CN1287171A - Human neuron calcium sensing protein and its code sequence, preparation and use - Google Patents
Human neuron calcium sensing protein and its code sequence, preparation and use Download PDFInfo
- Publication number
- CN1287171A CN1287171A CN 99118819 CN99118819A CN1287171A CN 1287171 A CN1287171 A CN 1287171A CN 99118819 CN99118819 CN 99118819 CN 99118819 A CN99118819 A CN 99118819A CN 1287171 A CN1287171 A CN 1287171A
- Authority
- CN
- China
- Prior art keywords
- ncs
- sequence
- polypeptide
- people
- protein
- 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.)
- Pending
Links
Abstract
The present invention provides the cDNA sequence of human neuronal calcium sensor protein 1 (NCS-1). The cDNA encoded protein is one member of neuronal calcium ion binding protein family and the homologue of human NCS-1. The present invention also relates to the nucleotide encoded polypeptide, the application of the polynucleotide and the polypeptide, and the production process of the polynucleotide and the polypeptide.
Description
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, (neuronal calcium sensor-1, cDNA sequence NCS-1), this cDNA encoded protein are the homologue of a member-NCS-1 in the people of neural calcium ion-binding protein family to the present invention relates to human neuron calcium sensing protein.The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.
The calcium ion signal is at many cellular activities, as in the processes such as cellular metabolism, genetic expression, cytoskeleton dynamic change, cell cycle, necrocytosis, signal conduction decisive role being arranged.In neural system, in such as processes such as neurotransmitter release, synaptic plasticity variation, neure growth and interneuronal correlations, calcium ion also is indispensable equally.In conjunction with the direct interaction of target enzymes such as the same proteolytic ferment of adjusting molecule of calcium ion, kinases, phosphohydrolase, phosphodiesterase just by the adjusting of calcium ion realize (
Cell Tissue Res.1999,295:1-12;
J.Neurochemistry1996,67:932-942).Calcium ion-binding protein combines Ca
2+Variation on later on can occurred conformation exposes hydrophobic surface, thus activate or suppress other proteic activity (
Biochem.Biophys.Res.Commun.1995,216 (1): 133-140).
In recent years, one the class new calcium is conjugated protein is found, they are specifically expressing in neurocyte, and its conservative amino acid shows that they are subfamilies independently in the EF hand regulation of calcium superfamily protein, are named as neural calcium ion-binding protein (neuronal Ca
2+Binding proteins, NCaPs) (J.Biol.Chem.1994,269 (52): 32807-32813).This family member comes from ancestors' derivation that four EF hand sites are arranged, in all member's sequences on the I of position EF hand site all be invalid, no longer in conjunction with calcium ion.Compare and distribution situation in neurocyte according to sequence, this family can be divided into two classes: the category-A member is arranged in photosensitive organ, as recoverin (recoverin) (the amphiblestroid calcium sensor protein of vertebrates) and visinin (visinin) (a kind of calcium binding protein), they contain two typical EF hand sites in position II and III; The category-B member all has expression in the various cells of retina and brain, comprise class visinin VILIP, neuron calcium sensing protein NCS-1 (
J.Biol.Chem.1994,269 (52): 32807-32813).
1993, Pongs etc. cloned the frequelin gene the earliest from fruit bat mutant V7, found that it in the expression of fruit bat neural system, is a new Ca
2+It is conjugated protein that (Neuron 1993,11:15-28).Nineteen ninety-five, Olafsson group is for research vertebrates frequenin function, in Xenopus laevis brain cDNA library, according to fruit bat frequelin sequences Design degenerated primer, cloned Xenopus laevis the frequelin gene (
Proc.Natl.Acad.Sci.1995,92:8001-8005).The same year, people such as Nef are that primer has found the NCS-1 sequence in chicken brain cDNA library with conserved amino acid sequence in the recoverin homologue, sequence finds that relatively the frequelin of chicken NCS-1 and fruit bat has the highest homology (72% is consistent), judge they be different plant species homologue (
J.Recept.Signal.Transduct. Res.1995,15 (1-4): 365-378).NCS-1 sequence in rat, the ox also in nineteen ninety-five cloned (
Histochem. J.1995,27:524-535).Up to now, still nobody's NCS-1 sequence is reported.
