CN1724664A - Human HOLF 402 protein and its coding sequence - Google Patents

Abstract

The invention supplies human HOLF402 albumen and code sequence expressing in plural organize of normal liver, the method to make the albumen and the code sequence, and the method to test human HOLF402 nucleic acid sequence and the polypeptide in sample.

Description

Human Olfactomedin-like protein 402 and encoding sequence thereof

Technical field

The present invention relates to molecular biology, genetic engineering, oncology, cytobiology field.Particularly, the present invention relates to a kind of Human Olfactomedin-like protein 402 (HOLF402) albumen and nucleotide sequences thereof of in multiple tissues such as human liver, expressing.The invention still further relates to the preparation method and the purposes of this albumen and nucleotide sequence.

Background technology

Before the present invention comes forth, any human HOLF402 albumen that discloses or reported is not arranged as yet.

That glaucoma has become at present is main, one of irreversible blinding illness in eye.In the non-selective crowd of China, the sickness rate of primary glaucoma is 0.52%, and the age surpasses among 40 years old the crowd, and the sickness rate of primary glaucoma is up to 1.2%.And the concealment of the pathogeny complexity of primary open angle glaucoma, onset, early stage subjective symptoms are few, have caused more serious visual function infringement just to obtain diagnosing often; On the other hand, because steroid hormone is widespread use and the abuse of glucocorticosteroid in ophthalmology and general disease especially, the sickness rate of corticosteroid glaucoma rises rapidly, and great majority see adolescents in male, its clinical manifestation is very similar to primary open angle glaucoma with the pathologic, physiologic feature, also because primary open angle glaucoma is extremely sensitive intraocular pressure rising reaction to glucocorticosteroid, the relation between the two comes into one's own simultaneously.Because glaucomatous blindness is irreversible, the burden on society that causes financial loss and increase therefrom is quite huge.Therefore, to primary open angle glaucoma and the pathogenetic research of corticosteroid glaucoma, become the important and urgent problem of field of ophthalmology.

Sense of smell mediation element is a kind of main component of the extracellular matrix of olifactory nerve epithelium.Sense of smell mediates plain structural domain and finds in sense of smell mediation element at first.The main member of this ectodomain family is many to be found in metazoan albumen, as latrophilins, and myocilins, and noelins.But they biological function still under study for action.The sudden change that sense of smell mediates plain structural domain among the myocilin causes primary open angle glaucoma more.Experiment in vitro has proved that also optimedin of rat and myocilin albumen mediate plain domain interaction by sense of smell.

Widespread use and development along with Protocols in Molecular Biology, the gene most possible relevant with the morbidity of primary open angle glaucoma and corticosteroid glaucoma is found, i.e. TIGR (Trabecular meshwork induced glucocurticoidresponse protein) gene.The TIGR gene has plain (OLFM) sample of sense of smell mediation district at C-terminal, mediate plain 40% homology with sense of smell, sense of smell mediation element is a kind of main component of the extracellular matrix of olifactory nerve epithelium, and all TIGR transgenations of having reported are contained in this zone, point out it relevant in conjunction with activity with cell.So, infer the TIGR gene this position sudden change can impede protein absorption and metabolism, cause proteic accumulation to stop aqueous humor to flow out and intraocular pressure raise, this unusual intraocular pressure is regulated the visual function infringement that will cause irreversible glaucoma at last.

So, infer that holf402 albumen may also have certain getting in touch with the especially glaucomatous pathogeny of illness in eye.

Summary of the invention

First purpose of the present invention just provides a kind of new people's gene HOLF402, and this gene is a people HOLF402 protein gene.

Second purpose of the present invention provides a kind of new people's albumen people HOLF402.

The 3rd purpose of the present invention provides a kind of recombinant technology that utilizes and produces the above-mentioned new Human Olfactomedin-like protein 402 and the method for nucleotide sequence.

The present invention also provides the application of this Human Olfactomedin-like protein 402's polypeptide and encoding sequence.

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the active polypeptide of Human Olfactomedin-like protein 402's matter, among the promptly described nucleotide sequence SEQ ID NO.1 from the nucleotide sequence of Nucleotide 395-1603 position dna molecular.

In another aspect of this invention, provide a kind of isolated Human Olfactomedin-like protein 402's matter polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ IDNO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

In another aspect of this invention, also provide a kind of carrier, it comprises above-mentioned dna molecular.

In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is intestinal bacteria in an example; In another example, this host cell is an eukaryotic cell.

In another aspect of this invention, the method that also provides a kind of generation to have the active polypeptide of Human Olfactomedin-like protein 402's matter, this method comprises:

(1) nucleotide sequence that coding is had an active polypeptide of Human Olfactomedin-like protein 402 operationally is connected in expression regulation sequence, forms Human Olfactomedin-like protein 402's expression vector;

(2) change the expression vector in the step (1) over to host cell, form Human Olfactomedin-like protein 402's reconstitution cell;

(3) under the condition that is fit to expressing human HOLF402 protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate and have the active polypeptide of Human Olfactomedin-like protein 402.

