quick.For the hind leg of blank, no matter be antisense oligonucleotide or missense tube nucleus thuja acid or the physiological saline of injection RSEP1, it is quick all can not to influence the quick and mechanical pain of heat pain.
Intrathecal injection RSEP1 antisense oligonucleotide, can either weaken the heat pain of CCI rat quick also can weaken the machinery pain quick (Fig. 5 C, 5D).Variance analysis shows to have the difference (p＜0.05) of tangible statistical significance in injection antisense oligonucleotide group and control group.And it is reversible acting on the specific effect of neuralgic antisense oligonucleotide, after stopping to inject antisense oligonucleotide, this pain that weakens is quick slowly return to former hyperpathia again and touch bring out pain (Fig. 5 C, 5D).The antisense oligonucleotide of RSEP1 is that CCI inductive neurodynia is special to the influence of pain behavior, the thresholding and the latent period of the contraction of its offside hind leg unaffected (Fig. 5 E, 5F).
Discuss:
In the present invention, the mRNA of the spinal cord of the L4-5 by extracting CCI rat, the reverse transcription label probe screens the cDNA library of rat brain, has identified the new gene of rat, RSEP1 (Fig. 1).Northern blot analysis has disclosed RSEP1 all has expression (Fig. 2) in some brain districts, spinal cord and peripheral tissues.In situ hybridization result (Fig. 3) shows, RSEP1 is at the CA1 of hippocampus, and all there is the expression of higher level in CA3 and DG district at the big motor neuron of the little Sensory neurone of cornu dorsale medullae spinalis and cornu ventrale medullae spinalis.At the dorsal horn of spinal cord, RSEP1 is expressed in I-II and V-VI layer, and the neurone of this position mainly is responsible for nocuous stimulation is made a response [Fyffe, 1984], shows that this gene may participate in the forming process of chronic neuralgia.On cell levels, the RSEP1 protein expression is (Fig. 4) in tenuigenin, and the function that shows this gene mainly is to participate in cell signal, rather than influences expression of gene in nucleus.
In the process that neurodynia forms after peripheral nerve injury, existing discovering, variation [Costigan M has taken place in a lot of expression of gene of different levels, Befort K, Karchewski L, et al.Replicate high-density rat genome oligonucleotide microarrays revealhundreds of regulated genes in the dorsal root
In sum, the inventor has identified a new gene-RSEP1 relevant with the chronic neuralgia behavior of rat.Because RSEP1 is being very conservative on the dna level or on the protein level, and the function of albumen homology analogue is not clear, therefore, RSEP1 may represent the new albumen of class mediation (or conduction) chronic neuralgia behavior.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Chinese Academy of Sciences Shanghai school of life and health sciences
＜120〉chronic neuralgia related albumen and gene thereof
<130>056616
<160>8
<170>PatentIn?version3.1
<210>1
<211>917
<212>DNA
＜213〉rat (rat)
<220>
<221>CDS
<222>(4)..(429)
<223>
<400>1
<210>2
<211>142
<212>PRT
＜213〉rat (rat)
<400>2
<210>3
<211>24
<212>DNA
＜213〉Oligonucleolide primers
<400>3
<210>4
<211>24
<212>DNA
＜213〉Oligonucleolide primers
<400>4
<210>5
<211>20
<212>DNA
＜213〉oligonucleotide
<220>
<221>misc_feature
＜223〉antisense nucleic acid
<400>5
<210>6
<211>20
<212>DNA
＜213〉oligonucleotide
<220>
<221>misc_feature
＜223〉missense nucleic acid
<400>6
<210>7
<211>20
<212>DNA
＜213〉Oligonucleolide primers
<400>7
<210>8
<211>28
<212>DNA
＜213〉Oligonucleolide primers
<400>8
Claims (8)
1. an isolated polypeptide is characterized in that, this polypeptide is:
Polypeptide shown in the SEQ ID NO:2 aminoacid sequence.
2. isolating polynucleotide is characterized in that, this nucleotide sequence is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
3. polynucleotide as claimed in claim 2 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) sequence of 4-429 position among the SEQ ID NO:1;
(b) sequence of 1-917 position among the SEQ ID NO:1.
4. a carrier is characterized in that, it contains the described polynucleotide of claim 2.
5. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 4.
