CN1259572A - New human protein and its code sequence, prepn. method and use thereof - Google Patents
New human protein and its code sequence, prepn. method and use thereof Download PDFInfo
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Abstract
The present invention relates to a new member of latexin gene family. It provides CDNA coding sequence of said new latexin and polypeptide of said sequential code and method of utilizing recombination technology to produce said new human latexin. Present invention also provides the application of said new human latexin.
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 a newcomer of latexin family by deduction.
Latexin is as instrument at first with the PC3.1 monoclonal antibody, antigen (the Proc.Natl.Acad.Sci.USA of 29 kilodaltons of a wide expression of in rat, finding in brain neopallium side (but not back side) internal granular layer neurone, 1992,89,8879-8883).All do not have significant homology between it and any known protein matter, thereby it is considered to belong to the brand-new protein of a class.
Discover, the expression of latexin has significant space-time specificity, and particularly the specific expressed characteristic of its space-time in brain provides certain clue (NeuroReport, 1995 for the regulation and control factor of further in depth studying the brain development process and participate, 6 (2), 281-283).In addition, there is research also to find to have restraining effect to Carboxypeptidase A 1, A2 and mastocyte Carboxypeptidase A by the latexin that is separated in the rat brain, thereby latexin is called as and organizes carboxypeptidase inhibition (tissue carboxypeptidase inhibitor sometimes, TCI) (Proc.Natl.Acad.Sci.USA, 1995,92,12225-12229).
Up to now, found latexin albumen in rat and mouse, wherein the latexin gene of rat is by clone (GenBank Accession NO.g543406 and u40260).Wherein, clone's process of this gene is as follows in the rat: because there is the transcript of Carboxypeptidase A in many studies show that in the brain of rat and non-pancreatic tissue, but do not have the activity of Carboxypeptidase A, infer thus to have soluble endogenous Carboxypeptidase A inhibition.By a series of separation and purification means, from rat cerebral tissue, isolated this inhibition, and its part of polypeptide sequence has been checked order.Utilize known peptide sequence design degenerated primer, after steps such as amplification, hybridization, obtain required dna fragmentation, obtain rat TCI full length sequence through order-checking.In addition, the latexin gene of mouse be located on No. 3 karyomit(e) (Genomics, 1997,39,419-421).
In the people, found the polypeptide of a plurality of clones codings and mouse latexin albumen homology, but the report that relevant as yet up to now philtrum latexin full-length gene is cloned.
An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding human Latexin gene family, the new Human genome of the present invention is named as human Latexin.
Another object of the present invention provides a kind of new latexin protein family member, and this albumen is named as human Latexin protein.
A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new human Latexin.
The invention still further relates to the application of this human Latexin nucleotide sequence 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 active polypeptide of human Latexin protein, shows at least 70% homology from the nucleotides sequence of Nucleotide 28-696 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 28-696 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has SEQ ID NO.3.
In another aspect of this invention, provide a kind of isolating human Latexin protein polypeptide, 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.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, the method that provides a kind of generation to have the active polypeptide of human Latexin protein, this method comprises:
(a) nucleotide sequence that coding is had an active polypeptide of human Latexin protein operationally is connected in expression regulation sequence, form the human Latexin protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 28-696 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 reconstitution cell of human Latexin protein;
(c) under the condition that is fit to expressing human latexin protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate and have the active polypeptide of human Latexin protein.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 776 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 28-696 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, refer to encode has the nucleotide sequence of the active polypeptide of human Latexin protein to term " human Latexin protein (or polypeptide) encoding sequence ", as 28-696 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 28-696 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 28-696 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, preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 28-696 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 28-696 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.3 sequence of human Latexin 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 " human Latexin protein or polypeptide " refers to have the active SEQ IDNO.4 of human Latexin protein polypeptide of sequence.This term also comprises having and the variant form human Latexin 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 the active fragments and the reactive derivative of human Latexin protein.
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 human Latexin DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-human Latexin polypeptide to obtain.The present invention also provides other polypeptide, as comprises human Latexin polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of human Latexin polypeptide.Usually, this fragment have the human Latexin 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 human Latexin protein or polypeptide.The difference of these analogues and natural human latexin 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, " human Latexin 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.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | ????Val |
| Arg(R) | Lys；Gln；Asn | ????Lys |
| Asn(N) | Gln；His；Lys；Arg | ????Gln |
| Asp(D) | Glu | ????Glu |
| Cys(C) | Ser | ????Ser |
| Gln(Q) | Asn | ????Asn |
| Glu(E) | Asp | ????Asp |
| Gly(G) | Pro；Ala | ????Ala |
| His(H) | Asn；Gln；Lys；Arg | ????Arg |
| Ile(I) | Leu; Val; Met; Ala; Phe; 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 human Latexin polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of human Latexin 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 human Latexin nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the human Latexin of encoding.