The nucleotide sequence of Shi Yonging has the sequence of 395-1603 position among the SEQ ID NO.1 in the method.

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 Olfactomedin-like protein 402 to term " Human Olfactomedin-like protein 402's (or polypeptide) encoding sequence ", as 395-1603 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 395-1603 position Nucleotide of SEQ ID NO.1 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 IDNO.1 in 395-1603 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 395-1603 position.This term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 395-1603 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.1 with natural people HOLF402 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 Olfactomedin-like protein 402 or polypeptide " refers to have the active SEQ ID of Human Olfactomedin-like protein 402 NO.2 polypeptide of sequence.This term also comprises having and variant form natural human HOLF402 identical function, SEQ ID NO.2 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 Human Olfactomedin-like protein 402's active fragments and reactive derivative.

The variant form of people HOLF402 polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of people HOLF402DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people HOLF402 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people HOLF402 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of people HOLF402 polypeptide.Usually, this fragment have people HOLF402 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.

In the present invention, " people HOLF402 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.2, 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 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	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Invention also comprises the analogue of Human Olfactomedin-like protein 402 or polypeptide.The difference of these analogues and natural human HOLF402 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing people HOLF402 polypeptide of the present invention, people HOLF402 encoding sequence operationally can be connected in expression regulation sequence, thereby form Human Olfactomedin-like protein 402's 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 adjacent, 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.

The present invention also provides the antibody special to people HOLF402, comprises polyclonal antibody and monoclonal antibody.

In the present invention, can use a series of methods known in the art to prepare special antibody at people HOLF402.For example, the people HOLF402 gene product or its antigen fragment of purifying is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human HOLF402 or its antigen fragment also can be used for animal is caused immunity and produces antibody.Antibody prepared in accordance with the present invention also can be monoclonal antibody, and these monoclonal antibodies can prepare (for example, Kohler et al., Nature 256:495,1975 with hybridoma technology; Kohler et al., Eur.J.Immunol.6:511,1976; Kohler et al., Eur.J.Immunol.6:292,1976).Antibody of the present invention comprises the antibody that can prevent people HOLF402 function, also can be the antibody that does not influence people HOLF402 function.Each antibody-like can produce by the fragment of people HOLF402 gene product or functional domain are caused immunity, and people HOLF402 gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the people HOLF402 gene product bonded antibody of non-modified forms, can come immune animal to obtain by being used in the gene product that prokaryotic cell prokaryocyte for example produces among the E.coli.With posttranslational modification form such as glycosylation or phosphorylated protein or polypeptide bonded antibody, can obtain by the immune animal that comes that is used in the gene product that produces in eukaryotic cell such as yeast or the insect cell.

People HOLF402 antibody of the present invention can be used for identifying the cell of expressing Human Olfactomedin-like protein 402 or polypeptide, as Jurkat T cell.For example, can with a kind of detectable molecule for example fluorescein isothiocyanic acid (FITC) come labelling human HOLF402 specific antibody, allow people HOLF402 specific antibody contact then, detect and people HOLF402 specific antibody bonded cell with fluorescent microscope or flow cytometer again with cell sample.

Except cell surface detects people HOLF402, can also analyze this protein with the Western engram technology.Cell pyrolysis liquid can from culturing cell or take from patient's tissue sample such as liver extract, and be dissolved in the lysis buffer that contains stain remover.Use sds polyacrylamide gel electrophoresis isolated cell extract (simultaneously with the people HOLF402 polypeptide of purifying as positive control) then, then it is transferred on the nitrocellulose by electrophoresis hybridization.In order to survey people HOLF402 polypeptide, can use typical antibodies detection method, for example radioautograph or alkaline phosphatase enzyme assay method with the immunity of Western trace.And can use the contrast of immunization serum or incoherent monoclonal antibody as non-specific responding.

Whether and quantity the expression of also available Nothern blotting technical Analysis people HOLF402 gene product, the i.e. existence of rna transcription thing in cell of analyst HOLF402.

The Western engram analysis of the Nothern engram analysis of people HOLF402DNA and people HOLF402 specific antibody can be united use, with the expression of confirmer HOLF402 in biological specimen.People HOLF402DNA can also be used for Southern engram analysis or in situ hybridization analysis, with this assignment of genes gene mapping on karyomit(e), and can carry out genetic linkage analysis to find out other possible disease related gene.

In addition, the present invention also provides a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of people HOLF402 nucleotide coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people HOLF402.

The present invention also provides the method that whether has people HOLF402 nucleotide sequence in the test sample, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to people HOLF402 nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

In addition, according to people HOLF402 nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening people's HOLF402 homologous gene or homologous protein.

