CN1390935A - Human hematolysis associated membrane protein and its coding sequence, preparing process and usage - Google Patents

Abstract

The present invention relates to the cDNA sequence of membrane protein (hHR) associated with human hematolysis, the polypeptide coded by said sequence, the process for preparing said polynucleotide sequence and polypeptide, and the application of said polynucleotide and polypeptide.

Description

A kind of new human hemolysis related, its encoding sequence, method for making and purposes

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to human hemolysis related's the cDNA sequence and the polypeptide of this sequence encoding.The invention still further relates to the production method of described polynucleotide sequence and described polypeptide, and the purposes of these polynucleotide and described polypeptide.

Background technology

Haemolysis is that oxyphorase only is left colourless matrix to outer effusion in red corpuscle.The erythrocytic ordinary life cycle is about 120 days, and promptly normal red corpuscle also can break about 120 days in aging, haemolysis occurred.The main effect of blood is transportation various nutritive substances, regulate body temperature, keep in environment constant etc., in case haemolysis is too much overrun above the hemopoietic system compensation ability, the body normal physiological activity just can not be carried out, and can cause various hemolytic disease even cause death.

Haemolysis can be divided into acute haemolysis and chronic hemolysis by the symptom tempo.The normal onset of acute haemolysis is hurried, and a large amount of haemolysis of short-term can have serious back and have aches in the limbs, and companion's headache, vomiting, shiver with cold are high heat, pale complexion and jaundice subsequently.This is because the red corpuscle considerable damage, and its degradation production is to due to the toxic action of human body, and severe patient can have peripheral circulatory failure more.Because hemolyzate causes the downright bad and tube chamber obstruction of renal tubular cell, causes acute renal failure at last.The chronic hemolysis onset is slow, and symptom is slight, and anaemia, jaundice, hepatosplenomegaly three big features are arranged.The chronic hemolysis patient is because secular hyperbilirubinemia can concurrent cholelithiasis and performance such as hepatic disorder.

Modal in the haemolysis disease is anaemia.Because the generative capacity of marrow blood cell is bigger, have 6～8 times of compensatory capacities of the normoerythrocyte of generation, as the erythrocytic lost of life, destroy and quicken, and marrow hemopoiesis anaemia can not occur still can be compensatory the time, is called haemolytic disease.When the mean corpuscular lifetime is shorter than 15～20d, hematoclasis speed then anaemia will occur considerably beyond the compensatory capacity of marrow.

The methods of treatment of hemolytic anemia roughly has following several method at present:

One, suprarenal gland glucocorticosteroid.Effective to the immunity anaemia, also can be applicable to paroxysmal nocturnal hemoglobinuria, invalid substantially to other types haemolysis.

Two, splenectomy.Be applicable to that mainly abnormal erythrocyte is mainly the spleen saboteur, as hereditary spherocytosis; The autoimmune hemolytic anemia that needs more heavy dose of adrenocortical hormone to keep, and the hemoglobinopathy of some type.

Three, immunosuppressor.As ciclosporin A, endoxan etc., only effective to the minority immune hemolytic anemia.

Four, blood transfusion.Though can temporarily improve patient's symptom, instead can bring severe reaction to some hemolytic anemia.As autoimmune hemolytic anemia,, and easily cause serious medical treatment anaemia because corresponding antigens can have strong positive reaction on himself antibody and the input erythrocyte membrane; Sometimes also can bring out haemolysis for paroxysmal nocturnal hemoglobinuria person blood transfusion.

Yet most hemolytic anemias still do not have the radical cure way at present.Along with development of molecular biology, people begin from gene and protein level research erythrocyte metabolism regulator control system.

HHR of the present invention has the type sequence of hemolysin III (Hly-III) family.At present this family member who finds is not following tens kinds, but hHR of the present invention also not the someone report.

Summary of the invention

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding translocator family, albumen of the present invention be named as the human hemolysis related ( hUman hEmolysis rElated pRotein, hHR).

Another object of the present invention provides a kind of new translocator family member, and this albumen is named as (hHR).

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described (hHR).

The present invention also provides the application of hHR gene order and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of (hHR) protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 97-753 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 97-753 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ IDNO.2.More preferably, this sequence has among the SEQ ID NO.1 nucleotide sequence from the 97-753 position.

