CN1978646A - Human hNmrr gene sequence, and its coded protein and preparing method - Google Patents

Abstract

The invention discloses the expression of hNmrr in human suprarenalis and the encoded protein sequence. The invention also discloses the preparation methods of the protein and nucleic acid, and antibodies of hNmrr. The hNmrr nucleic acid (coding sequence or antisense sequences) can be introduced into cells to increase the expression level or inhibit over expression of hNmrr. hNmrr protein or polypeptide fragments can be used on patients to treat or alleviate diseases caused by missing, or non-functional abnormalities of hNmrr. In addition, it can be used for the diagnosis or prognosis based on the nucleic acid sequence or antibodies.

Description

Human hNmrr gene sequence, its proteins encoded and preparation method

Technical field

The present invention relates to a kind of people's gene and albumen, relate in particular to nucleotide sequence and the proteins encoded thereof of a kind of hNmrr that in human suprarenal gland, expresses; In addition, the invention still further relates to the preparation method of this albumen and nucleotide sequence.

Background technology

Fungi can utilize the nitrogen resource in the mixture of wide scope, but utilizes the required enzyme of nitrogen resource often to be regulated by specific abduction mechanism.Modal regulation mechanism is the regulatory mechanism of so-called " nitrogen metabolism suppress (nitrogen metaboliterepression, Nmr) ".Discover that the nitrogen metabolism of being cloned into suppresses regulatory gene, and (nitrogen metabolite repression regulator Nmrr) has played very key in nitrogen metabolism suppresses to reply in Emericella nidulans.(J Bacteriol.1998 Apr；180(7)：1973-7).

Among the present invention, we are cloned into the homologous gene hNmrr of Emericella nidulans Nmrr gene in human suprarenal gland.

In view of hNmrr gene and proteic critical function, hNmrr is significant in research and development.

Summary of the invention

One of the technical problem to be solved in the present invention provides a kind of people's gene hNmrr (Genbank AccessionNo.AF225419), and this gene is a human hNmrr protein gene.

Two of the technical problem to be solved in the present invention provides a kind of people's albumen hNmrr.

Three of the technical problem to be solved in the present invention provides the preparation method of a kind of above-mentioned human hNmrr albumen and nucleotide sequence.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the polypeptide of human hNmrr protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 241-1140 position dna molecular among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 241-1140 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.More preferably, described sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 241-1140 position.

In another aspect of this invention, provide a kind of isolated human hNmrr protein and peptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.7 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, also provide a kind of preparation method with polypeptide of human hNmrr protein active, its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of human hNmrr protein-active operationally is connected in expression regulation sequence, form the human hNmrr protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 241-1140 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of human hNmrr;

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

(4) isolate polypeptide with human hNmrr protein-active.

Preferably, the nucleotide sequence that uses in the method has the sequence of 241-1140 position among the SEQ ID NO.6.

In the present invention, " isolating " DNA is meant, this DNA or fragment have been arranged in the sequence of its both sides under native state separates, and refers to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separates with the protein of following it in cell.

In the present invention, term " human hNmrr albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with human hNmrr protein-active is as 241-1140 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.6.This degenerate sequence is meant, is arranged in the encoder block 241-1140 position Nucleotide of SEQ ID NO.6 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.6 in 241-1140 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEO ID NO.7 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.6 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 241-1140 position.Wherein, " stringent condition " is meant that nucleotides sequence is listed in the film condition of washing after the hybridization on the film.For example, in the art, low stringency is washed film can pour 150ml left and right sides washing lotion in hybrid pipe, put into Hybond membrane, room temperature continued to shake about 20 minutes, and high tight degree to wash film can be to pour 200ml left and right sides washing lotion in hybrid pipe into, put into Hybond membrane, continue to shake about 20 minutes in 50 ℃ of shaking tables.This term also comprise with SEQ ID NO.6 in from the homology of nucleotide sequence at least 70% of Nucleotide 241-1140 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.6 with natural human hNmrr 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, term " human hNmrr albumen or polypeptide " refers to have the SEQ ID NO.7 polypeptide of sequence of human hNmrr protein-active.This term also comprises having and variant form natural human hNmrr identical function, SEQ ID NO.7 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of human hNmrr and reactive derivative.

The variant form of human hNmrr 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 human hNmrr DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-human hNmrr polypeptide to obtain.The present invention also provides other polypeptide, as comprises human hNmrr polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of human hNmrr polypeptide.Usually, this fragment have the human hNmrr 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, " human hNmrr conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.7, 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

The present invention also comprises the analogue of human hNmrr albumen or polypeptide.The difference of these analogues and natural human hNmrr 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 human hNmrr polypeptide of the present invention, the human hNmrr encoding sequence operationally can be connected in expression regulation sequence, thereby form the human hNmrr 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 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 and hNmrr protein polypeptide specificity bonded antibody, and it comprises polyclonal antibody and monoclonal antibody.