Research shows that neuron calcium sensing protein is relevant with some diseases, and therefore, to research and develop human neuron calcium sensing protein significant for therapeutic purpose.
An object of the present invention is to provide a kind of new polynucleotide, a newcomer of the neural calcium ion-binding protein of this polynucleotide encoding family, the newcomer of neural calcium ion-binding protein of the present invention family is named as human neuron calcium sensing protein, abbreviates " people NCS-1 " as.
Another object of the present invention provides a kind of new neural calcium ion-binding protein family member, and this albumen is named as people NCS-1 albumen.
A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's NCS-1 polypeptide.
The present invention also provides this people's the NCS-1 nucleotide sequence and the application of polypeptide.
In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people NCS-1 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 3-575 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 3-575 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQID NO.3 nucleotide sequence from Nucleotide 3-575 position.
In another aspect of this invention, provide a kind of isolating people NCS-1 protein polypeptide, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQID NO.4 aminoacid sequence.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.
In another aspect of this invention, provide a kind of described carrier transformed host cells.
In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people NCS-1 protein-active, this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people NCS-1 protein-active operationally is connected in expression regulation sequence, form people NCS-1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 3-575 position among described nucleotide sequence and the SEQ ID NO.3;
(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people NCS-1;
(c) under the condition that is fit to expressing human NCS-1 protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people NCS-1 protein-active.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 627 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 3-575 position Nucleotide.
In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, term " people NCS-1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people NCS-1 protein-active is as 3-575 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 3-575 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 3-575 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 3-575 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 3-575 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people NCS-1 identical function.These variant forms comprise (but being not limited to): disappearance, insertion and/or the replacement of several (be generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) Nucleotide, and add several Nucleotide at 5 and/or 3 ends.Encoding sequence of the present invention can be DNA or RNA, can be strand or two strands.
In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.
In the present invention, term " people NCS-1 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people NCS-1 protein-active.This term also comprises having and variant form people NCS-1 albumen identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are 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 people NCS-1 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 people NCS-1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people NCS-1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people NCS-1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people NCS-1 polypeptide.Usually, this fragment have people NCS-1 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 1 00 continuous amino acids.
Invention also provides the analogue of people NCS-1 albumen or polypeptide.The difference of these analogues and natural human NCS-1 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, " people NCS-1 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, 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
The present invention also comprises people NCS-1 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people NCS-1 in the cell.
The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of people NCS-1 polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people NCS-1.
The present invention also comprises the method that detects people NCS-1 nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of people NCS-1 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
On the other hand, the present invention also comprises people NCS-1 DNA 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 people NCS-1 gene product or fragment.Preferably, refer to that those can combine with people NCS-1 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 people NCS-1, comprise that also those do not influence the antibody of people NCS-1 protein function.The present invention also comprise those can with modify or without the people NCS-1 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 people NCS-1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human NCS-1 or its have 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 people NCS-1 function and the antibody that does not influence people NCS-1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people NCS-1 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 people NCS-1 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).
People NCS-1 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 come 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 dna molecular 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.
The proteic fragment of the present invention, except producing with recombination method, also available solid phase technique is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.
The proteic encoding sequence of the present invention also can be used for the assignment of genes gene mapping.For example,, the cDNA clone is hybridized with the karyomit(e) of metaphase, can carry out chromosomal localization exactly by fluorescence in situ hybridization technique (FISH).This technology can be used the cDNA that is as short as about 500bp; Also can use and grow to about 2000bp or longer cDNA.For this technology, can be referring to people such as Verma, Human Chromosomes:A Manual ofBasic Techniques, Pergamon Press, New York (1988).
In case sequence is located in certain exact position on the karyomit(e), the physical location of sequence on karyomit(e) can be associated with the genetic map data.These genetic map data can obtain, for example by Mendelian (Mendelian) people genetic database (can obtain on the net by Johns Hopkins University Welch MedicalLibrary).Then, come identified gene by linkage analysis and be positioned dependency between the disease of same chromosomal region.