In another aspect of this invention, provide a kind of isolating hHR protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.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, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, the method that provides a kind of generation to have the polypeptide of hHR protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hHR protein-active operationally is connected in expression regulation sequence, form the hHR protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 97-753 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hHR;

(c) be fit to express under the condition of hHR protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hHR protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 2013 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 97-753 position Nucleotide.

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

In the present invention, term " hHR albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with hHR protein-active is as 97-753 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 97-753 position Nucleotide of SEQID 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 ID NO.1 in 97-753 position nucleotide sequence homology be low to moderate about 90% 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 97-753 position.Also term also comprise with SEQ ID NO.1 in from the nucleotide sequence of the homology of nucleotide sequence at least 90% of Nucleotide 97-753 position.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.1 with (HHR) 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 " hHR protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hHR protein-active.This term also comprises having and (hHR) variant form 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 art end and also can not change proteinic function usually.This term also comprises proteic active fragments of hHR and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of hHR DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hHR polypeptide to obtain.The present invention also provides other polypeptide, as comprises hHR polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of hHR polypeptide.Usually, this fragment have the hHR peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of hHR albumen or polypeptide.The difference of these analogues and natural hHR 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.

The present invention also comprises the antisense sequences of hHR polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hHR in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of hHR polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the hHR that encodes.

The present invention also comprises the method that detects the hHR nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of hHR polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises hHR DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into hHR gene product or fragment.Preferably, refer to that those can combine with hHR gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of hHR, comprise that also those do not influence the antibody of hHR protein function.The present invention also comprise those can with modify or without the hHR gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ＇ or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the hHR gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hHR or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,2013; People such as Kohler, Eur.J.Immunol.6:292,2013; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the hHR function and the antibody that does not influence the hHR function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hHR gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of hHR gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

(hHR) of the present invention nucleotide sequence full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

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

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Certainly, by first synthetic a plurality of small segments, and then connect and obtain the very long fragment of sequence.

In the present invention, the cDNA nucleotide sequence of hHR is so to obtain: with people's testis λ gt10cDNA library (Clontech company) is template, is that primer A1:5 ＇-catcatgt ctggctaccg ccg-3 ＇ is a forward primer with two pairs of oligonucleotide; Oligonucleotide A2:5 ＇-CCTTTCA GTCAGTTTGG GAGG-3 ' is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product finds that the gained fragment length is about 700 bases.Above-mentioned amplified production is connected with pGEM-T  carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Check order with disappearance of SequiTherm EXCELTMDNA sequencing kit (EpicentreTechnologies) to brachymemma successively, obtain full length cDNA sequence at last, be total to 1780bp, detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 97-753 position Nucleotide.Derive the aminoacid sequence of hHR according to the full length cDNA sequence that obtains, totally 218 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Find to have the type sequence that has hemolysin 3 (Hly-III) family in the protein sequence that found that hHR in the hHR protein sequence of the present invention by the homology retrieval.The group membership of this family is an inner membrane protein, owing to the albumen member with hemolysin activity that is separated from Bacillus cereus in this family gains the name.Nineteen ninety-five, gene that obtains from Bacillus cereus VKM-B164 separation of people such as Baida GE has been cloned into the intestinal bacteria and has been checked order, and this hemolysin is called hemolysin III again.Therefore, hHR of the present invention may be relevant with coagulation process, and by to further investigation of the present invention, one finds the relation of it and many haemolysis relative diseases surely, and final for addressing these problems developing new thinking and method.The present invention (hHR) nucleic acid (encoding sequence or antisense sequences) is introduced cell, can improve the expression level of (hHR) or the overexpression of inhibition (hHR).(hHR) albumen of the present invention or its active polypeptide fragment also can be applied to patient, with treatment or alleviate because of (hHR) disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hHR

1. primer amplification

In the present invention, the cDNA nucleotide sequence of hHR is so to obtain: with people's testis λ gt10cDNA library (Clontech company) is template, is that primer A1:5 ＇-CATCATGT CTGGCTACCG CCG-3 ＇ is a forward primer with two pairs of oligonucleotide; Oligonucleotide A2:5 ＇-CCTTTCA GTCAGTTTGG GAGG-3 ' is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product finds that the gained fragment length is about 700 bases.