In the present invention, can use a series of methods known in the art to prepare at the special antibody of human hNmrr.For example, the human hNmrr gene product or its antigen fragment of purifying is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human hNmrr 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 the human hNmrr function, also can be the antibody that does not influence the human hNmrr function.Each antibody-like can produce by the fragment of human hNmrr gene product or functional domain are caused immunity, and human hNmrr gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the human hNmrr 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.

Human hNmrr antibody of the present invention can be used for identifying the cell of expressing human hNmrr albumen or polypeptide, as the JurkatT cell.For example, can with a kind of detectable molecule for example fluorescein isothiocyanic acid (FITC) come labelling human hNmrr specific antibody, allow the human hNmrr specific antibody contact then, detect and human hNmrr specific antibody bonded cell with fluorescent microscope or flow cytometer again with cell sample.

Except cell surface detects human hNmrr, 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 suprarenal gland extract, and be dissolved in the lysis buffer that contains stain remover.Use sds polyacrylamide gel electrophoresis isolated cell extract (simultaneously with the human hNmrr polypeptide of purifying as positive control) then, then it is transferred on the nitrocellulose by electrophoresis hybridization.In order to survey the human hNmrr 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 human hNmrr gene product, the i.e. existence of rna transcription thing in cell of analyst hNmrr.

The Nothern engram analysis of human hNmrr DNA and the Western engram analysis of human hNmrr specific antibody can be united use, with the expression of confirmer hNmrr in biological specimen.Human hNmrr DNA 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 continuous nucleotide of human hNmrr nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people bNmrr.

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

In order to obtain and the people cDNAs of human hNmrr 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, use ³²P human hNmrr all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from human adrenal gland.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 human hNmrr.

Can finish as follows according to Nucleotide similarity screening human hNmrr homologue.Human acth cDNA library, for example (Clontech, Palo Alto Cal.) can use one section all or part of random primer dna probe screening that comprises human hNmrr gene sequence to Clontech Cat.#7430-1.Finish having clone's the evaluation of the DNA insertion sequence of 70% homology at least with the human hNmrr 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 human hNmrr 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.

The human hNmrr homologue also can be used at the antibody of human hNmrr albumen 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 organize for example adrenal expression library to screen.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 human hNmrr antibody.Identify the DNA insertion sequence among the clone in this way, can be further analyze to estimate the similarity of it and human hNmrr 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.

Human hNmrr 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 hNmrr.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 MedicalLibrary).Then, come identified gene by linkage analysis and be positioned dependency between the disease of same chromosomal region.

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

Utilize human hNmrr albumen of the present invention,, can filter out with human hNmrr interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.

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

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

In addition, inventor hNmrr nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves human hNmrr or the overexpression that suppresses human hNmrr.Human hNmrr albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of human hNmrr 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 hNmrr albumen 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 Emericella nidulans), 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.

Description of drawings

Fig. 1 is that the homology of human hNmrr albumen of the present invention and the proteic nucleotide sequence of Emericella nidulansNmrr (GenBank Accession No.AF041976) compares (GAP) figure;

Fig. 2 is that the homology of human hNmrr albumen of the present invention and the proteic aminoacid sequence of Emericella nidulansNmrr (GenPept Accession No.AAC39442) compares (FASTA) figure.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

Embodiment

The present invention is further detailed explanation below in conjunction with drawings and Examples.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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of human hNmrr gene

1. separate tissue (suprarenal gland isolation)

Suprarenal gland derives from 5 normal adult male sex operation patients, and suprarenal gland is taken out in operation, places the freezing preservation of liquid nitrogen immediately.

2.mRNA separation (mRNA isolation)

Take out tissue, grind, add the 50ml pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to 50ml after the homogenate and newly manage, and extracted total RNA (TRIzol Reagents, Gibco, NY, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis.Cellulose column with band Oligod (T) separates mRNA among total RNA, quantitatively.

3.cDNA the structure in library (Construction of cDNA library)

With mRNA is template, and synthetic double chain cDNA, reverse transcription primer are seen SEQ ID NO.1.After mending flat end, add the joint that contains the EcoRI point of contact, joint sequence is seen SEQ ID NO.2 and 3 respectively.Behind the phosphorylation EcoRI end, use XhoI digestion with restriction enzyme 1.5 hours, carry out fragment again and separate.Cross the fragment of post screening length＞500bp, use the phenol-chloroform extracting, ethanol sedimentation, the sterilized water dissolving, be connected to Uni-ZAP XR carrier (Stratagene, CA9203, USA), with Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene, CA9203 USA) packs, and the host bacterium is used XL 1-Blue MRF ' (Stratagene, CA9203, USA) bacterium.Coated plate is also measured titre.

4. order-checking and database are set up (Seqencing and Database Constructing)

Select the clone who has the external source fragment to insert in the library, amplification back extracting plasmid (Qiagen, Germany), hold as 3 ' and the 5 ' universal primer of holding with T3 and T7, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer carries out the EST large scale sequencing on USA) at ABI 377 sequenators.Sequencing result is removed the carrier sequence with FACTURA software, is transferred to the processing of carrying out next step on SUN Ultra 450 Server.All sequence informations are used the GCG software package again, and (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL) are lower than 95% sequence with no homology or homology and are considered as new gene and set up database.

5. 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 (consensus sequence), 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 (OpenReading 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 human hNmrr 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 suprarenal gland 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 total RNA of suprarenal gland 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 the proteic complete encoding sequence of human hNmrr.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further R1:5 '-CACCTCGCTCATTTTGGTCT-3 ' (SEQ ID NO.4) is a forward primer to the design primer, oligonucleotide R2:5 '-TTCAGATGTTGGTGCCTCTG-3 ' (SEQ ID NO.5) is a reverse primer, with adrenal total RNA is template, carry out the RT-PCR amplification, the PCR condition of R1/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 1 minute with 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ thereupon, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1207bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQID NO.6.

Embodiment 2

The sequence information of human hNmrr gene and homology analysis:

Human hNmrr full-length cDNA (the GenBank Accession No.AF225419 that the present invention is new.Unexposed mistake before the application) length is 1207bp, and detailed sequence is seen SEQ ID NO.6, and wherein open reading frame is positioned at 241-1140 position Nucleotide.Derive the aminoacid sequence of human hNmrr according to full-length cDNA, totally 299 amino-acid residues, molecular weight 33344.38, pI are 7.06.Detailed sequence is seen SEQ ID NO.7.

The full length cDNA sequence of hNmrr 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 Nmrr gene of it and Emericella nidulans.On nucleotide level, the mRNA whole coding sequence (GenBank Accession No.AF041976) of it and Emericella nidulansNmrr gene has 54.2% homogeny (as shown in Figure 1), on amino acid levels, the 1-222 amino acids residue of it and EmericellanidulansNmrr albumen (GenPept Accession No.AAC39442) has 29.3% homogeny and 60.8% similarity (as shown in Figure 2).Therefore all there are higher homology in hNmrr gene and EmericellanidulansNmrr gene on nucleic acid still is protein level, can think that both also have very high similarity on function.

Human hNmrr 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, human hNmrr of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.For example proteic N end of human hNmrr of the present invention and the proteic N end of EmericellanidulansNmrr are exchanged, to produce the albumen that new activity is higher or have new features.

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

Embodiment 3

The distribution expression pattern of human hNmrr gene

Electronics Northern express spectra.Press people's such as Ton C. method (Ton C et al., Biochem Biophys ResCommun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation 1997 Dec16; 96 (12): 4146-4203), the BLAST retrieval will be done in the humanEST database of human hNmrr cDNA sequence in the GCG software package, in the human EST that obtains, the EST of probable value＜10e-10, homogeny＞95% has 76, can be considered the transcriptional expression of this gene in tissue originally, draw the tissue spectrum of expressing this gene thus, find that it has expression in pancreas, kidney, parathyroidoma, placenta, baby's heart, fetus liver, testis, tire brain or the like tissue, show that it is bringing into play important effect in these histoorgans of human body.

Embodiment 4

The preparation of human hNmrr polypeptide and purification

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

The human hNmrr polypeptide is carried out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.

1. construction of prokaryotic expression vector, and transformed into escherichia coli

Complete encoding sequence (SEQ ID NO.6) according to human hNmrr, 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-2T 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 the human hNmrr gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-2T 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-2T-hNmrr of pGEX-2T-hNmrr expression vector.

2. express the isolation identification of the engineering bacteria of GST-hNmrr recombinant protein

DH5 α-pGEX-2T-hNmrr 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 LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD ₆₀₀After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/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 12%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-hNmrr fusion rotein.