In order to obtain and the people cDNAs of people HOLF402 gene-correlation or the dot matrix of genomic dna s, can screen people cDNA or genome dna library with dna probe, these probes are under low stringent condition, with 32P people HOLF402 all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from human liver organization.Also can be used for screening purpose from the cDNA library that participates in endocrine other tissue or specific human body cell strain.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Palo Alto, Cal..This screening method can be discerned the nucleotide sequence of the gene family relevant with people HOLF402.

Can finish as follows according to Nucleotide similarity screening people HOLF402 homologue.Human liver cDNA library, for example (Clontech, Palo Alto Cal.) can use one section all or part of random primer dna probe screening that comprises people HOLF402 gene order to Clontech Cat.#7429-1.Finish having clone's the evaluation of the DNA insertion sequence of 70% homology at least with people HOLF402 sequence, can use hybridization temperature is 55 ℃ hybridization solution, uses 0.5 * SSC and 0.1%SDS to clean then.Shi Bie clone's DNA insertion sequence can be further estimated the similarity of it and people HOLF402 gene with DNA restriction endonuclease analysis and dna sequencing in this way.The distribution of tissue expression can be with above-mentioned Northern blotting technical Analysis.

People HOLF402 homologue also can be used at the antibody of Human Olfactomedin-like protein 402 or polypeptide and discern.For example, can be with the method for standard to commercial or make up with currently known methods, from cell or for example organize that the expression library of liver screens.Pour the library into plate, on colony lift to a nitrocellulose membrane, the recombinant protein of expression is attached on the film.Just can carry out typical antibodies and detection then with specific people HOLF402 antibody.Identify the DNA insertion sequence among the clone in this way, can be further analyze to estimate the similarity of it and people HOLF402 gene with DNA restriction endonuclease analysis and dna sequencing.The tissue expression of the gene of new identification distributes and can similarly analyze as stated above.

People HOLF402 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book contriver HOLF402.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

The proteic encoding sequence of the present invention can be used for the assignment of genes gene mapping.For example,, the cDNA clone is hybridized with the karyomit(e) of metaphase, can carry out chromosomal localization exactly by fluorescence in situ hybridization technique (FISH).This technology can be used the cDNA that is as short as about 500bp; Also can use and grow to about 2000bp or longer cDNA.For this technology, can be referring to people such as Verma, Human Chromosomes:A Manual of Basic Techniques, Pergamon Press, New York (1988).

In case sequence is located in certain exact position on the karyomit(e), the physical location of sequence on karyomit(e) can be associated with the genetic map data.These genetic map data can obtain, for example by Mendelian (Mendelian) people genetic database (can obtain on the net by Johns Hopkins University Welch Medical Library).Then, come identified gene by linkage analysis and be positioned dependency between the disease of same chromosomal region.

Then, be necessary to determine the cDNA between diseased individuals and the healthy individual or the difference of genome sequence aspect.Be not present in normal individual if a certain sudden change is present in part or all of diseased individuals, this sudden change may be exactly the paathogenic factor of this disease so.

Utilize Human Olfactomedin-like protein 402 of the present invention, by various conventional screening methods, can filter out with people HOLF402 take place interactional material or, as acceptor, inhibitor or antagonist etc.

Human Olfactomedin-like protein 402 of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is generally about 5-8, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

With Human Olfactomedin-like protein 402 of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Human Olfactomedin-like protein 402 of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When Human Olfactomedin-like protein 402's polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Find that by the homology retrieval new gene of the present invention has and delivers and be confirmed to be the proteic gene height of house mouse HOLF402 homologous sequence, and new albumen of the present invention has the aminoacid sequence of house mouse HOLF402 albumen high conservative.So people HOLF402 of the present invention is a homologous gene of house mouse HOLF402 protein gene and have similar function.

Description of drawings

Fig. 1 is that the homology of people HOLF402 of the present invention and house mouse HOLF402 gene nucleic acid sequence (GenBank Accession No.AK078505) compares (FASTA) figure.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is that the homology of Human Olfactomedin-like protein 402 of the present invention and the proteic aminoacid sequence of house mouse HOLF402 (SwissProtAccession No.BAC37312.1) compares (FASTA) figure.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

Embodiment

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 Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of people HOLF402 gene

1. the full-length clone of gene (Cloning of Full-length cDNA)

On the new gene fragment order information basis that obtains, carry out the cDNA full-length clone, carry out in two stages:

(1) " electronic cloning " (Electronic Cloning)

Search the dbEST database with new gene fragment order as probe, with overlap＞50bp, homology is at (the Expressed Sequence Tag of the expressed sequence tag more than 98%, being called for short " EST ") sequence thinks same sequence (consensussequence), take out and splice with AUTOASSEMBLER software, part EST can the extension probes sequence.Whether the sequence that is extended with the STRIDER software analysis has complete open reading frame (Open Reading Frame again, ORF), on Nucleotide and amino acid levels, whether homology is arranged with definite this sequence with BLAST search Genbank or SwissProt, to help how differentiate resulting full length gene integrity with other species.By the method for electronic cloning, can obtain the full length sequence of people HOLF402 gene usually.