Then, be necessary to determine the cDNA between diseased individuals and the healthy individual or the difference of genome sequence aspect.Be not present in normal individual if a certain sudden change is present in part or all of diseased individuals, this sudden change may be exactly the paathogenic factor of this disease so.
Utilize albumen of the present invention,, can filter out with people NCS-1 interactional material takes place, as acceptor, inhibitor or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation 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, subcutaneous, intracutaneous or topical.
With people NCS-1 albumen of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.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.People NCS-1 albumen 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 people NCS-1 protein polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose 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.
Because people NCS-1 of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.
In one embodiment of the invention, the cDNA nucleotide sequence of people NCS-1 is so to obtain, with human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5-GGATGGGGAAATCCAACAGCAAG-3 ' is a forward primer, oligonucleotide B:5 '-ATGGCACAGAAGGAGGTGAGTGG-3 is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after amplified production checked order.
People NCS-1 albumen of the present invention is as a member of neural calcium ion-binding protein family, and the signal pathway that participates in for the variation of regulating small calcium ion concn and other calcium ion has a very important role.
In the accompanying drawings, Fig. 1 be people NCS-1 of the present invention (hNCS-1) with rat NCS-1 (rNCS-1) (L27421), chicken NCS-1 (galNCS-1) (L27420), mouse NCS-1 (mNCS-1) (AF020184) and Africa xenopus NCS-1 (xlNCS-1) 5 kinds of proteic amino acid sequence homologous comparison diagrams such as (U27274).Wherein, identical amino acid marks with " * " below sequence, and similar amino acid marks with ": ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.
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:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone and the mensuration of the cDNA sequence of people NCS-1
1. primer amplification
With human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer--A:5-GGATGGGGAAATCCAACAGCAAG-3 (SEQ ID NO:1) is a forward primer, oligonucleotide B:5-ATGGCACAGAAGGAGGTGAGTGG-3 (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A/B be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 650bp.
2.PCR the order-checking of product
Above-mentioned pcr amplification product A/B is connected with pGEM-T carrier (Promega), and transformed into escherichia coli JM103 extracts plasmid with QIAprep Plasmid test kit (QIAGEN), uses SequiTherm EXCEL
TMDna sequencing kit (Epicentre Technologies) checks order to extractive plasmid, with computer software splicing order, obtain full length cDNA sequence at last, altogether 627bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 3-575 position Nucleotide.
Derive the aminoacid sequence of people NCS-1 according to the full length cDNA sequence that obtains, totally 190 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.
Embodiment 2
Homology relatively
Full length cDNA sequence and proteins encoded thereof with people NCS-1 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that they and neural calcium ion-binding protein family member have very high homology, particularly the neuron calcium sensing protein NCS-1 with different sources has significant homology.Homology with PCGENE software analysis finder NCS-1 albumen and rat NCS-1 (L27421), chicken NCS-1 (L27420), mouse NCS-1 (AF020184) encoded protein is 98%, similarity is 99%, with the homology of Africa xenopus NCS-1 (U27274) encoded protein be 97%, similarity is 98% (Fig. 1).And people NCS-1 albumen of the present invention contain the N-myristoylation site that all neural calcium ion-binding proteins all have at nearly N end place (J.Biol.Chem.1998,273 (35): 22768-22772):
G｛E/D/R/K/H/P/F/Y/W｝X
2[S/T/A／G/C/N]｛P｝，
Wherein braces is represented any a kind of amino acid except that drawing together amino acid, square brackets represent draw together in the amino acid any one, X
nRepresent any n amino acid, sequence corresponding among the present invention is
41GTMDAA
46
And the category-B member on position II, III, IV, contain three typical EF hand sites (
J.Biol.Chem.1994,269 (52): 32807-32813)
DX[D/N/S]｛I/L/V/F/Y/W｝[D/E/N/S/T/G][D/N/Q/G/H/R/K]｛G/P｝[L/I/V/M/C][D/E/N/Q/S/T/A/G/C]X
2[D/E][L/I/V/M/F/Y/W]，
Wherein brace is represented any a kind of amino acid except that drawing together amino acid, square brackets represent draw together in the amino acid any one, X
nRepresent any n amino acid, sequence corresponding among the present invention is respectively
73DENKDGRIEFSEF
85,
109DLDNDGYITRNEM
121,
157DKNADGKLTLQEF
169Therefore can determine that it is people's neuron calcium sensing protein NCS-1, and have the general utility functions of neuron calcium sensing protein NCS-1.
