CN1251860A - Human gene sequence and its coded polypeptide, preparing process and application - Google Patents
Human gene sequence and its coded polypeptide, preparing process and application Download PDFInfo
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Abstract
The present invention relates to the human NP25 protein as homogeneous protein of rat NP25 protein in human body. The cDNA coding sequence of said protein, the polypeptide coded with said sequence, the process for preparing new human NP25 protein by recombination technique, and the application of said human NP25 protein are disclosed.
Description
The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, polypeptide of the present invention is accredited as the homologous protein of rat NP25 albumen in human body by deduction.
1994, people such as Ren Wen-Zhi isolated a kind of brand-new cDNA (Ren Wen-Zhi, etal, Mol.Brain Res., 22 (1994) 173-185) from rat brain.This of cDNA coding comprises 219 amino acid whose albumen.Calculate to such an extent that its molecular weight is about 25kDa according to proteic amino acid composition, thereby with this albumen called after NP25.
Primary structure to this NP25 protein polypeptide studies show that, contains the phosphorylation site of a casein kinase i I: Thr in this amino acid sequence of polypeptide
138-Lys-Asp-Asp
142(Kuenzel, E.A.et al, J.Biol.Chem., 262 (1987) 9136-9140), two possible protein kinase C phosphorylation site: Ser
180-Asn-Lys-Gly
183Ser
198-Cys-Lys-Met
201(Woodget, J.R.et al, Eur.J.Biochem., 161 (1986) 177-184), and ATP binding sequence conservative in the protein kinase C family member a: Gly
191-X-Gly-X-X-Gly-Arg-Ser-Cys-Lys
200(Nisizuka, Y., et al, Nature, 334 (1988) 661-665) and an EF palmistry calcium are in conjunction with (EF hand-likecalcium binding) structure.
Especially, this NP25 peptide C end also contains a tumor-necrosis factor glycoproteins conservative in Calponin family: [LIVM]-X-[LS]-Q-[MAS]-G-[STY]-[NT]-[KRQ]-X (2)-[STN]-Q-X-G-X (3,4)-G.This sequence has repeated (Maguchi three times in people calponin, M., et al, Biochem.Biophy.Res.Commu., 217 (1995) 238-244), this external vertebrates unstriated muscle Protein S M-22 alpha (transgelin) (Prinjha, R.K., CellMotility and the cytoskeleton 28 (1994) 243-255) and among the synchronous flight muscle albumen of the fruit bat mp20 (Ayme-Southgate et al., J.Cell Biol.108 (1989) 521-531) also respectively there is a copy.The existence prompting of this conserved sequence in rat NP25 albumen comprises that the above-mentioned albumen of people NP25 and calponin may constitute a multigene family.The member of this multigene family may come from same ancestors, and their activity is subjected to calcium ion or/and the adjusting of phosphorylation.In this multigene family, calponin is that a kind of thin filament is conjugated protein, is regulating the contraction of unstriated muscle.Calponin has suppressed the activity (Winder, S.J., etal, J.Biol.Chem., 265 (1990) 10148-10155) of actomyosin MgATPase with the interaction of Actin muscle.SM22 alpha also is a kind of and Actin muscle bonded albumen, and its function relates to the gelification (Shapland, C., et al, J.CellBiol.121 (1993) 1065-1073) of Actin muscle.The proteic function of fruit bat mp20 is not clear, but the distribution prompting of this albumen in synchronous flight muscle, and this protein function may relevant with the Muscle contraction frequency (Ayme-Southgate et al., J.Cell Biol.108 (1989) 521-531).
Studies show that of immunocytochemistry that rat NP25 albumen is carried out and in situ hybridization, rat NP25 albumen is only expressed in well differentiated axoneuron.This points out this albumen may not be structural protein but has the function of eggcase, and play an important role in specific neuron system (Ren Wen-Zhi, et al, Mol.BrainRes., 22 (1994) 173-185).
Though utilize rat NP25 cDNA probe in human brain tissue, to detect the mRNA (RenWen-Zhi of similar size, et al., Mol.Brain Res., 22 (1994) 173-185), however before the present invention the still unexposed cDNA sequence of delivering remarkable NP25.
An object of the present invention is to provide a kind of new polynucleotide, the homologous protein of this polynucleotide encoding rat NP25 albumen in human body, homologous protein gene of the present invention is named as people NP25.