2.PCR the order-checking of product

Above-mentioned amplified production is connected with pGEM-T  carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprepPlasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1780bp, detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 97-753 position Nucleotide.Derive the aminoacid sequence of hHR according to the full length cDNA sequence that obtains, totally 218 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

Structural analysis

Full length cDNA sequence and proteins encoded thereof with hHR carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database.Found that the type sequence that has hemolysin 3 (Hly-III) family in the protein sequence of hHR.

Meet hemolysin III among the hHR of the present invention, (Hly-III) part of family's conserved sequence is SALDCVLSSFQMTNETVNIWTHFLPTWYFLWRLLALAGGPGFRAEPYHWPLLVFLL PACLYPFASCCAHTFSSMSPRMRHICYFLDYGALSLYSLGCAFPYAAYSMPASWLH GHLHQFFVPAAALNSFLCTGLSCYSRFLELESPGLSKVLRTGAFAYPFLFDNLPLF YRLGLCWGRGHGCGQEALSTSHGYHLFCALLTGFL, (among the SEQ ID NO 2 from the 9-212 position), (http://smart.embl-heidelberg.de/smart/show_motifs.pl).The group membership of this family is an inner membrane protein, owing to the albumen member with hemolysin activity that is separated from Bacillus cereus in this family gains the name.Nineteen ninety-five, gene that obtains from Bacillus cereus VKM-B164 separation of people such as BaidaGE has been cloned into the intestinal bacteria and has been checked order.Infer that by this sequence the aminoacid sequence that obtains contains 219 amino-acid residues, molecular weight is about 24.4kDa., and this hemolysin is called hemolysin III (Biochim Biophys Acta 1995 again; 1264 (2): 151-4).Therefore, hHR of the present invention may be relevant with coagulation process, and by to further investigation of the present invention, one finds the relation of it and many clinical cases surely, and final for addressing these problems developing new thinking and method.

HHR of the present invention is used for further functional study except can be used as hemolysin III (Hly-III) family a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, hHR of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.At the antibody of hHR of the present invention, be used to screen other albumen with similar structures territory, perhaps be used for the affinity purification associated protein.

For example, hHR nucleic acid of the present invention (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves hHR or the overexpression that suppresses hHR.HHR albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of hHR disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3:

The expression of hHR in intestinal bacteria

In this embodiment, use corresponding to 5 ＇ of cDNA sequence of coding hHR and the PCR Oligonucleolide primers of 3 ＇ end and increase, the cDNA that obtains hHR is as inserting fragment.

The 5 ＇ Oligonucleolide primers sequences of using in the PCR reaction are:

5＇-AGAT?GGATCCATGT?CTGGCTACCG?CCGCC-3＇

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is the N-terminal partial nucleotide sequence of hHR encoding sequence;

3 ＇ end primer sequence is:

5’-CAGCAAGCTT?GTCAGTTTGG?GAGGCGAAG?A-3’

This primer contains the restriction enzyme site of HindIII restriction enzyme, the partial nucleotide sequence of the encoding sequence of translation termination and hHR.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, and the cDNA fragment of sequence verification hHR has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when reaching 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved hHR from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hHR from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 4

The expression of hHR in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, use corresponding to 5 ＇ of cDNA sequence of coding hHR and the PCR Oligonucleolide primers of 3 ＇ end and increase, obtain hHR cDNA as inserting fragment.

5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:

5＇-AGAT?AAGCTTATGT?CTGGCTACCG?CCGCC-3＇

This primer contains the restriction enzyme site of HindIII restriction enzyme, is the aminoterminal partial nucleotide sequence of hHR encoding sequence after this restriction enzyme site;

3 ' end primer sequence is:

5’-CAGCGGATCC?GTCAGTTTGG?GAGGCGAAG-3’

This primer contains the partial nucleotide sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hHR.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3 (Invitrogen company), this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With BamHI and HindIII digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, and the cDNA fragment of sequence verification hHR has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured expression of peptides translocator vigor.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mM TrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 4 and 5 is used for immune animal to produce antibody, specific as follows.Recombinant protein is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation (hHR) gene translation product with it.

Example 6

The assignment of genes gene mapping

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

The physical location of sequence on karyomit(e) is 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.