The present invention also comprises the method that detects the human Latexin 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 human Latexin 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 human Latexin DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into human Latexin gene product or fragment.Preferably, refer to that those can combine with human Latexin 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 molecule of human Latexin protein, comprise that also those do not influence the antibody of human Latexin protein function.The present invention also comprise those can with modify or without the human Latexin 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 human Latexin gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human latexin 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 the human Latexin function and the antibody that does not influence the human Latexin function.Each antibody-like of the present invention can utilize the fragment or the functional zone of human Latexin 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 human Latexin 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).
Human Latexin 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 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, polynucleotide total length of the present invention is 776 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 106-252 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gtllcDNA library (available from Clontech company) is template, synthetic forward primer A1:5 '-AAGCCCTGAAGTCACTGCTCATC-3 ' and reverse primer B1:5 '-TGGCCTCATAAGCTAGATGC CAG-3 ' carry out PCR, obtain the purpose fragment of 776bp.Obtain the full length cDNA sequence of SEQ IDNO.3 after the order-checking.
According to homology result relatively, the latexin of nucleotide sequence of the present invention and encoded protein matter sequence and different sources has shown significant homology, therefore, this shows that it is a newcomer of latexin family, and has some functions of latexin family protein.
Latexin presents the temporal and spatial specificity in growth course, particularly in cerebral tissue, it only has expressed in pallium, thereby it is used as a kind of molecular marked compound of the specific region of discerning cerebral tissue.In addition, it also can be used as a kind of inhibition of Carboxypeptidase A.
In the accompanying drawings,
Fig. 1 is the homology comparison diagram of the nucleotide sequence of human Latexin of the present invention and mouse latexin (Mlatexin).Wherein, identical Nucleotide marks with " | ".
Fig. 2 is the homology comparison diagram of the aminoacid sequence of human Latexin of the present invention and mouse latexin (Mlatexin).Wherein, identical amino acid marks with " | " between two sequences.
Fig. 3 is the homology comparison diagram of the nucleotide sequence of human Latexin of the present invention and rat latexin (Rlatexin).Wherein, identical Nucleotide marks with " | ".
Fig. 4 is the homology comparison diagram of the aminoacid sequence of human Latexin of the present invention and rat latexin (Rlatexin).Wherein, identical amino acid marks with " | " between two sequences.
Fig. 5 is the homology comparison diagram of the aminoacid sequence of human Latexin of the present invention and mouse latexin (Mlatexin) and rat latexin (Rlatexin).Wherein, identical amino acid marks with " * " below three sequences.Similar amino acid marks with " ".
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 Spring HarborLaboratory 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 human Latexin
1. primer amplification
With human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-AAGCCCTGAAGTCACTGCTCATC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-TGGCCTCATAAGCTAGATGCCAG-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 780bp.
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 776bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 28-696 position Nucleotide.
Derive the aminoacid sequence of human Latexin according to the full length cDNA sequence that obtains, totally 222 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.
Homology relatively
Full length cDNA sequence and proteins encoded thereof with human Latexin of the present invention, in Non-redundant+GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST.The latexin that found that they and mouse and rat has shown higher homology.Use the PCGENE software analysis, it and mouse and rat have shown high homology on nucleic acid and protein level, are 82.7% (Fig. 1) with the identity of mouse on nucleic acid level wherein, with the identity of rat be 83.5% (Fig. 3); Identity with mouse on protein level is 84.7% (Fig. 2), and with the identity of rat be 84.2% (Fig. 4).In addition, on protein level, 13% the amino acid similarity (Fig. 5) of also having an appointment in addition.This shows, the latexin of latexin of the present invention and mouse and rat belongs to same family, and according to the structures shape function, the intimate Biological Principles of structural similitude can be inferred the character of latexin of the present invention from the character of mouse and rat latexin.
Many researchs find that all latexin expression in vivo has the space-time specificity.In rat, studies show that it only has expression in the neuronal cell of adult rats side pallium part.Utilize immunological method that latexin is grown early stage distribution situation in cerebral tissue at rat embryo and analyze, found that latexin comes across the diencephalon back side midline of fetal development the 11 day (E11) the earliest.In fetal development the 12nd day (E12), latexin then is expressed in the back side midline of diencephalon and midbrain simultaneously.In fetal development the 14th day (E14), its expression zone more is restricted in the time of the 12nd day than fetal development.The strongly expressed of latexin only limits to pineal gland, does not then express latexin with pineal gland opposing backside surface midline cell.Above true prompting, latexin plays a role in the formation of the different specific region of brain, and may be at forebrain, (Developmental Neuroscience works during particularly the form of its back side centerline construction (comprising pineal gland) takes place, 1995,6 (2), 281-283).The characteristic of this specifically expressing of latexin makes it might become a kind of molecule marker, for the mechanism of studying the formation of its different structure in the still unclear up to now brain development process provides research tool (NeuroscienceResearch, 1994,20,131-135).