(2) the terminal rapid amplifying of cDNA (Rapid Amplification of cDNA Ends, RACE)

If do not obtain complete cDNA total length yet by " electronic cloning " method, then at 5 ' or 3 ' end design primer of existing sequence, (Clontech Lab, Inc carry out the long range PCR reaction in USA) in human liver Marathon-Ready cDNA library.Then to PCR product cloning, order-checking.The sequence that is extended with AUTOASSEMBLER and STRIDER software analysis has or not complete ORF, as not having, repeats said process until obtaining total length.

(3)RT-PCR

For 5 ' and 3 ' end known sequences,, can consider to adopt the method for RT-PCR if the centre still has an intersegmental crack (gap) to obtain from existing public database or its data storehouse.At sequence 5 ' end design primer, 3 ' end primer adopts Oligo-dT, increases in the liver total RNA storehouse.Then product is cloned, checked order.Splice at last and obtain total length.

By being used in combination above-mentioned 3 kinds of methods, obtained candidate's Human Olfactomedin-like protein 402's complete encoding sequence.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further R1:5 '-CCGGCTAGCAGAAGAGCCACAGCATAAG-3 ' (SEQ ID NO.3) is a forward primer to the design primer, oligonucleotide R2:5 '-GCCGGTACCCTTCAGAGGCAGCTTTCTC-3 ' (SEQ ID NO.4) is a reverse primer, mixing cDNA (spleen with 9 kinds of tissues of people, heart, brain, thymus, small intestine, testis, lung, kidney liver) is template, carry out pcr amplification, the PCR condition of R1/R2 be 94 ℃ 4 minutes, thereupon with 94 ℃ 40 seconds, 58 ℃ of 30 seconds and 72 ℃ carried out 35 circulations in 1.5 minutes, extended 7 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1252bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.1.

Embodiment 2

The sequence information and the homology analysis of people HOLF402 gene:

The length of the people HOLF402 full-length cDNA that the present invention is new is 2320bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 395-1603 position Nucleotide.Derive the aminoacid sequence of people HOLF402 according to full-length cDNA, totally 402 amino-acid residues, molecular weight 45950.89, pI are 8.29.Detailed sequence is seen SEQ ID NO.2.

The full length cDNA sequence of people HOLF402 and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that there is certain homology in the gene of it and house mouse.On nucleotide level, the 1152-1373 bit base of the mRNA whole coding sequence (GenBank Accession No.NM_172907) of it and house mouse unnamed protein gene has 83% homogeny, the 186-238 bit base has 78% homogeny (Fig. 1), on amino acid levels, the 1-233 amino acids residue of it and house mouse HOLF402 albumen (SwissProtAccession No.BC047207.1) has 80% homogeny and 85% similarity (Fig. 2).Therefore all there are higher homology in people HOLF402 gene and house mouse HOLF402 gene on nucleic acid still is protein level, belong to same family and both also have very high similarity on function.

People HOLF402 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, people HOLF402 of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end with Human Olfactomedin-like protein 402 of the present invention exchanges with the proteic N end of house mouse HOLF402, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor HOLF402, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor HOLF402 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people HOLF402 or the overexpression that suppresses people HOLF402.Human Olfactomedin-like protein 402 of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people HOLF402 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.

Because Human Olfactomedin-like protein 402 of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species (as house mouse), estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

Embodiment 3

Human Olfactomedin-like protein 402's 26S Proteasome Structure and Function research:

With Human Olfactomedin-like protein 402's aminoacid sequence the PROSITE database (network address is: retrieval motif (motif) http://expasy.hcuge.ch/sprot/scnpsitl.btml) obtains following result:

1 MMVALRGASA LLVLFLAAFL PPPQCTQDPA MVHYIYQRFR VLEQGLEKCT

51 QATRAYIQEF QEFSKNISVM LGRCQTYTSE YKSAVGNLAL RVERAQREID

101 YIQYLREADE CIESEDKTLA EMLLQEAEEE KKIRTLLNAS CDNMLMGIKS

151 LKIVKKMMDT HGSWMKDAVY NSPKVYLLIG SRNNTVWEFA NIRAFMEDNT

201 KPAPRKQILT LSWQGTGQVI YKGFLFFHNQ ATSNEIIKYN LQKRTVEDRM

251 LLPGGVGRAL VYQHSPSTYI DLAVDEHGLW AIHSGPGTHS HLVLTKIEPG

301 TLGVEHSWDT PCRSQDAEAS FLLCGVLYVV YSTGGQGPHR ITCIYDPLGT

351 ISEEDLPNLF FPKRPRSHSM IHYNPRDKQL YAWNEGNQII YKLQTKRKLP LK

(1) in aminoacid sequence, there is following function motif:

Black matrix district (162-397): Olfactomedin-like domain

(2) functional analysis:

Olfactomedin-like domain belongs to olfactomedin family, and at multiple protein such as olfactomedin, myocilin exists among pancortin and the latrophilin.

Embodiment 4

The preparation and the purification of people HOLF402 polypeptide

In this embodiment, the people HOLF402 encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.

1. people HOLF402 polypeptide is carried out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.

Construction of prokaryotic expression vector, and transformed into escherichia coli

Complete encoding sequence (SEQ ID NO.1) according to people HOLF402, design amplifies complete coding and reads the primer of frame (corresponding respectively to about 20 above Nucleotide of encoding sequence 5 ' and 3 ' end), and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pGEX-4T carrier of selecting for use) respectively, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with people HOLF402 gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-4T-1 carrier (Pharmacia, Piscataway, NJ).Identify that good expression vector utilizes CaCl ₂Method changes bacillus coli DH 5 alpha over to, and Screening and Identification obtains containing the engineering bacteria DH5 α-pGEX-4T-1-HOLF402 of pGEX-4T-1-HOLF402 expression vector.

Express the isolation identification of the engineering bacteria of GST-HOLF402 recombinant protein

DH5 α-pGEX-4T-1-HOLF402 the engineering bacteria of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draw nutrient solution by 1: 100 concentration and in new YT substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD ₆₀₀After reaching 0.5, adding IPTG continues at 30 ℃ to final concentration 0.2mol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 15%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-HOLF402 fusion rotein.

The extraction purifying of GST-HOLF402 fusion rotein

The proteic engineering bacteria DH5 of abduction delivering GST-HOLF402 amalgamation and expression α-pGEX-4T-HOLF402 as stated above.Bacterium centrifugation after inducing adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid centrifuging and taking of broken bacterium supernatant adds 50% saturated Triptide Sepharose 4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines GST-HOLF402 in centrifugal 10 minutes, abandoned supernatant.The amount that adds 10ml PBS by every milliliter of ultrasonic liquid cleans twice, then adds 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid, 4 ℃ of wash-outs that spend the night, and centrifugal 10 minutes of 10000rpm, supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 40kDa place is the Human Olfactomedin-like protein 402.

Embodiment 5

Human Olfactomedin-like protein 402 or polypeptide carry out eucaryon transfection proof secretion property in Chinese hamster ovary celI

1. the structure of people HOLF402 rhabdovirus expression vector and transfection cell strain

According to the complete encoding sequence (SEQ ID NO.1) of people HOLF402, design amplifies the primer that complete coding is read frame, and introduces restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively on positive anti-primer, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with people HOLF402cDNA under the prerequisite that guarantees reading frame, be cloned into the pcDNA3.1A carrier (Invitrogen, Carlsbad, CA).Identify good expression vector 2 μ g, pcDNA3.1A DNA (BaculoGold ^TMACMNPV DNA, Pharmingen, San Diego, CA) 1 μ g and Lipofection (Gibco-BRL, NY) 25 μ l add in the DMEM substratum of 1ml serum-free, the 15 seconds mixings that vibrate, incubated at room 15 minutes is standby.Get 1ml (2 * 10 ⁶) the Chinese hamster ovary celI suspension is in 60mm tissue culturing plate, change transfection media after adherent 1 hour, incubated at room was abandoned substratum after 15 minutes, add the dna vector transfection mixture for preparing previously, Parafilm seals culture plate, cultivated 4 hours in 27 ℃ of joltings of room temperature, then change perfect medium and cultivated 3 days, cleer and peaceful cell pyrolysis liquid is standby in the collection.

2. change the Screening and Identification of the Chinese hamster ovary celI strain of recombinant expression vector over to

The Chinese hamster ovary celI of transfection after 3 days carries out Western to be identified.The SDS-PAGE electrophoresis will be carried out after the lysis, glue behind the electrophoresis prints to protein transduction on the nitrocellulose membrane in the half-dried electrotransfer instrument of the Multiphor of Pharmacia II, nitrocellulose membrane is placed confining liquid sealing 2 hours, then in the c-myc antibody-solutions, sealed 1 hour, the jolting of PBST liquid is cleaned 10 minutes 3 times totally, then film is placed fluorescently-labeled D﹠amp; Jolting is 1 hour in the R second antibody solution, and PBST cleans, and adds fluorescent color-developing agent reaction 1 minute, develops and observes protein band.