Example 7

Substrate as micromatrix

Micromatrix (Microarrays) claims the DNA chip again.Chip can obtain on the substrate by using ink-jet technology and a series of chemical process such as ray, chemistry, thermodynamics, mechanical means etc. that sample is fixed on, and the element of formation has point-like, bar shaped or the like.A typical chip contains the element of some amount usually, can be by preparing by hand or with suitable plant and instrument.Behind the hybridization, the unconjugated probe of flush away detects the element of generation hybridization and the degree of reaction with scanner then.The complementarity of probe and each chip component and binding capacity all can be judged by the analysis to the image that scans.

Full-length cDNA, EST or gene fragment can be as the samples that is fixed on the substrate.The fragment that is suitable for hybridizing can be selected by with some famous biosoftwares (as LASERGENE SOFTWARE (DNASTAR)).The fragment of selecting at random in these full-length cDNAs, EST, the segment relevant with nucleotide sequence of the present invention or the cDNA storehouse relevant with this invention is arranged on the carrier such as glass by orderly.The method that cDNA is bonded to slide is: ultraviolet-crosslinkable, calorifics, chemical treatment, drying (Schena, M.et al. (1995) Science 270:467-470; And Shalon, D.et al. (1996) GenomeRes.6:639-645.) etc.With probe with fluorescent mark after, again with the hybridization of set ground response element.Results of hybridization can be used for inferring gene function, understands the gene basis that disease produces, and diagnoses the illness, and can improve and monitor the activity of medicament.

HHR nucleotide sequence of the present invention or its full length fragment can be made the object of micromatrix, detect genovariation, sudden change and polymorphism, thereby the diagnoses and treatment of relative disease is helped out.

Embodiment 8

The preparation of test kit

Test kit 1:

It is right that it contains the primer of (1) specific amplification hHR, and operation instruction.This test kit also can contain or not contain the required reagent that the mRNA reverse transcription is become cDNA.Wherein, the sequence of this Auele Specific Primer is derived from the sequence of SEQ ID NO:1, and length is 15-35.The cDNA that reverse transcription is become carries out specific amplification, whether to contain the nucleotide sequence of hHR in the test sample.This test kit also can contain and carries out other required reagent of PCR reaction, for example damping fluid etc.

Test kit 2:

This test kit contains the specific antibody (for example polyclonal antibody of preparation among the embodiment 5, the perhaps monoclonal antibody of the anti-hHR of the hybridoma technology generation of usefulness standard) at hHR, and operation instruction.This test kit is used for direct test sample and whether has hHR albumen.Form immunocomplex by specific immune response earlier, detect immunocomplex with routine techniques then.

Test kit 3:

This test kit contains can be specifically and the probe of the mRNA hybridization of hHR.It also can contain or not contain hybridization buffer.This test kit comes whether to exist in the test sample nucleic acid molecule of hHR by the hybridization between nucleic acid molecule and the hHR specific probe.

Test kit also can be used for detecting genovariation, sudden change and polymorphism, and detects the relevant gene of hHR a series of and of the present invention, thereby diagnosis, the treatment of relative disease helped out.

Embodiment 9

Make medicine

HHR albumen of the present invention and antibody, inhibitor, antagonist or acceptor etc. can be used as medicine, are used to diagnose and treatment and haemolysis diseases associated etc. have the small-molecule drug of important value.

Albumen of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is 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.

In addition, hHR nucleic acid of the present invention (encoding sequence or antisense sequences) can directly be introduced cell, with expression level that improves hHR or the overexpression that suppresses hHR.HHR albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of hHR 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.

When hHR protein polypeptide of the present invention is used as medicine, the treatment effective dose is 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-about 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.

Sequence table＜210〉1＜211〉1780＜212〉Nucleotide＜213〉human＜220〉＜221〉encoding sequence＜222〉(97) .. (753)＜223〉＜400〉1cagcagcctt ggactccgcc cgtggagccc tgggcctgtt gacccaccag cttaggagca 60cccaccaagc tctgggtgtt ctgggaagat ggcatc atg tct ggc tac cgc cgc 114

Met?Ser?Gly?Tyr?Arg?Arg

1 5ccc?acc?agc?tcg?gct?ttg?gac?tgt?gtc?ctc?agc?tcc?ttc?cag?atg?acc 162Pro?Thr?Ser?Ser?Ala?Leu?Asp?Cys?Val?Leu?Ser?Ser?Phe?Gln?Met?Thr