Other researchs find that also the latexin that is separated to also has the inhibition activity to Carboxypeptidase A 1, A2, mastocyte Carboxypeptidase A from rat cerebral tissue, then not obvious to the inhibition ability of protaminase.As everyone knows, proteolytic enzyme has participated in the various kinds of cell process.The activity of protein kinase is subjected to strict regulation and control in vivo, and for example, proteolytic enzyme can exist with inactive precursor forms, only competence exertion function after being cut one section (or several sections) polypeptide to become sophisticated proteolytic enzyme; Or there is the inhibition of proteolytic enzyme in the body.The proteic discovery of the present invention, in the research body to the active mechanism of regulating of Carboxypeptidase A provide clue (Proc.Natl.Acad.Sci.USA, 1995,92,12225-12229).
Human Latexin of the present invention is used for also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig) the further functional study and the molecule that serves as a mark except can be used as this family's a member.In addition, inventor latexin can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor latexin and the N end of mouse latexin are exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor latexin, 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 latexin of rat).
For example, inventor latexin nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves human Latexin or the overexpression that suppresses human Latexin.Human Latexin protein of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of human Latexin 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 human Latexin in intestinal bacteria
The PCR Oligonucleolide primers of the cDNA sequence of coding human Latexin corresponding to 5 of this dna sequence dna ' and 3 ' end is that template increases with the pcr amplification product that obtains among the embodiment 1, with synthetic insertion fragment.
5 ' Oligonucleolide primers sequence is
5′-TTGCGGATCCATGGAAATCCCGCCGACC-3′(SEQ?ID?NO.5)，
This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 18 Nucleotide of the human Latexin encoding sequence that begun by initiator codon;
3 ' Oligonucleolide primers sequence is
5’-GTTCGTCGACTTATTCCAGTTGTACTCCT-3’(SEQ?ID?NO.6)，
This primer contains the restriction enzyme site of SalI restriction enzyme, the encoding sequence of translation termination and human Latexin.
The restriction enzyme site of restriction enzyme 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.
Digest the pQE-9 carrier, insert fragment with BamHI and SalI, 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 is cut with the HindIII enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the cDNA of human Latexin 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 human Latexin from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out human Latexin 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 26KDa.
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 human Latexin in eukaryotic cell (Chinese hamster ovary celI strain)
In this embodiment, the cDNA sequence of coding human Latexin is used PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end, the pcr amplification product that obtains among the embodiment 1 is that template increases, with the synthetic fragment of inserting.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TTGCGAGCTCATGGAAATCCCGCCGACC-3′(SEQ?ID?NO.7)，
This primer contains the restriction enzyme site of SacI restriction enzyme, and what connect is 18 Nucleotide of the human Latexin encoding sequence that begun by initiator codon;
3 ' end primer sequence is:
5’-GTTCGGATCCTTATTCCAGTTGTACTCCT-3’(SEQ?ID?NO.8)
This primer contains the encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and human Latexin.
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 SacI, BamHI digestion pcDNA3 carrier, 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 human Latexin 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 26KDa.
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.