6. the preparation method of the described polypeptide of claim 1 is characterized in that, this method comprises:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 5;
(b) from culture, isolate the described polypeptide of claim 1.
7. energy and the described polypeptid specificity bonded of claim 1 antibody.
8. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1, the antisense oligonucleotide shown in the SEQ ID NO:5 or the antibody of the described polypeptide of claim 1 of safe and effective amount, and pharmaceutically acceptable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005100291135A CN1919868B (en) | 2005-08-26 | 2005-08-26 | Chronic neuralgia related albumen and gene thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005100291135A CN1919868B (en) | 2005-08-26 | 2005-08-26 | Chronic neuralgia related albumen and gene thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1919868A CN1919868A (en) | 2007-02-28 |
| CN1919868B true CN1919868B (en) | 2011-09-21 |
Family
ID=37777741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005100291135A Expired - Fee Related CN1919868B (en) | 2005-08-26 | 2005-08-26 | Chronic neuralgia related albumen and gene thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1919868B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002083847A2 (en) * | 2001-04-11 | 2002-10-24 | Ortho Mcneil Pharmaceutical, Inc. | Cdna encoding the human alpha2 delta4 calcium channel subunit |
-
2005
- 2005-08-26 CN CN2005100291135A patent/CN1919868B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002083847A2 (en) * | 2001-04-11 | 2002-10-24 | Ortho Mcneil Pharmaceutical, Inc. | Cdna encoding the human alpha2 delta4 calcium channel subunit |
Non-Patent Citations (2)
| Title |
|---|
| 万丽 等.鞘内注射反义蛋白激酶Cr寡核苷酸对慢性神经痛大鼠对镇痛作用.中华麻醉学杂志25 1.2005,25(1),全文. |
| 万丽 等.鞘内注射反义蛋白激酶Cr寡核苷酸对慢性神经痛大鼠对镇痛作用.中华麻醉学杂志25 1.2005,25(1),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1919868A (en) | 2007-02-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE69836131T2 (en) | NEW HUMAN DELTA3 COMPOSITION AND ITS THERAPEUTIC AND DIAGNOSTIC USES | |
| KR20000029877A (en) | A tumor necrosis factor related ligand | |
| JPH08502168A (en) | Trophic factors with ion channel-inducing activity in neuronal cells | |
| CN102134275A (en) | Epidermal growth factor receptor variant | |
| CN1919868B (en) | Chronic neuralgia related albumen and gene thereof | |
| CN102443056B (en) | Exon deleted variant of epidermal growth factor receptor | |
| CN100443501C (en) | Mitochondria traffic protein molecule for human marrow stromal cell and sequence encoding same and use thereof | |
| CN100408597C (en) | Human calcium ion / calmodulin deopendent protein kinase II inhibitory protein alpha, its coded sequence and use | |
| CN100400658C (en) | New type human death resist protein, its coded sequence and use | |
| US7544485B2 (en) | Baldness related gene and the polypeptide encoded thereby, and uses | |
| CN1477117A (en) | Novel human phosphotidylethanolamine binding protein, its coding sequence and application | |
| CN100358917C (en) | Tumor tag and the use thereof | |
| CN100425695C (en) | Novel human Rab GTP enzyme, its coding sequence and application | |
| DE60124910T2 (en) | HUMAN TWIK-8 MOLECULES AND THEIR USE | |
| CN100480264C (en) | Earthworm protein suppressing cancer cell accretion by road spectrum and coding sequence thereof | |
| CN100381466C (en) | Human pifl gene, its coding protein and use thereof | |
| CN100523189C (en) | Function and use of human immune cell inhibition acceptor KLRL1 | |
| CN100543134C (en) | Phosphokinase and application thereof | |
| CN1778816B (en) | Human cell surface membrane molecule and use thereof | |
| CN100480263C (en) | Immunosuppressive receptor derived from dendritic cell, coding sequence and uses thereof | |
| CN100355782C (en) | New liver cancer up expressing gene and its encoded protein and application | |
| CN100543133C (en) | Phosphokinase and application thereof | |
| CN1325874A (en) | Human C-type lectin-like receptor and its coding sequence and application | |
| CN1394873A (en) | Human calcium ion/calmodulin dependent protein kinase II inhibitory protein and its application | |
| CN1299828A (en) | New cell factor acceptor and its code sequence and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110921 Termination date: 20160826 |