10 15 20aac?gag?acg?gtc?aac?atc?tgg?act?cac?ttc?ctg?ccc?acc?tgg?tac?ttc 210Asn?Glu?Thr?Val?Asn?Ile?Trp?Thr?His?Phe?Leu?Pro?Thr?Trp?Tyr?Phe

25 30 35ctg?tgg?cgg?ctc?ctg?gcg?ctg?gcg?ggc?ggc?ccc?ggc?ttc?cgt?gcg?gag 258Leu?Trp?Arg?Leu?Leu?Ala?Leu?Ala?Gly?Gly?Pro?Gly?Phe?Arg?Ala?Glu

40 45 50ccg?tac?cac?tgg?ccg?ctg?ctg?gtc?ttc?ctg?ctg?ccc?gcc?tgc?ctc?tac 306Pro?Tyr?His?Trp?Pro?Leu?Leu?Val?Phe?Leu?Leu?Pro?Ala?Cys?Leu?Tyr55 60 65 70ccc?ttc?gcg?tcg?tgc?tgc?gcg?cac?acc?ttc?agc?tcc?atg?tcg?ccc?cgc 354Pro?Phe?Ala?Ser?Cys?Cys?Ala?His?Thr?Phe?Ser?Ser?Met?Ser?Pro?Arg

75 80 85atg?cgc?cac?atc?tgc?tac?ttc?ctc?gac?tac?ggc?gcg?ctc?agc?ctc?tac 402Met?Arg?His?Ile?Cys?Tyr?Phe?Leu?Asp?Tyr?Gly?Ala?Leu?Ser?Leu?Tyr

90 95 100agt?ctg?ggc?tgc?gcc?ttc?ccc?tat?gcc?gcc?tac?tcc?atg?ccg?gcc?tcc 450Ser?Leu?Gly?Cys?Ala?Phe?Pro?Tyr?Ala?Ala?Tyr?Ser?Met?Pro?Ala?Ser

105 110 115tgg?ctg?cac?ggc?cac?ctg?cac?cag?ttc?ttt?gtg?cct?gcc?gcc?gca?ctc 498Trp?Leu?His?Gly?His?Leu?His?Gln?Phe?Phe?Val?Pro?Ala?Ala?Ala?Leu

120 125 130aac?tcc?ttc?ctg?tgc?acc?ggc?ctc?tcc?tgc?tac?tcc?cgt?ttc?ctg?gag 546Asn?Ser?Phe?Leu?Cys?Thr?Gly?Leu?Ser?Cys?Tyr?Ser?Arg?Phe?Leu?Glu135 140 145 150ctg?gaa?agc?cct?ggg?ctc?agt?aag?gtc?ctc?cgc?aca?gga?gcc?ttc?gcc 594Leu?Glu?Ser?Pro?Gly?Leu?Ser?Lys?Val?Leu?Arg?Thr?Gly?Ala?Phe?Ala

155 160 165tat?cca?ttc?ctg?ttc?gac?aac?ctc?cca?ctc?ttt?tat?cgg?ctc?ggg?ctg 642Tyr?Pro?Phe?Leu?Phe?Asp?Asn?Leu?Pro?Leu?Phe?Tyr?Arg?Leu?Gly?Leu

170 175 180tgc?tgg?ggc?agg?ggc?cac?ggc?tgt?ggg?cag?gag?gcc?ctg?agc?acc?agc 690Cys?Trp?Gly?Arg?Gly?His?Gly?Cys?Gly?Gln?Glu?Ala?Leu?Ser?Thr?Ser

185 190 195cat?ggc?tac?cat?ctc?ttc?tgc?gcg?ctg?ctc?act?ggc?ttc?ctc?ttc?gcc 738His?Gly?Tyr?His?Leu?Phe?Cys?Ala?Leu?Leu?Thr?Gly?Phe?Leu?Phe?Ala