Another object of the present invention provides a kind of new homologous protein of rat NP25 albumen in human body, and this albumen is named as people NP25 albumen.
A further object of the present invention provides a kind of recombinant technology that utilizes and produces the described new proteic method of people NP25.
The invention still further relates to the application of this people NP25 gene order and 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 NP25 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 66-731 position among described nucleotide sequence and the SEQIDNO.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 66-731 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence is the nucleotide sequence that has among the SEQ ID NO.3 from Nucleotide 66-731 position.
In another aspect of this invention, provide a kind of isolating people NP25 protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.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 NP25 protein-active, this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people NP25 protein-active operationally is connected in expression regulation sequence, form people NP25 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 66-731 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 NP25;
(c) under the condition that is fit to expressing human NP25 protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people NP25 protein-active.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 761 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 66-731 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 NP25 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people NP25 protein-active is as 66-731 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 66-731 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 66-731 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 66-731 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 66-731 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people NP25 identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
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 NP25 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people NP25 protein-active.This term also comprises having and variant form people NP25 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 NP25 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 NP25 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people NP25 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people NP25 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of people NP25 polypeptide.Usually, this fragment have people NP25 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 people NP25 albumen or polypeptide.The difference of these analogues and natural human NP25 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 can also comprise 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 NP25 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.In addition, these conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
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; Nor-leucine | Leu |
| Leu(L) | Nor-leucine; 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; Nor-leucine | Leu |
The present invention also comprises people NP25 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people NP25 in the cell.
The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people NP25 nucleotide 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 NP25.
The present invention also comprises the method that detects people NP25 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 can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of people NP25 polypeptide.Primer length is generally 20-50 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 NP25 DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people NP25 gene product or fragment.Preferably, refer to that those can combine with people NP25 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 NP25, comprise that also those do not influence the antibody of people NP25 protein function.The present invention also comprise those can with modify or without the people NP25 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 NP25 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human NP25 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 to prepare that (see people such as Kohler, Nature 256; 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 NP25 function and the antibody that does not influence people NP25 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people NP25 gene product, obtains by immunological technique.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 NP25 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 NP25 nucleotide sequence 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about 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.
In one embodiment of the invention, the polynucleotide total length that the present invention obtains is 761 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 66-731 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A1:5 '-TGAGAAGCGG GAAGAAACGT CCG-3 ' and reverse primer B1:5 '-GTTCAAGCTT CTATTTCTTT TCGTAGAGA-3 ' carry out PCR, obtain the purpose fragment of 761bp.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.
The homology comparative result shows that albumen of the present invention is the homologous protein of rat NP25 albumen in human body, therefore the invention provides to the research of NP25 albumen newly by way of.
Also there are higher homology in albumen of the present invention and calponin and SM22 alpha, and this homology discloses and comprises that the above-mentioned albumen of people NP25 and SM-22 alpha, calponin may constitute a multigene family.The member of this multigene family may come from same ancestors.Calponin and SM22 are all mytolin: calponin is that a kind of thin filament is conjugated protein, regulating the contraction of unstriated muscle, and SM22 alpha is a kind of actin binding protein, and its function relates to the gelification of Actin muscle.People NP25 albumen of the present invention and above-mentioned albumen homology have disclosed the evolutionary relationship of nervous tissue and muscle tissue, for the evolutionary relationship of studying two kinds of tissues provides new way.In addition, the present invention provides new material and approach for research comprises the multigene family of NP25 albumen and SM22 alpha, calponin.
In the accompanying drawings,
Fig. 1 is the homology comparison diagram of the nucleotide sequence of people NP25 of the present invention and rat NP25.Wherein, identical Nucleotide marks with " | ".
Fig. 2 is the homology comparison diagram of the aminoacid sequence of people NP25 of the present invention and rat NP25.Wherein, identical amino acid marks with " | " between two sequences, 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.
Fig. 3 is the homology comparison diagram of people NP25 of the present invention and the proteic nucleotide sequence of people SM22 alpha.Wherein, identical Nucleotide marks with " | ".
Fig. 2 is the homology comparison diagram of people NP25 of the present invention and the proteic aminoacid sequence of people SM22 alpha.Wherein, identical amino acid marks with " | " between two sequences, 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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The clone and the mensuration of the cDNA sequence of people NP25
1. primer amplification
With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-TGAGAAGCGG GAAGAAACGT CCG-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-AGAAAGAGGA GAAGGTCAGA AGG-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is about the purpose fragment of 770bp.