NCS-1 albumen in biologies such as fruit bat, Aplysia biology, Xenopus laevis and chicken, rat, ox, all have expression (
Cell Tissue Res.1999,295:1-12).NCS-1 and Ca
2+Cohesive process in demonstrate the height positive cooperativity, it also can and Mg
2+In conjunction with, but do not show the plus or minus synergy.NCS-1 is to Ca
2+High susceptibility is arranged, work as Ca
2+When 0.15 μ mol/L was changed to 1.5 μ mol/L, the saturation ratio of NCS-1 can be increased to 90% from 10%, and if other neural calcium ion-binding protein NcaP will reach same saturation ratio, Ca
2+Concentration need improve 100 times (
J.Biol.Chem.1994,269 (52): 32807-32813).In the body that the chicken brain is carried out, experiment in vitro shows, the function of the alternative calmodulin CaM of NCS-1 in neural system, enzyme and ionic channel to many important calmodulin dependent forms all have effect, as phosphodiesterase, calcineurin, nitric oxide synthetase etc.Investigators infer that except the activity of NCS-1 and CaM, content and ubcellular distributing position are different NCS-1 is because it is to Ca
2+Hypersensitivity, for neurone provides a kind of meticulous switch, to Ca
2+The subtle change of concentration can be made reaction rapidly, and CaM is then to high density Ca
2+Change and to react (
Proc.Natl.Acad.Sci.1996,93:9253-9258).
Other member in NCS-1 albumen and the family also demonstrate g protein coupled receptor phosphorylations such as the inhibition of calcium dependent form such as Visual purple function (
Biochem.Biophys.Res.Commun.1995,216 (1): 133-140).They suppress receptor phosphorylation by suppressing the g protein coupled receptor kinase activity, thereby the realization desensitization (
FASEB J.1999,13:1-8;
J.Recept.Signal.Transduct.Res.1995,15 (1-4): 365-378).In the fruit bat the proteic homologue frequenin of NCS-1 external as recoverin as calcium sensitive guanylate cyclase (GC) performance activator function (
Neuron1993,11:15-28).The GC of the single-minded activation retinal rod of recoverin outside, and the GC gene is a multigene family, frequenin may act on the GC at other position of neural system.Pheochromocyte of ox and PCI2 cell expressing NSC-1 albumen show that it is not limited to express in neurone, this research find first NCS-1 to the dense-core granule exocytosis process of neurosecretory cell have regulating effect (
J.Biol.Chem.1998,273 (35): 22768-22772).In the Xenopus laevis embryo development procedure, NCS-1 appeared at for 21 stages (nerve-muscle begins contact) the earliest, and 40 stages (but tadpole of free swimming) expression amount rises, the developmental phase of it and neuromuscular synapse is consistent, and external source NCS-1 injects the presynaptic spinal nerves unit cynapse transmission that can cause developmental neuromuscular junction and is increased sharply, this point out it may the cynapse transmission of vertebrates neuromuscular synapse be played an important role (
Proc.Natl.Acad.Sci.1995,92:8001-8005).
People NCS-1 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor NCS-1 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor NCS-1 and the N end of rat NCS-1 are exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor NCS-1, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).
In addition, inventor NCS-1 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people NCS-1 or the overexpression that suppresses people NCS-1.People NCS-1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people NCS-1 disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.
Embodiment 3
The expression of people NCS-1 in intestinal bacteria
In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people NCS-1 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.5 and 6) of 3 ' end increase, obtain people NCS-1 cDNA as inserting fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TCAGGGATCCATGGGGAAATCCAACAGC-3′(SEQ?ID?NO.5)，
This primer contains the restriction enzyme site of BamH I restriction enzyme, is 18 Nucleotide of the people NCS-1 encoding sequence that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5-TTGGAAGCTTCTATACCAGCCCGTCGTAG-3(SEQ?ID?NO.6)，
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people NCS-1 of Hind III restriction enzyme.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp
r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.