200 205 210tcc caa act gac tga aaggctggca ccaggacgct ttgattacat cggccacagc 793Ser Gln Thr Asp215caccagttat tccacatctg tgcagtgctg ggcacccact tccagctgga ggcagtgctg 853gctgatatgg gatcacgcag agcctggctg gccacacagg aacctgccct gggcctggca 913ggcacagtgg ccacactggt cttggctgca gctgggaacc tactcattat tgctgctttc 973acagccaccc tgcttcgggc ccccagtaca tgccctctgc tgcagggtgg cccactggag 1033gggggtaccc aggccaaaca acagtgaggc cccatccctg accctgtcct ggagggggca 1093gaggccaggc cccagtgctg acgaggagcc cagatttggg cctaatcagg tggggacgca 1153tctcagcctg gaaccaacag gggctgagga gagagggcac aggagagagg gcagagaaga 1213ggaggggtgt ctagggggac tggcagagtg tgagagggac cgtgaggggg ctcttgatgg 1273gagtggaaga agtgctgagg gtctgagagg ggagatgcat gcgtgtccag gctgaagatg 1333cccctatatt ctgtcaaagg ttggcggggg gaggtgttgg ggtcctttca tctggctccg 1393tttctggtgc ttctggaagt ctctgctcag cacagggaag aactaacacg actaacctag 1453gcctaccctg aatgcttctt gctaaccagg ccgagaggcc acacacttgc ccccccatcc 1513ccacaaacca ggtaatgcca gtttgccagc agctatttgc ctatagagat gagtctgtcc 1573tggtcataac tgtgtgctca aggtgtccag gcttttgggg gtgggcctat ctgggtgcat 1633tatggatggt ttggtggatt gaggtgtggg gaggagggtc ctaggctaga gggggtatcc 1693ctagttagac tttgggaagc caccttcaac gttttctgga acaaggcagg tacaaataaa 1753aaaataaaac tttaaaaaaa aaaaaaa＜210〉2＜211〉218＜212〉＜213〉＜400〉2Met Ser Gly Tyr Arg Arg Pro Thr Ser Ser Ala Leu Asp Cys Val Leu1 5 10 15Ser Ser Phe Gln Met Thr Asn Glu Thr Val Asn Ile Trp Thr His Phe

20 25 30Leu?Pro?Thr?Trp?Tyr?Phe?Leu?Trp?Arg?Leu?Leu?Ala?Leu?Ala?Gly?Gly

35 40 45Pro?Gly?Phe?Arg?Ala?Glu?Pro?Tyr?His?Trp?Pro?Leu?Leu?Val?Phe?Leu

50 55 60Leu?Pro?Ala?Cys?Leu?Tyr?Pro?Phe?Ala?Ser?Cys?Cys?Ala?His?Thr?Phe65 70 75 80Ser?Ser?Met?Ser?Pro?Arg?Met?Arg?His?Ile?Cys?Tyr?Phe?Leu?Asp?Tyr

85 90 95Gly?Ala?Leu?Ser?Leu?Tyr?Ser?Leu?Gly?Cys?Ala?Phe?Pro?Tyr?Ala?Ala

100 105 110Tyr?Ser?Met?Pro?Ala?Ser?Trp?Leu?His?Gly?His?Leu?His?Gln?Phe?Phe

115 120 125Val?Pro?Ala?Ala?Ala?Leu?Asn?Ser?Phe?Leu?Cys?Thr?Gly?Leu?Ser?Cys

130 135 140Tyr?Ser?Arg?Phe?Leu?Glu?Leu?Glu?Ser?Pro?Gly?Leu?Ser?Lys?Val?Leu145 150 155 160Arg?Thr?Gly?Ala?Phe?Ala?Tyr?Pro?Phe?Leu?Phe?Asp?Ash?Leu?Pro?Leu

165 170 175Phe?Tyr?Arg?Leu?Gly?Leu?Cys?Trp?Gly?Arg?Gly?His?Gly?Cys?Gly?Gln

180 185 190Glu?Ala?Leu?Ser?Thr?Ser?His?Gly?Tyr?His?Leu?Phe?Cys?Ala?Leu?Leu

195 200 205Thr?Gly?Phe?Leu?Phe?Ala?Ser?Gln?Thr?Asp

210 215

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of (hHR) protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 97-753 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 97-753 position.

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

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has the sequence of Nucleotide 97-753 position among the SEQ ID NO.1.

4. isolating hHR protein polypeptide is characterized in that it comprises: have 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 contains the described DNA of claim 1.

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

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of hHR protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hHR protein-active operationally is connected in expression regulation sequence, form the hHR protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 97-753 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hHR;

(c) be fit to express under the condition of hHR protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hHR protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 97-753 position among the SEQ ID NO.1.

12. energy and the described hHR protein polypeptide of claim 4 specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

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