3.GST-hNmrr the extraction purifying of fusion rotein

The proteic engineering bacteria DH5 of abduction delivering GST-hNmrr amalgamation and expression α-pGEX-2T-hNmrr as stated above.Bacterium centrifugation after inducing adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose4B 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-hNmrr in centrifugal 10 minutes, abandoned supernatant.Clean twice by the amount that every milliliter of ultrasonic liquid gained precipitation adds 100 μ l PBS, then add 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000rpm, and 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 33kDa place is human hNmrr albumen.

Embodiment 5

Human hNmrr albumen or polypeptide carry out eukaryotic cell expression in insect cell

1. the structure of human hNmrr rhabdovirus expression vector and transfection Sf 9 insect cell strain

According to the complete encoding sequence (SEQ ID NO.6) of human hNmrr, 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 human hNmrr cDNA under the prerequisite that guarantees reading frame, be cloned into the pVL1392 carrier (Invitrogen, Carlsbad, CA).Identify good expression vector 3 μ g, wild-type linearized baculovirus dna (BaculoGold ^TMACMNPV DNA, Pharmingen, SanDiego, CA) 1 μ g and Lipofection (Gibco-BRL, NY) 25 μ l add in the insect substratum of 1ml serum-free, the 15 seconds mixings that vibrate, incubated at room 15 minutes is standby.Get 1ml (2 * 10 ⁶) Sf9 insect cell 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, it is standby to collect supernatant.

2. change the Screening and Identification of the insect cell line of recombinant expression vector over to

The insect cell of transfection after 3 days made cell suspension (2 * 10 with fresh culture ⁶/ 1ml), get the 1ml cell suspension and place 60mm tissue culture ware, add the 3ml substratum, the culture supernatant that 100 μ l collect, adherent 1 hour, abandon the 2ml substratum, continue incubated at room temperature 1 hour, and discarded all substratum, add the 3ml semisolid medium that contains 20 μ l4%X-gal of preheating, cultivate after 5-7 days picking white cell clone and in 96 well culture plates, cultivated 3-5 days, then draw supernatant infection Sf9 insect cell.

Collect the cell that infects and carry out the Western evaluation.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 1 hour, then in the antibody-solutions of anti-human hNmrr, sealed 1 hour, the jolting of TBS liquid is cleaned 5 minutes 2 times totally, then film is placed the anti-second antibody solution jolting of biotin labeled anti-human hNmrr one 1 hour, TBS cleans, adding avidin-alkaline phosphatase enzyme complex reacted 30 minutes, TBS cleans 2 times, adds freshly prepared colour developing liquid colour developing and observes protein band.

The Sf9 cell clone of picking high expression level human hNmrr.

3. the proteic extraction purifying of human hNmrr

Supernatant with the Sf9 cell clone of high expression level human hNmrr infects the Sf9 cell in a large number, infects collecting cell after 48 hours, the PBS washing.Per 2 * 10 ⁸Cell adds 20ml cell pyrolysis liquid (0.5%Triton X-100,20mMNa ₃PO ₄(sodium phosphate, pH7.8), 500mM NaCl, 1mM Na ₃VO ₄(vanadic acid sodium), 1mM Pefabloc, 1 μ g/ml pepstatin, leupeptin and aprotinin) broken cell, the centrifugal 20min of 12000 * g removes cell debris, and supernatant is by per 2 * 10 ⁸Cell add 2ml NTA-agarose (Qiagen, Germany), 4 ℃ of absorption 1 hour.Then with containing the His damping fluid washed twice of 100nM imidazoles, with containing 20mM N, N '-two piperazine, 500mM NaCl, the buffer solution elution of 300mM imidazoles is to obtain the albumen of purifying.Elutriant is stored in 4 ℃, and carries out the proteic purity of human hNmrr that the SDS-PAGE electrophoresis detection is extracted.Protein band at the 33kDa place is human hNmrr albumen.

Embodiment 6

The preparation of anti-human hNmrr antibody

1. the preparation of immune mouse and splenocyte: it is standby that the human hNmrr albumen that obtains among embodiment 5 and the embodiment 6 is separated the back 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 human hNmrr albumen and do the preliminary screening of 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 hNmrr gene sequence, its proteins encoded and preparation method

<130>NP-10240

<160>7

<210>1

<211>50

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>1

GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT 50

<210>2

<211>13

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉joint sequence

<400>2

AATTCGGCAC GAG 13

<210>3

<211>9

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉joint sequence

<400>3

GCCGTGCTC 9

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

CACCTCGCTCATTTTGGTCT 20

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>5

TTCAGATGTTGGTGCCTCTG 20

<210>6

<211>1207

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(241)..(1140)

<400>6

1 CACCTCGCTC ATTTTGGTCT AACTGGATTT ACACAAACTC ACTGTGCACG

51 TGCCTGGGGC GGGCAAACCC GGAAACGACG CCGAGTCCCG CGGGATCCAG

101 GAGGAGCCAA CTGCGCCGGA GGAGGGGTTT CGGCCGACAC GTCGGGATTG

151 GCGGCTGCAG CCAGGGGTCC TCCGACGCTG GGCTTCCGTG AGCGGCGCTC

201 TGCCAGATCT CTGGACCGGA TTCGTCCCAT TCTCGTCCTC ATGGTGGACA

251 AGAAACTGGT GGTGGTTTTC GGAGGCACAG GTGCCCAGGG TGGCTCCGTG

301 GCCCGCACAC TCCTGGAAGA TGGGACATTC AAGGTTCGAG TGGTGACCCG

351 AAACCCTAGG AAGAAGGCAG CAAAGGAGCT GAGGCTGCAA GGTGCAGAAG

401 TAGTGCAGGG AGACCAAGAT GACCAGGTCA TCATGGAGCT GGCCCTGAAT

451 GGGGCTTACG CCACCTTCAT CGTGACCAAT TACTGGGAGA GCTGCAGCCA

501 GGAGCAGGAG GTCAAGCAGG GGAAGCTGCT CGCTGATCTG GCCAGGCGCC

551 TGGGCCTCCA CTATGTGGTC TACAGCGGCC TGGAGAACAT CAAGAAGCTG

601 ACGGCAGGGA GATTGGCCGC CGCGCACTTT GACGGCAAAG GGGAGGTGGA

651 GGAATATTTC CGGGACATTG GCGTTCCCAT GACCAGTGTG CGGCTGCCCT

701 GCTATTTTGA GAACCTCCTC TCCCACTTCT TGCCCCAGAA AGCCCCAGAC

751 GGAAAGAGCT ACTTGCTGAG CTTGCCCACA GGTGACGTTC CCATGGATGG

801 CATGTCCGTG TCTGACCTGG GTCCTGTGGT GCTCAGCCTT TTGAAGATGC

851 CAGAAAAATA CGTCGGCCAG AACATCGGGC TGAGCACTTG CAGGCACACG

901 GCCGAGGAGT ACGCTGCCCT GCTCACCAAG CACACCCGCA AGGTCGTGCA

951 CGATGCCAAG ATGACTCCTG AGGACTACGA AAAGCTTGGC TTTCCCGGTG

1001 CCCGGGACCT GGCCAACATG TTCCGTTTCT ATGCCCTGAG ACCCGACCGT

1051 GACATCGAGC TGACCCTGAG ACTCAACCCC AAGGCCCTGA CGCTGGACCA

1101 GTGGCTGGAA CAGCACAAAG GGGACTTCAA CCTGCTGTGA CCTGCCCGCC

1151 TCGCGGCCCC TTGTGGGGAT CGGGGGCACC AGAGGGGCAG AGGCACCAAC

1201 ATCTGAA

<210>7

<211>299

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>7

1 MVDKKLVVVF GGTGAQGGSV ARTLLEDGTF KVRVVTRNPR KKAAKELRLQ

51 GAEVVQGDQD DQVIMELALN GAYATFIVTN YWESCSQEQE VKQGKLLADL

101 ARRLGLHYVV YSGLENIKKL TAGRLAAAHF DGKGEVEEYF RDIGVPMTSV

151 RLPCYFENLL SHFLPQKAPD GKSYLLSLPT GDVPMDGMSV SDLGPVVLSL

201 LKMPEKYVGQ NIGLSTCRHT AEEYAALLTK HTRKVVHDAK MTPEDYEKLG

251 FPGARDLANM FRFYALRPDR DIELTLRLNP KALTLDQWLE QHKGDFNLL

Claims (10)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of human hNmrr protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 241-1140 position among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 241-1140 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.

3. dna molecular as claimed in claim 1 is characterized in that, described sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 241-1140 position.

4. isolated human hNmrr protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, described polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

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

7. a host cell is characterized in that, described host cell is transformed by the described carrier of claim 6, and it comprises prokaryotic cell prokaryocyte and eukaryotic cell.

8. the preparation method with polypeptide of human hNmrr protein active is characterized in that, comprises the steps:

(1) nucleotide sequence that coding is had a polypeptide of human hNmrr protein-active operationally is connected in expression regulation sequence, form the human hNmrr protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 241-1140 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of human hNmrr;

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

(4) isolate polypeptide with human hNmrr protein-active.

9. an antibody is characterized in that, described antibody capable combines with the described human hNmrr protein polypeptide of claim 4 specificity, and it comprises polyclonal antibody and monoclonal antibody.

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

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