Find that this albumen all has existence in last cleer and peaceful cell pyrolysis liquid.

Embodiment 6

The preparation of anti-people HOLF402 antibody

1. the preparation of immune mouse and splenocyte: separate the Human Olfactomedin-like protein 402 of above-mentioned acquisition back standby with chromatography, also can separate with the SDS-PAGE gel electrophoresis, electrophoretic band is cut off from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Get the female mouse of 6-8 week Balb/C in age, the albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, with the same antigen of non-complete Freund ' s adjuvant emulsion to mouse with the dosage of 50-100 μ g/0.2ml again booster immunization once be used for after 3-5 days merging.Wherein, E Zheng chief editor, " tissue culture and molecular cell learn a skill ", Beijing Publishing House, the 210th page are seen in the splenocyte preparation.

2. by " tissue culture and molecular cell learn a skill " (the same), the method in the 371st page, preparation feeder cell.

3. by " tissue culture and molecular cell learn a skill " (the same), the method in the 213rd page is carried out cytogamy.

4. detection of antibodies: after cytogamy 10-15 days, need to check by the hole, in case find vigorous hybrid cell colony growth, just use the preliminary screening that the Human Olfactomedin-like protein 402 does antibody activity, method commonly used has: immunofluorescent test, emission immunity test (RIA), enzyme linked immunosorbent assay (ELISA).After checking out the hole of antibody activity, clone cultivation at once, and isolate antibody.

Sequence table

＜110〉Research Center of Shanghai Human Genome

＜120〉Human Olfactomedin-like protein 402 and encoding sequence thereof

<130>NP-1375

<160>4

<170>PatentIn version 3.2

<210>1

<211>2320

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(395)..(1603)

<400>1

gggggaaggg aacgggggga agccatcttc accccccacc ccaatgcaca cacaattaag 60

ccaggaagca gcttgcaacc actagcctgg ggagggtccg catgtgtcaa gggtgagggc 120

aacagatgct ggacccaggg agctctctgc cacaggtcag tctacaaggc ctcagggacc 180

aacttgccaa cagctggact tgatcactag ctggcaaact gagctcacgt atcgggtgga 240

ataacaagcg gactttgctc tctgctgtgc aaaacgctgt ttttagagga tttgccacag 300

cagcggatag agcaggagag caccaccgga gcccttgaga catccttgag aagagccaca 360

gcataagaga ctgccctgct tggtgttttg cagg atg atg gtg gcc ctt cga gga 415

Met Met Val Ala Leu Arg Gly

1 5

gct tct gca ttg ctg gtt ctg ttc ctt gca gct ttt ctg ccc ccg ccg 463

Ala Ser Ala Leu Leu Val Leu Phe Leu Ala Ala Phe Leu Pro Pro Pro

10 15 20

cag tgt acc cag gac cca gcc atg gtg cat tac atc tac cag cgc ttt 511

Gln Cys Thr Gln Asp Pro Ala Met Val His Tyr Ile Tyr Gln Arg Phe

25 30 35

cga gtc ttg gag caa ggg ctg gaa aaa tgt acc caa gca acg agg gca 559

Arg Val Leu Glu Gln Gly Leu Glu Lys Cys Thr Gln Ala Thr Arg Ala

40 45 50 55

tac att caa gaa ttc caa gag ttc tca aaa aat ata tct gtc atg ctg 607

Tyr Ile Gln Glu Phe Gln Glu Phe Ser Lys Asn Ile Ser Val Met Leu

60 65 70

gga aga tgt cag acc tac aca agt gag tac aag agt gca gtg ggt aac 655

Gly Arg Cys Gln Thr Tyr Thr Ser Glu Tyr Lys Ser Ala Val Gly Asn

75 80 85

ttg gca ctg aga gtt gaa cgt gcc caa cgg gag att gac tac ata caa 703

Leu Ala Leu Arg Val Glu Arg Ala Gln Arg Glu Ile Asp Tyr Ile Gln

90 95 100

tac ctt cga gag gct gac gag tgc atc gaa tca gag gac aag aca ctg 751

Tyr Leu Arg Glu Ala Asp Glu Cys Ile Glu Ser Glu Asp Lys Thr Leu

105 110 115

gca gaa atg ttg ctc caa gaa gct gaa gaa gag aaa aag atc cgg act 799

Ala Glu Met Leu Leu Gln Glu Ala Glu Glu Glu Lys Lys Ile Arg Thr

120 125 130 135

ctg ctg aat gca agc tgt gac aac atg ctg atg ggc ata aag tct ttg 847

Leu Leu Asn Ala Ser Cys Asp Asn Met Leu Met Gly Ile Lys Ser Leu

140 145 150

aaa ata gtg aag aag atg atg gac aca cat ggc tct tgg atg aaa gat 895

Lys Ile Val Lys Lys Met Met Asp Thr His Gly Ser Trp Met Lys Asp

155 160 165

gct gtc tat aac tct cca aag gtg tac tta tta att gga tcc aga aac 943

Ala Val Tyr Asn Ser Pro Lys Val Tyr Leu Leu Ile Gly Ser Arg Asn

170 175 180

aac act gtt tgg gaa ttt gca aac ata cgg gca ttc atg gag gat aac 991

Asn Thr Val Trp Glu Phe Ala Asn Ile Arg Ala Phe Met Glu Asp Asn

185 190 195

acc aag cca gct ccc cgg aag caa atc cta aca ctt tcc tgg cag gga 1039

Thr Lys Pro Ala Pro Arg Lys Gln Ile Leu Thr Leu Ser Trp Gln Gly

200 205 210 215

aca ggc caa gtg atc tac aaa ggt ttt cta ttt ttt cat aac caa gca 1087

Thr Gly Gln Val Ile Tyr Lys Gly Phe Leu Phe Phe His Asn Gln Ala

220 225 230

act tct aat gag ata atc aaa tat aac ctg cag aag agg act gtg gaa 1135

Thr Ser Asn Glu Ile Ile Lys Tyr Asn Leu Gln Lys Arg Thr Val Glu

235 240 245

gat cga atg ctg ctc cca gga ggg gta ggc cga gca ttg gtt tac cag 1l83

Asp Arg Met Leu Leu Pro Gly Gly Val Gly Arg Ala Leu Val Tyr Gln

250 255 260

cac tcc ccc tca act tac att gac ctg gct gtg gat gag cat ggg ctc 1231

His Ser Pro Ser Thr Tyr Ile Asp Leu Ala Val Asp Glu His Gly Leu

265 270 275

tgg gcc atc cac tct ggg cca ggc acc cat agc cat ttg gtt ctc aca 1279

Trp Ala Ile His Ser Gly Pro Gly Thr His Ser His Leu Val Leu Thr

280 285 290 295

aag att gag ccg ggc aca ctg gga gtg gag cat tca tgg gat acc cca 1327

Lys Ile Glu Pro Gly Thr Leu Gly Val Glu His Ser Trp Asp Thr Pro

300 305 310

tgc aga agc cag gat gct gaa gcc tca ttc ctc ttg tgt ggg gtt ctc 1375

Cys Arg Ser Gln Asp Ala Glu Ala Ser Phe Leu Leu Cys Gly Val Leu

315 320 325

tat gtg gtc tac agt act ggg ggc cag ggc cct cat cgc atc acc tgc 1423

Tyr Val Val Tyr Ser Thr Gly Gly Gln Gly Pro His Arg Ile Thr Cys

330 335 340

atc tat gat cca ctg ggc act atc agt gag gag gac ttg ccc aac ttg 1471

Ile Tyr Asp Pro Leu Gly Thr Ile Ser Glu Glu Asp Leu Pro Asn Leu

345 350 355

ttc ttc ccc aag aga cca aga agt cac tcc atg atc cat tac aac ccc 1519

Phe Phe Pro Lys Arg Pro Arg Ser His Ser Met Ile His Tyr Asn Pro

360 365 370 375

aga gat aag cag ctc tat gcc tgg aat gaa gga aac cag atc att tac 1567

Arg Asp Lys Gln Leu Tyr Ala Trp Asn Glu Gly Asn Gln Ile Ile Tyr

380 385 390

aaa ctc cag aca aag aga aag ctg cct ctg aag taa tgcattacag 1613

Lys Leu Gln Thr Lys Arg Lys Leu Pro Leu Lys

395 400

ctgtgagaaa gagcactgtg gctttggcag ctgttctaca ggacagtgag gctatagccc 1673

cttcacaata tagtatccct ctaatcacac acaggaagag tgtgtagaag tggaaatacg 1733

tatgcctcct ttcccaaatg tcactgcctt aggtatcttc caagagctta gatgagagca 1793

tatcatcagg aaagtttcaa caatgtccat tactccccca aacctcctgg ctctcaagga 1853