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 human Latexin 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): new human protein and encoding sequence thereof reach (iii) sequence number of method for making and purposes: 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:AAGCCCTGAA GTCACTGCTC ATC 23 (2) SEQ ID NO.2
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2:TGGCCTCATA AGCTAGATGC CAG 23 (2) SEQ ID NO.3: (i) sequence signature:
(A) length: 776bp
(B) type: nucleic acid
(C) chain: strand
( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3AAGCCCTGAA GTCACTGCTC ATCCGGAATG GAAATCCCGC CGACCAACTA CCCAGCCTCC 60AGGGCGGCCT TGGTGGCACA GAACTACATC AACTACCAGC AGGGGACCCC GCACAGGGTG 120TTTGAGGTGC AGAAGGTCAA ACAAGCCAGC ATGGAGGATA TTCCAGGAAG AGGACATAAG 180TATCGCCTTA AATTTGCTGT TGAAGAAATT ATACAAAAAC AAGTTAAGGT GAACTGCACA 240GCTGAAGTAC TTTACCCTTC AACGGGACAA GAAACTGCAC CAGAAGTCAA CTTCACATTT 300GAAGGAGAAA CTGGAAAGAA TCCAGATGAA GAAGACAACA CATTTTATCA AAGACTTAAG 360TCCATGAAGG AACCGCTAGA AGCACAAAAT ATTCCAGACA ATTTTGGAAA TGTATCTCCA 420GAAATGACGC TCGTTCTACA TTTAGCCTGG GTTGCCTGTG GTTATATAAT ATGGCAAAAT 480TCTACTGAAG ACACATGGTA TAAAATGGTA AAAATTCAAA CTGTCAAGCA AGTGCAAAGA 540AATGATGACT TTATTGAATT AGACTACACC ATTCTACTTC ATAATATAGC ATCTCAGGAG 600ATTATTCCCT GGCAAATGCA AGTTCTCTGG CATCCACAAT ACGGCACTAA AGTAAAACAT 660AATAGCCGTC TGCCAAAGGA AGTACAACTG GAATAAACAA AAACCCTAAC ACTGGAAGTG 720TAAACATGTC TATTGATGTG TATGCCAATT TCACTGGCAT CTAGCTTATG AGGCCA 776 ( 2 ) SEQ ID NO.4： ( i ) ：
(A) length: 222 amino acid
(B) type: amino acid
( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4Met Glu Ile Pro Pro Thr Asn Tyr Pro Ala Ser Arg Ala Ala Leu 15Val Ala Gln Asn Tyr Ile Asn Tyr Gln Gln Gly Thr Pro His Arg 30Val Phe Glu Val Gln Lys Val Lys Gln Ala Ser Met Glu Asp Ile 45Pro Gly Arg Gly His Lys Tyr Arg Leu Lys Phe Ala Val Glu Glu 60Ile Ile Gln Lys Gln Val Lys Val Asn Cys Thr Ala Glu Val Leu 75Tyr Pro Ser Thr Gly Gln Glu Thr Ala Pro Glu Val Asn Phe Thr 90Phe Glu Gly Glu Thr Gly Lys Asn Pro Asp Glu Glu Asp Asn Thr 105Phe Tyr Gln Arg Leu Lys Ser Met Lys Glu Pro Leu Glu Ala Gln 120Asn Ile Pro Asp Asn Phe Gly Ash Val Ser Pro Glu Met Thr Leu 135Val Leu His Leu Ala Trp Val Ala Cys Gly Tyr Ile Ile Trp Gln 150Asn Ser Thr Glu Asp Thr Trp Tyr Lys Met Val Lys Ile Gln Thr 165Val Lys Gln Val Gln Arg Asn Asp Asp Phe Ile Glu Leu Asp Tyr 180Thr Ile Leu Leu His Asn Ile Ala Ser Gln Glu Ile Ile Pro Trp 195Gln Met Gln Val Leu Trp His Pro Gln Tyr Gly Thr Lys Val Lys 210His Asn Ser Arg Leu Pro Lys Glu Val Gln Leu Glu 222 ( 2 ) SEQ ID NO.5 ( i )
(A) length: 28 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.5:TTGCGGATCC ATGGAAATCC CGCCGACC 28 (2) SEQ ID NO.6
(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.6:GTTCGTCGAC TTATTCCAGT TGTACTCCT 29 (2) SEQ ID NO.7
(A) length: 28 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:TTGCGAGCTC ATGGAAATCC CGCCGACC 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:GTTCGGATCC TTATTCCAGT TGTACTCCT 29
Claims (14)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the active polypeptide of human Latexin protein,
Show at least 70% homology from the nucleotides sequence of Nucleotide 28-696 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 28-696 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 28-696 position.
4. isolating human Latexin 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 active polypeptide of human Latexin protein, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had an active polypeptide of human Latexin protein operationally is connected in expression regulation sequence, form the human Latexin protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 28-696 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 reconstitution cell of human Latexin protein;
(c) under the condition that is fit to expressing human latexin protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate and have the active polypeptide of human Latexin protein.
11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 28-696 position among the SEQ ID NO.3.
12. energy and the described human Latexin protein polypeptid specificity of claim 4 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98123797A CN1259572A (en) | 1998-11-02 | 1998-11-02 | New human protein and its code sequence, prepn. method and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98123797A CN1259572A (en) | 1998-11-02 | 1998-11-02 | New human protein and its code sequence, prepn. method and use thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1259572A true CN1259572A (en) | 2000-07-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98123797A Pending CN1259572A (en) | 1998-11-02 | 1998-11-02 | New human protein and its code sequence, prepn. method and use thereof |
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| Country | Link |
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| CN (1) | CN1259572A (en) |
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1998
- 1998-11-02 CN CN98123797A patent/CN1259572A/en active Pending
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