With BamH I and Hind III digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed lac I repressor and carries kalamycin resistance (Kan
r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people NCS-1 has correctly been inserted carrier.
Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD
600) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lac I repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation culture, the collecting cell lysate also is diluted in it in 6M Guanidinium hydrochloride.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people NCS-1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people NCS-1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.
SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 22KDa.
In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 4
The expression of people NCS-1 in eukaryotic cell (Chinese hamster ovary celI strain)
In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people NCS-1 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.7 and 8) of 3 ' end increase, obtain people NCS-1 cDNA as inserting fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TCAGAAGCTTATGGGGAAATCCAACAGC-3′(SEQ?ID?NO.7)
This primer contains the restriction enzyme site of Hind III restriction enzyme, is 18 Nucleotide of the people NCS-1 encoding sequence that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5-TTGGGAATTCCTATACCAGCCCGTCGTAG-3(SEQ?ID?NO.8)
This primer contains the part encoding sequence of the restriction enzyme site of EcoR I restriction enzyme, translation termination and people NCS-1.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp
rAnd Neo
r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.
With Hind III and EcoR I digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, the cDNA fragment of sequence verification people NCS-1 has correctly been inserted carrier.
The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.
SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 22KDa.
In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 5
Preparation antibody
The recombinant protein that obtains in embodiment 3 and 4 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 people NCS-1 gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.
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 (1) general information: (ⅱ) denomination of invention: human neuron calcium sensing protein and encoding sequence thereof and method for making and purposes (ⅲ) sequence number: information (ⅰ) sequence signature of 8 (2) SEQ ID NO.1
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.1:GGATGGGGAA ATCCAACAGC AAG 23 (2) SEQ ID NO.2
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:ATGGCACAGA AGGAGGTGAG TGG 23 (2) SEQ ID NO.3: (ⅰ) sequence signature:
(A) length: 627bp
(B) type: nucleic acid
(C) chain: two strands
( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO.3GGATGGGGAA ATCCAACAGC AAGTTGAAGC CCGAAGTTGT GGAGGAGCGG ACCAGGAAGA 60CCTACTTTAC CGAGAAGGAG GTCCAGCAGT GGTACAAAGG CTTCATCAAG GACTGCCCCA 120GTGGCACCAT GGATGCGGCA GGCTTCCAGA AGATCTACAA GCAATTCTTC CCGTTCGGAG 180ACCCCACCAA GTTTGCCACA TTTGTTTTCA ACGTCTTTGA TGAAAACAAG GACGGGCGAA 240TTGAGTTCTC CGAGTTCATC CAGGCGCTGT CGGTGACCTC ACGGGGAACC CTGGATGAGA 300AGCTACGGTG GGCCTTCAAG CTCTACGACT TGGACAATGA TGGCTACATC ACCAGGAATG 360AGATGCTGGA CATTGTGGAT GCCATTTACC AGATGGTGGG GAATACCGTG GAGCTCCCAG 420AGGAGGAGAA CACTCCTGAG AAGAGGGTGG ACCGGATCTT TGCCATGATG GATAAGAATG 480CCGACGGGAA GCTGACCCTG CAGGAGTTCC AGGAGGGTTC CAAGGCAGAC CCGTCCATTG 540TGCAGGCGCT GTCCCTCTAC GACGGGCTGG TATAGTCCCA GGCTTGGAGC TGGATGCCTG 600GGAACCACTC ACCTCCTTCT GTGCCAT 627 ( 2 ) SEQ ID NO.4： ( ⅰ ) ：
(A) length: 190 amino acid
(B) type: amino acid
( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO.4Met Gly Lys Ser Asn Ser Lys Leu Lys Pro Glu Val Val Glu Glu 15Arg Thr Arg Lys Thr Tyr Phe Thr Glu Lys Glu Val Gln Gln Trp 30Tyr Lys Gly Phe Ile Lys Asp Cys Pro Ser Gly Thr Met Asp Ala 45Ala Gly Phe Gln Lys Ile Tyr Lys Gln Phe Phe Pro Phe Gly Asp 60Pro Thr Lys Phe Ala Thr Phe Val Phe Asn Val Phe Asp Glu Asn 75Lys Asp Gly Arg Ile Glu Phe Ser Glu Phe Ile Gln Ala Leu Ser 90Val Thr Ser Arg Gly Thr Leu Asp Glu Lys Leu Arg Trp Ala Phe 105Lys Leu Tyr Asp Leu Asp Asn Asp Gly Tyr Ile Thr Arg Asn Glu 120Met Leu Asp Ile Val Asp Ala Ile Tyr Gln Met Val Gly Asn Thr 135Val Glu Leu Pro Glu Glu Glu Asn Thr Pro Glu Lys Arg Val Asp 150Arg Ile Phe Ala Met Met Asp Lys Asn Ala Asp Gly Lys Leu Thr 165Leu Gln Glu Phe Gln Glu Gly Ser Lys Ala Asp Pro Ser Ile Val 180Gln Ala Leu Ser Leu Tyr Asp Gly Leu Val 190 ( 2 ) SEQ ID NO.5 ( ⅰ )
(A) length: 28 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.5:TCAGGGATCC ATGGGGAAAT CCAACAGC 28 (2) SEQ ID NO.6
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.6:TTGGAAGCTT CTATACCAGC CCGTCGTAG 29 (2) SEQ ID NO.7
(A) length: 28 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.7:TCAGAAGCTT ATGGGGAAAT CCAACAGC 28 (2) SEQ ID NO.8
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: SEQ ID NO.8:TTGGGAATTC CTATACCAGC CCGTCGTAG 29
Claims (10)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people NCS-1 protein-active,
Show at least 70% homology from the nucleotides sequence of Nucleotide 3-575 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps
Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 3-575 position.
2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.
3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 3-575 position.
4. an isolating people NCS-1 protein polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO.4 aminoacid sequence.
5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
6. a carrier is characterized in that, it contains the described DNA of claim 1.
7. one kind with the described carrier transformed host cells of claim 6.
8. a generation has the method for the polypeptide of people NCS-1 protein-active, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people NCS-1 protein-active operationally is connected in expression regulation sequence, form people NCS-1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 3-575 position among described nucleotide sequence and the SEQ ID NO.3;
(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people NCS-1;
(c) under the condition that is fit to expressing human NCS-1 protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people NCS-1 protein-active.
9. energy and the described people NCS-1 of claim 4 protein polypeptide specificity bonded antibody.
10. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99118819 CN1287171A (en) | 1999-09-07 | 1999-09-07 | Human neuron calcium sensing protein and its code sequence, preparation and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99118819 CN1287171A (en) | 1999-09-07 | 1999-09-07 | Human neuron calcium sensing protein and its code sequence, preparation and use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1287171A true CN1287171A (en) | 2001-03-14 |
Family
ID=5280567
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99118819 Pending CN1287171A (en) | 1999-09-07 | 1999-09-07 | Human neuron calcium sensing protein and its code sequence, preparation and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1287171A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1250931A1 (en) * | 2001-04-17 | 2002-10-23 | F. Hoffmann-La Roche Ag | Novel use of neuronal calcium sensor-1 (NCS-1) in therapy of CNS disorders and in the development of therapeutic agents |
| FR2824478A1 (en) * | 2001-04-17 | 2002-11-15 | Hoffmann La Roche | NEW USE OF NCS-1 (NEURON SPECIFIC CALCIUM SENSOR-1) FOR THE TREATMENT OF CNS DISORDERS AND THE DEVELOPMENT OF THERAPEUTIC AGENTS |
| CN103958684A (en) * | 2012-03-19 | 2014-07-30 | 瑞健生物医药（苏州）有限公司 | Redox-sensitive calcium-sensor proteins and methods of use thereof |
| WO2017162798A1 (en) * | 2016-03-23 | 2017-09-28 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Targeting the neuronal calcium sensor 1 for treating wolfram syndrome |
| CN112852976A (en) * | 2021-03-17 | 2021-05-28 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof |
-
1999