2.PCR the order-checking of product
With pcr amplification product and the pGEM-T that as above obtains
Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL
TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 761bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 66-731 position Nucleotide.
Derive the aminoacid sequence of people NP25 according to the full length cDNA sequence that obtains, totally 221 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 NP25 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that the present invention and rat NP25 have high homology on albumen and nucleotide level, as use PCGENE software analysis, it and rat NP25 (GeneBank accession No.:M84725) on Nucleotide and protein level, have an appointment respectively 87% and 95% identity and similarity (Fig. 1 and Fig. 2); In addition, be 69% and 66.2% (Fig. 3 and 4) with the identity of people SM22alpha (GenBank accession No.D17409) on Nucleotide and protein level.In addition, the identity with people alkalescence calponin (GenBank accessionNo.P51911) has also surpassed 40%.
Discover, the same with the NP25 protein polypeptide of rat, in inventor NP25 albumen, also contain the phosphorylation site of a position and the identical casein kinase i I of sequence: Thr
138-Lys-Asp-Asp
142(138-142 position among the SEQ IDNo.4); Position and the identical protein kinase C phosphorylation site of sequence: Ser
180-Asn-Lys-Gly
183(180-183 position among the SEQ ID No.4) and the protein kinase C phosphorylation site that the position is identical, sequence is more similar a: Ser
198-Cys-Arg-Thr
201(in rat NP25, this site is Ser
198-Cys-Lys-Met
201); And one with the protein kinase C family member in conservative ATP binding sequence: Gly
191-X-Gly-X-X-Gly-Arg-Ser-Cys-Lys
200Extremely similar sequence, this sequence are Gly
191-Tyr-Gly-Ile-Pro-Gly-Arg-Ser-Cys-Arg
200(191-200 position among the SEQ ID No.4), wherein only the 200th Arg and the Lys in the ATP binding sequence are different, but, be the ATP binding sequence so can affirm 191-200 of the present invention position because Arg and Lys are structure and the extremely close amino acid of character.
In addition, the C-terminal of people NP25 polypeptide of the present invention also contains a tumor-necrosis factor glycoproteins conservative in calponin family: [LIVM]-X-[LS]-Q-[MAS]-G-[STY]-[NT]-[KRQ]-X (2)-[STN]-Q-X-G-X (3,4)-G.
This sequence has repeated (Maguchi three times in people calponin, M., et al, Biochem.Biophy.Res.Commu., 217 (1995) 238-244), this external vertebrates unstriated muscle Protein S M-22 alpha (transgelin) (Prinjha, R.K., Cell Motility and the cytoskeleton 28 (1994) 243-255) and among the synchronous flight muscle albumen of the fruit bat mp20 (Ayme-Southgate et al., J.Cell Biol.108 (1989) 521-531) also respectively there is a copy.This conserved sequence also exists in people NP25 albumen of the present invention, and concrete sequence is: I
174GLQMGSNKGASQAGMTGYG
193(174-193 position among the SEQ ID No.4).This prompting comprises that people NP25 and the above-mentioned albumen of SM-22 alpha may constitute a multigene family.The member of this multigene family may come from same ancestors.
Rat NP25 albumen is a kind of albumen that distributes in specific axoneuron, and its concrete function is unclear.The invention provides new way to NP25 protein structure and functional study.Rat NP25 albumen only is distributed in the situation hint in the specific neurone in addition, people NP25 albumen of the present invention also has similar distribution character, this specific character makes albumen of the present invention might be used as specific neuronic labelled protein, thereby is playing a significant role aspect the evaluation of central nervous system disease and the treatment.
Studies show that, Caplonin and SM22 are all mytolin: caplonin is that a kind of thin filament is conjugated protein, regulating the contraction of unstriated muscle, SM22 alpha is a kind of actin binding protein, its function relates to the gelification of Actin muscle, people NP25 albumen of the present invention and above-mentioned albumen homology have disclosed the evolutionary relationship of nervous tissue and muscle tissue, for the evolutionary relationship of studying two kinds of tissues provides new way.In addition, the present invention provides new material and approach for research comprises the multigene family of NP25 albumen and SM22 alpha, caplonin.
People NP25 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 NP25 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the C end of inventor NP25 and the C end of rat NP25 are exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor NP25, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family, as the NP25 albumen of rat).
For example, inventor NP25 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people NP25 or the overexpression that suppresses people NP25.People NP25 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people NP25 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.
The expression of people NP25 in intestinal bacteria
The cDNA sequence of coding people NP25 is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, with human brain λ gt11cDNA library (available from Clontech company) for template increases, to synthesize the insertion fragment.
5 ' Oligonucleolide primers sequence is
5′-TTGCGTCGAC?ATGGCTAACA?GGGGCCCGA-3′(SEQ?ID?NO.5)，
This primer contains the restriction enzyme site of SalI restriction enzyme, and what connect is 19 Nucleotide of the people NP25 encoding sequence that begun by initiator codon;
3 ' Oligonucleolide primers sequence is
5’-GTTCAAGCTT?CTATTTCTTT?TCGTAGAGA-3’(SEQ?ID?NO.6)，
This primer contains the restriction enzyme site of HindIII restriction enzyme, the encoding sequence of translation termination and people NP25.
The restriction enzyme site of restriction enzyme is corresponding to bacterial expression vector pQE-9 (QiagenInc., 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 SalI and HindIII 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 the lacI repressor and carries kalamycin resistance (Kan
r).Screen transformant containing on the LB culture dish of Amp and Kan, 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).The extracting plasmid, sequence verification result shows that the cDNA of people NP25 inserts the fragment carrier of correctly packing into.
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 lacI 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 inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people NP25 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people NP25 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, 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 25KDa.
In addition, the amino acid of expressing 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 NP25 in eukaryotic cell (Chinese hamster ovary celI strain)
In this embodiment, the cDNA sequence of coding people NP25 is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, with human brain λ gt11cDNA library (available from Clontech company) for template increases, to synthesize the insertion fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TTGCAAGCTT?ATGGCTAACA?GGGGCCCGA-3′(SEQ?ID?NO.7)，
This primer contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 19 Nucleotide of the people NP25 encoding sequence that begun by initiator codon;
3 ' end primer sequence is:
5’-GTTCGAATTC?CTATTTCTTT?TCGTAGAGA-3’(SEQ?ID?NO.8)
This primer contains the encoding sequence of the restriction enzyme site of EcoRI restriction enzyme, translation termination and people NP25.
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 HindIII, EroRI 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, sequence verification result shows that the cDNA of people NP25 inserts the fragment carrier of correctly packing into.
Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (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 50mM TrisHCl (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 50mM TrisHCl (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 25KDa.
In addition, the amino acid of expressing 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 as above obtains is used for immune animal to produce antibody, specific 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 NP25 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: (i) applicant: Fudan University is denomination of invention (ii): a kind of new people's gene sequence, its encoded polypeptides and method for making and purposes be the sequence number (iii): 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: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:TGAGAAGCGG GAAGAAACGT CCG 23 (2) SEQ ID NO.2
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：2AGAAAGAGGA GAAGGTCAGA AGG 23 ( 2 ) SEQ ID NO.3： ( i ) ： ( A ) ：761bp ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3TGAGAAGCGG GAAGAAACGT CCGCCACCCA TCCCCCTTGC TGCCTGGGGG TTCAGACTTG 60ATTAGATGGC TAACAGGGGC CCGAGCTATG GCTTAAGCCG AGAGGTGCAG GAGAAGATCG 120AGCAGAAGTA TGATGCGGAC CTGGAGAACA AGCTGGTGGA CTGGATCATC CTGCAGTGCG 180CCGAGGACAT AGAGCACCCG CCCCCCGGCA GGGCCCATTT TCAGAAATGG TTAATGGACG 240GGACGGTCCT GTGCAAGCTG ATAAATAGTT TATACCCACC AGGACAAGAG CCCATACCCA 300AGATCTCAGA GTCAAAGATG GCTTTTAAGC AGATGGAGCA AATCTCCCAG TTCCTAAAAG 360CTGCGGAGAC CTATGGTGTC AGAACCACCG ACATCTTTCA GACGGTGGAT CTATGGGAAG 420GGAAGGACAT GGCAGCTGTG CAGAGGACCC TGATGGCTTT AGGCAGCGTT GCAGTCACCA 480AGGATGATGG CTGCTATCGG GGAGAGCCAT CCTGGTTTCA CAGGAAAGCC CAGCAGAATC 540GGAGAGGCTT TTCCGAGGAG CAGCTTCGCC AGGGACAGAA CGTAATAGGC CTGCAGATGG 600GCAGCAACAA GGGAGCCTCC CAGGCGGGCA TGACAGGGTA CGGGATCCCA GGCAGATCAT 660GTAGGACGGG GCACCTGCCC CTGGTAGAGA GGACGAATGT TCCACACCAT GGTCTCTACG 720AAAAGAAATA GTTAGTCACC TTCTGACCTT CTCCTCTTTC T 761
(2) information of SEQ ID NO.4:
(i) sequence signature:
(A) length: 221 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
( xi ) ：SEQ ID NO.4Met Ala Asn Arg Gly Pro Ser Tyr Gly Leu Ser Arg Glu Val Gln 15Glu Lys Ile Glu Gln Lys Tyr Asp Ala Asp Leu Glu Asn Lys Leu 30Val Asp Trp Ile Ile Leu Gln Cys Ala Glu Asp Ile Glu His Pro 45Pro Pro Gly Arg Ala His Phe Gln Lys Trp Leu Met Asp Gly Thr 60Val Leu Cys Lys Leu Ile Asn Ser Leu Tyr Pro Pro Gly Gln Glu 75Pro Ile Pro Lys Ile Ser Glu Ser Lys Met Ala Phe Lys Gln Met 90Glu Gln Ile Ser Gln Phe Leu Lys Ala Ala Glu Thr Tyr Gly Val 105Arg Thr Thr Asp Ile Phe Gln Thr Val Asp Leu Trp Glu Gly Lys 120Asp Met Ala Ala Val Gln Arg Thr Leu Met Ala Leu Gly Ser Val 135Ala Val Thr Lys Asp Asp Gly Cys Tyr Arg Gly Glu Pro Ser Trp 150Phe His Arg Lys Ala Gln Gln Asn Arg Arg Gly Phe Ser Glu Glu 165Gln Leu Arg Gln Gly Gln Asn Val Ile Gly Leu Gln Met Gly Ser 180Asn Lys Gly Ala Ser Gln Ala Gly Met Thr Gly Tyr Gly Ile Pro 195Gly Arg Ser Cys Arg Thr Gly His Leu Pro Leu Val Glu Arg Thr 210Asn Val Pro His His Gly Leu Tyr Glu Lys Lys 221
(2) information of SEQ ID NO.5
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.5:TTGCGTCGAC ATGGCTAACA GGGGCCCGA 29 (2) SEQ ID NO.6
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: information (i) sequence signature of SEQ ID NO.6:GTTCAAGCTT CTATTTCTTT TCGTAGAGA 29 (2) SEQ ID NO.7
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TTGCAAGCTT ATGGCTAACA GGGGCCCGA 29 (2) SEQ ID NO.8
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:GTTCGAATTC CTATTTCTTT TCGTAGAGA 29
Claims (14)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people NP25 protein-active,
Show at least 70% homology from the nucleotides sequence of Nucleotide 66-731 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 66-731 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 66-731 position.
4. isolating people NP25 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.
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. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.
9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.
10. a generation has the method for the polypeptide of people NP25 protein-active, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people NP25 protein-active operationally is connected in expression regulation sequence, form people NP25 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 66-731 position among described nucleotide sequence and the SEQIDNO.3;
(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people NP25;
(c) under the condition that is fit to expressing human NP25 protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people NP25 protein-active.
11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 66-731 position among the SEQ ID NO.3.
12. energy and the described people NP25 of claim 4 protein polypeptide specificity bonded antibody.
13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.
14. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the described dna molecular of claim 1.
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|---|---|---|---|
| CN98121528A CN1251860A (en) | 1998-10-20 | 1998-10-20 | Human gene sequence and its coded polypeptide, preparing process and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121528A CN1251860A (en) | 1998-10-20 | 1998-10-20 | Human gene sequence and its coded polypeptide, preparing process and application |
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| Publication Number | Publication Date |
|---|---|
| CN1251860A true CN1251860A (en) | 2000-05-03 |
Family
ID=5227146
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98121528A Pending CN1251860A (en) | 1998-10-20 | 1998-10-20 | Human gene sequence and its coded polypeptide, preparing process and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11979106B2 (en) * | 2019-01-30 | 2024-05-07 | Sdmo Industries S.A.S. | Apparatus and method for tracking the usage time of a generator set |
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1998
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11979106B2 (en) * | 2019-01-30 | 2024-05-07 | Sdmo Industries S.A.S. | Apparatus and method for tracking the usage time of a generator set |
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