tgaccacatt ctgatacagc ctacttcaag ccttttgttt tactgctccc cagcatttac 1913

tgtaactctg ccatcttccc tcccacaatt agagttgtat gccagcccct aatattcacc 1973

actggctttt ctctcccctg gcctttgctg aagctcttcc ctctttttca aatgtctatt 2033

gatattctcc cattttcact gcccaactaa aatactatta atatttcttt cttttctttt 2093

cttttttttg agacaaggtc tcactatgtt gcccaggctg gtctcaaact ccagagctca 2153

agagatcctc ctgcctcagc ctcctaagta cctgggatta caggcatgtg ccaccacacc 2213

tggcttaaaa tactatttct tattgaggtt taacctctat ttcccctagc cctgtccttc 2273

cactaagctt ggtagatgta ataataaagt gaaaatatta acatttg 2320

<210>2

<211>402

<212>PRT

<213>Homo sapiens

<400>2

Met Met Val Ala Leu Arg Gly Ala Ser Ala Leu Leu Val Leu Phe Leu

l 5 10 15

Ala Ala Phe Leu Pro Pro Pro Gln Cys Thr Gln Asp Pro Ala Met Val

20 25 30

His Tyr Ile Tyr Gln Arg Phe Arg Val Leu Glu Gln Gly Leu Glu Lys

35 40 45

Cys Thr Gln Ala Thr Arg Ala Tyr Ile Gln Glu Phe Gln Glu Phe Ser

50 55 60

Lys Asn Ile Ser Val Met Leu Gly Arg Cys Gln Thr Tyr Thr Ser Glu

65 70 75 80

Tyr Lys Ser Ala Val Gly Asn Leu Ala Leu Arg Val Glu Arg Ala Gln

85 90 95

Arg Glu Ile Asp Tyr Ile Gln Tyr Leu Arg Glu Ala Asp Glu Cys Ile

100 105 110

Glu Ser Glu Asp Lys Thr Leu Ala Glu Met Leu Leu Gln Glu Ala Glu

115 120 125

Glu Glu Lys Lys Ile Arg Thr Leu Leu Asn Ala Ser Cys Asp Asn Met

130 135 140

Leu Met Gly Ile Lys Ser Leu Lys Ile Val Lys Lys Met Met Asp Thr

145 150 155 160

His Gly Ser Trp Met Lys Asp Ala Val Tyr Asn Ser Pro Lys Val Tyr

165 170 175

Leu Leu Ile Gly Ser Arg Asn Asn Thr Val Trp Glu Phe Ala Asn Ile

180 185 190

Arg Ala Phe Met Glu Asp Asn Thr Lys Pro Ala Pro Arg Lys Gln Ile

195 200 205

Leu Thr Leu Ser Trp Gln Gly Thr Gly Gln Val Ile Tyr Lys Gly Phe

210 215 220

Leu Phe Phe His Asn Gln Ala Thr Ser Asn Glu Ile Ile Lys Tyr Asn

225 230 235 240

Leu Gln Lys Arg Thr Val Glu Asp Arg Met Leu Leu Pro Gly Gly Val

245 250 255

Gly Arg Ala Leu Val Tyr Gln His Ser Pro Ser Thr Tyr Ile Asp Leu

260 265 270

Ala Val Asp Glu His Gly Leu Trp Ala Ile His Ser Gly Pro Gly Thr

275 280 285

His Ser His Leu Val Leu Thr Lys Ile Glu Pro Gly Thr Leu Gly Val

290 295 300

Glu His Ser Trp Asp Thr Pro Cys Arg Ser Gln Asp Ala Glu Ala Ser

305 310 315 320

Phe Leu Leu Cys Gly Val Leu Tyr Val Val Tyr Ser Thr Gly Gly Gln

325 330 335

Gly Pro His Arg Ile Thr Cys Ile Tyr Asp Pro Leu Gly Thr Ile Ser

340 345 350

Glu Glu Asp Leu Pro Asn Leu Phe Phe Pro Lys Arg Pro Arg Ser His

355 360 365

Ser Met Ile His Tyr Asn Pro Arg Asp Lys Gln Leu Tyr Ala Trp Asn

370 375 380

Glu Gly Asn Gln Ile Ile Tyr Lys Leu Gln Thr Lys Arg Lys Leu Pro

385 390 395 400

Leu Lys

<210>3

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

ccggctagca gaagagccac agcataag 28

<210>4

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

gccggtaccc ttcagaggca gctttctc 28

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the active polypeptide of Human Olfactomedin-like protein 402's matter, and

Show at least 70% homology from the nucleotides sequence of Nucleotide 395-1603 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 395-1603 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of sequence shown in the SEQ ID NO.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 395-1603 position.

4. isolated Human Olfactomedin-like protein 402's polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 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.2 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. a generation has the method for the active polypeptide of Human Olfactomedin-like protein 402's matter, it is characterized in that this method comprises:

(1) nucleotide sequence that coding is had an active polypeptide of Human Olfactomedin-like protein 402 operationally is connected in expression regulation sequence, form Human Olfactomedin-like protein 402's expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 395-1603 position among described nucleotide sequence and the SEQ ID NO.1;

(2) change the expression vector in the step (1) over to host cell, form Human Olfactomedin-like protein 402's reconstitution cell;

(3) under the condition that is fit to expressing human HOLF402 protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate and have the active polypeptide of Human Olfactomedin-like protein 402.

9. energy and the described Human Olfactomedin-like protein 402's polypeptid specificity of claim 7 bonded antibody.

10. a nucleic acid molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

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