- 1999-09-07 CN CN 99118819 patent/CN1287171A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1250931A1 (en) * | 2001-04-17 | 2002-10-23 | F. Hoffmann-La Roche Ag | Novel use of neuronal calcium sensor-1 (NCS-1) in therapy of CNS disorders and in the development of therapeutic agents |
| FR2824478A1 (en) * | 2001-04-17 | 2002-11-15 | Hoffmann La Roche | NEW USE OF NCS-1 (NEURON SPECIFIC CALCIUM SENSOR-1) FOR THE TREATMENT OF CNS DISORDERS AND THE DEVELOPMENT OF THERAPEUTIC AGENTS |
| GB2377635B (en) * | 2001-04-17 | 2005-10-19 | Hoffmann La Roche | Novel use of neuronal calcium sensor-1 (NCS-1) in therapy of CNS disorders and in the development of therapeutic agents |
| CN103958684A (en) * | 2012-03-19 | 2014-07-30 | 瑞健生物医药（苏州）有限公司 | Redox-sensitive calcium-sensor proteins and methods of use thereof |
| WO2017162798A1 (en) * | 2016-03-23 | 2017-09-28 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Targeting the neuronal calcium sensor 1 for treating wolfram syndrome |
| CN112852976A (en) * | 2021-03-17 | 2021-05-28 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof |
| CN112852976B (en) * | 2021-03-17 | 2023-10-31 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to later egg laying characteristics in NCS1 gene of laying hen and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1287171A (en) | Human neuron calcium sensing protein and its code sequence, preparation and use | |
| CN1287169A (en) | New human G protein protomer and its code sequence | |
| CN1249347A (en) | Human calcineurin regulatory subunit, its coding sequence and its preparing process | |
| CN1277259A (en) | Homologous protein of late embryo ample-protein and its code sequence | |
| CN1401770A (en) | Human amino acid transporter, its coding sequence, preparing method and use | |
| CN1287170A (en) | New human nerve mass-transferring protein and its code sequence | |
| CN1287172A (en) | Human uridine kinase and its code sequence, preparation and application | |
| CN1290747A (en) | New human metal thioalbumen and its coding sequence | |
| CN1287173A (en) | Human 3-phosphoserine transferase and its code sequence, preparation and application | |
| CN1366046A (en) | Coding sequence, preparing process and application of human disease associated epidermal growth factor | |
| CN1287168A (en) | New human hair protein and its sequence | |
| CN1374405A (en) | Relative protein coding sequence, preparation and use of human adenylate cyclase | |
| CN1246532A (en) | Coding sequence of human heat shock associated protein, its encoded polypeptide and its preparing process | |
| CN1279290A (en) | Human signal recognition particle receptor beta and its coding sequence, preparing process and application | |
| CN1376796A (en) | Coding sequence of novel human protein and its preparing process and application | |
| CN1431307A (en) | Derivatization growth factor 4 of human liver cancer, its coding sequence, preparing method and usage | |
| CN1390935A (en) | Human hematolysis associated membrane protein and its coding sequence, preparing process and usage | |
| CN1246529A (en) | Coding sequence of human translation initiation factor subunit, its encoded polypeptide and its preparing process | |
| CN1257921A (en) | Human melanoma growth correlation factor and its coding sequence, preparing process and usage | |
| CN1250097A (en) | New human gene coding series and the polypeptide therewith and its preparation | |
| CN1394955A (en) | Human cytokine receptor protein, its code sequence, preparation method and application | |
| CN1431306A (en) | Derivatization growth factors 5 of human liver cancer, its coding sequence, preparing method and usage | |
| CN1371995A (en) | Human peptide transport protein coding sequence, preparation method and use thereof | |
| CN1303940A (en) | Human charcot-leyden crystal 2, its code sequence and preparation method and application | |
| CN1246537A (en) | Coding sequence of human serine/threonine kinase II, its encoded polypeptide and its preparing process |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |