CN1276380A - Human reduced nicotinamide adenine nucleotie-cytochrome b5 reductase and its coding sequence - Google Patents

Abstract

The present invention discloses the cDNA sequence of human NADH-cytochrome b5 reductase (humNCb5Rh). The protein coded with said sequence is the member in NCb5R family. The polypeptide coded by the nucleotide sequence, the application of said polynucleotides and polypeptide and the process for preparing said polynucleotide and polypeptide are also disclosed.

Description

People's reduced form nicotinamide adenine nucleotide-cytochrome b5 reductase and encoding sequence thereof

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 the cDNA sequence of people's reduced form nicotinamide adenine nucleotide-cytochrome b5 reductase (human NADH-cytochrome b5 reductase abbreviates humNCb5Rh as), this albumen is the member of NCb5R family.The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

(NADH-cytochrom b5 reductare NCb5R) is one of electron transit mediator set member in the cytomicrosome to reduced form nicotinamide adenine nucleotide (NADH)-cytochrome b5 (cytb5) reductase enzyme.With flavin adenine Nucleotide (FMN) is prothetic group, and the reduction reaction of catalysis cytochrome b5 participates in important building-up reactions such as unsaturated fatty formation in the body, and 7-dehydrocholesterol synthetic also participates in the reduction of methemoglobin and regulate O ₂Important physiological function such as transportation.This enzyme is formed (Spatz, L.et al, J.Biol.Chem.248:793-799,1973) by a big wetting ability catalyst structure domain and a little hydrophobic film binding domains.

According to the difference that distributes in cell NCb5R is divided into two types: a kind of is the soluble-type that is distributed in the red corpuscle matter, main reduction reaction (the Hultquist that participates in the red corpuscle methemoglobin, D.E., et al.Nature New Biol.299:252-254,1971); Another kind is the film mating type that combines with cytoplasmic membrane, mainly participates in prolongation and desaturation (Oshino, N., et al.J.Biochem.69:155-167,1971 of lipid acid; Keyes, S.R., et al.J.Biol.Chem.255:11357-11364), the biosynthesizing (Reddy.V.V.R. of cholesterol, etal.J.Biol.Chem.252:2797-2801,1977), the redox reaction (Hildebrandt in Green Tea Extract and the drug metabolism processes, A., et al.Arch.Biochem.Biophys.143:66-79,1971).

Confirmed that the NADH-cytb5 reductase gene is the candidate gene of genotype methemoglobinemia.The characteristics of I type NCb5R defective are NCb5R defectives and cause methemoglobinemia in the red corpuscle, cause cyanosis (Scott, E.M.et al.Biochem.Biophys.Acta.34:584,1959); The characteristics of II type NCb5R defective are NCb5R defectives in all cells, cause methemoglobinemia, and follow intelligence beneath (Lerou, A., et al.Nature 258:619,1975); The characteristics of III type NCb5R defective are NCb5R defectives in white corpuscle, thrombocyte, the red corpuscle, the methemoglobinemia that causes but do not follow (Tanishima.K., et al.Blood 66:1288-1291,1985) under the intelligence.

1989, the cDNA sequence of people NCb5R and genome sequence obtained (Shunji.T.et al.Gene 80:353-361,1989) by methods such as restriction enzyme mapping and order-checkings.The cDNA of the NCb5R of mouse and genome sequence are that the cDNA with people NCb5R is a probe, and the clone obtains (Shuhei.Z., et al.J.Biochem.107:810-816,1990) from the liver cDNA library of mouse.All or part of DNA or the aminoacid sequence of the NCb5R of other species such as ox etc. also have report.

Yet, before the present invention, also do not report new people's reduced form nicotinamide adenine nucleotide-cytochrome b5 reductase or its encoding sequence of the present invention.

An object of the present invention is to provide a kind of new polynucleotide, this polynucleotide encoding people reduced form nicotinamide adenine nucleotide-cytochrome b5 reductase, it is a newcomer of NCb5R family, and NCb5R family member of the present invention is named as people humNCb5Rh.

Another object of the present invention provides a kind of new people's NCb5R family member, and this albumen is named as people humNCb5Rh albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's NCb5R polypeptide.

The present invention also provides this people's the NCb5R nucleotide sequence and the application of polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people humNCb5Rh protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 39-956 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 39-956 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 39-956 position.

In another aspect of this invention, provide a kind of isolating people humNCb5Rh protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

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

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people humNCb5Rh protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people humNCb5Rh protein-active operationally is connected in expression regulation sequence, form people humNCb5Rh protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 39-956 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people humNCb5Rh;

(c) under the condition that is fit to expressing human humNCb5Rh protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people humNCb5Rh protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1633 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 39-956 position Nucleotide.

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

In the present invention, term " people humNCb5Rh albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people humNCb5Rh protein-active is as 39-956 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 39-956 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 39-956 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 39-956 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 39-956 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people humNCb5Rh identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people humNCb5Rh protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people humNCb5Rh protein-active.This term also comprises having and variant form people humNCb5Rh identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people humNCb5Rh and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people humNCb5Rh DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people humNCb5Rh polypeptide to obtain.The present invention also provides other polypeptide, as comprises people humNCb5Rh polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people humNCb5Rh polypeptide.Usually, this fragment have people humNCb5Rh peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people humNCb5Rh albumen or polypeptide.The difference of these analogues and natural human humNCb5Rh 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, " people humNCb5Rh conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	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 people humNCb5Rh polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people humNCb5Rh 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 people humNCb5Rh 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 coding people humNCb5Rh.

The present invention also comprises the method that detects people humNCb5Rh 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 people humNCb5Rh polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

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

On the other hand, the present invention also comprises people humNCb5RhDNA 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 people humNCb5Rh gene product or fragment.Preferably, refer to that those can combine with people humNCb5Rh gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people humNCb5Rh, comprise that also those do not influence the antibody of people humNCb5Rh protein function.The present invention also comprise those can with modify or without the people humNCb5Rh gene product bonded antibody of modified forms.

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

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people humNCb5Rh gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human humNCb5Rh or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people humNCb5Rh function and the antibody that does not influence people humNCb5Rh function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people humNCb5Rh 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 people humNCb5Rh gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People humNCb5Rh 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 synthetic is synthesized relevant sequence.Usually, can synthesize a plurality of small segments earlier, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the cDNA nucleotide sequence of people humNCb5Rh is so to obtain, cDNA library of molecules (available from Clontech company) with tissues such as people body-centered, brain, placenta, liver, kidney, marrow, skeletal muscle, peripheral blood leucocyte and testis is a template, with a pair of oligonucleotide is that primer-A:5 '-GTTCGCTGCAGTTGTTCCCCATC-3 ' is a forward primer, oligonucleotide B:5 '-TGCCATCTCC ATAAGACCAC CTCTG-3 ' is a reverse primer, carry out PCR, successfully amplify the fragment of 446bp.With this 446bp fragment is probe, and after the TESTIS cDNA library was carried out sieving just, again, we had obtained 7 clones.Wherein, insert fragment and be 3 of the clones of 1.6kb, 4 of the clones of 1.2kb.After series disappearance, two-way order-checking and splicing, we have obtained the full-length cDNA order (SEQID NO.3) of a 1633bp.

Because humNCb5Rh and unsaturated fatty acids acid oxidase, drug metabolism and apoptosis and tumour take place, development has substantial connection, therefore for organism, has important biological significance.In addition, because humNCb5Rh of the present invention has the natural acid sequence that is derived from the people, therefore, estimate to have high reactivity and low side effect (for example in the intravital immunogenicity of people very low or do not have) being applied to man-hour.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people humNCb5Rh albumen of the present invention and people NCB5R (SP|P00387).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people humNCb5Rh

1, primer amplification

With the people body-centered, brain, placenta, liver, kidney, marrow, skeletal muscle, the cDNA library of molecules (available from Clontech company) of tissue such as peripheral blood leucocyte and testis is a template, with a pair of oligonucleotide is that primer-A:5 '-GTTCGCTGCAGTTGTTCCCCATC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-TGCCATCTCC ATAAGACCAC CTCTG-3 ' (SEQ ID NO.2) is a reverse primer, carry out PCR, the PCR condition be 93 ℃ 4 minutes, thereupon with 93 ℃ 1 minute, 66 ℃ of 1 minute and 72 ℃ carried out 35 circulations in 1 minute, last 72 ℃ were extended 5 minutes, and the result successfully amplifies the 446bp fragment.With this 446bp sequence be probe (through α- ³²The P mark), to the TESTIS cDNA library carry out just, behind the multiple sieve, we have obtained 7 clones.Insert fragment and be respectively two kinds of 1.6kb and 1.2kb, each 3 and 4 clone, wherein 3 clones contain complete ORF.After carrying out series disappearance, two-way order-checking and splicing with double-stranded nested type disappearance test kit (Pharmacia), we have obtained the cDNA order of a 1633bp length, obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking, wherein open reading frame is the 39-956 position.

Derive the aminoacid sequence of people humNCb5Rh according to the full-length gene group sequence that obtains, totally 305 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.

2, the order-checking of PCR product

According to above-mentioned sequencing result, design PCR primer C:5 '-ATGGGGATCCAGACGAGCC-3 ' (SEQ ID NO.9) and D:5 '-TCAGTAGGTGAATCGCATC-3 ' (SEQ ID NO.10).CDNA library of molecules (available from Clontech company) with tissues such as people body-centered, brain, placenta, liver, kidney, marrow, skeletal muscle, peripheral blood leucocyte and testis is a template, to carrying out pcr amplification, obtains the amplified fragments of about 900bp with above-mentioned C/D primer.With SequiTherm EXCELTMDNA sequencing kit (EpicentreTechnologies) amplified fragments is checked order, at last with computer software splicing order, obtain the cDNA sequence, about altogether 900bp, the corresponding part in the full length cDNA sequence of comparison and SEQ ID NO.3 is consistent.

3, RH location

It is identical with SEQ ID NO.2 to be used for the used SEQ ID NO.1 of the localized primer of RH and embodiment 1.RH cell strain DNA is available from Research Genetics company.Carry out PCR by G4 Panel method, condition is: 93 ℃ of sex change 3min, and then with 93 ℃ of 1min, 62 ℃ of 1min, 72 ℃ of 1min carry out 35 circulations, last 72 ℃ of incubation 5min.The result send Sanger center and WI to handle.Analytical results shows that the assignment of genes gene mapping of the present invention is between 1pter-DIS504-NCb5Rg1--WI-9647-1qter, and through STS figure, genetic map, physical map confluence analysis, humNCb5Rh is positioned 1q23-32 the most at last.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people humNCb5Rh of the present invention, in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that 5 ' end 151bp-1006bp has higher homology with people's (emb|Y09501,67%), mouse (dbj|D00636,67%), ox (gb|M83104,66%) etc. respectively on nucleotide level.Homologous protein is more also supported the correct of this sequence, on amino acid levels humNCB5Rh derive proteic 36-305 amino acid and people (SP|P00387) (Fig. 1), the homogeny of mouse (SP|P20070), ox (SP|P07514) etc. reaches 64%, 63%, 64% respectively, positive rate is all up to more than 80%.

Behind the PRODOM software analysis, confirm that humNCB5Rh derivation albumen has the catalysis territory of the NCB5R that is made up of two subdomains in flavine (nicotinamide) adenylic acid (AMP) (F (N) AD) land and cytochrome b5 reductase catalysis territory.Derive the 27th the-the 303rd of proteic amino-acid residue of humNCB5Rh is the cytochrome b5 reductase functional domain, wherein the 176th the-the 290th is F (N) AD land, the 27th the-the 149th is cytochrome b5 reductase catalysis territory, especially the 55th, 69, four histidine residues of 77,117 may be the sites that combines with protoheme in the cytochrome b5 molecule.Therefore, humNCB5Rh derivation albumen has NADH-cytochrome b5 reductase basic molecular structure, and may have similar oxydo-reductase function.

Studies show that, NCB5R participates in plastosome external oxidation reduction reaction, at present, known NCB5R is the gene of methemoglobinemia, and mutational site difference, due to the clinical symptom weight different and be divided into I, II, III type, based on the multiple biochemical reaction of its participation of autoploid that has various kinds of cell pigment b5 in the body, may exist the NCB5R homologue in the body.Therefore, humNCb5Rh of the present invention is exactly one of NCB5R homologue, is the candidate gene of methemoglobinemia.

HumNCB5Rh derivation albumen n end has the bigger peptide section of hydrophobicity, thereby has and membrane-bound possibility, and NCB5R such as known person, mouse can be combined into film mating type with the cell sarcolemma, so humNCB5Rh also may become film mating type; Or also may be after protein processing, remove the hydrophobic peptide section and form lysotype, participate in intracytoplasmic redox, make the molecule of other similar methemoglobin such as important substance such as O-2, oxidation lipid to be reduced, thereby removed the toxic action of oxidizing substance pair cell.So humNCB5Rh may have very important physiological action.

In addition, also the someone thinks relevant (the Gruis.N.A.et al.Bull.Cancer 85:627-30 with flavoprotein class redox enzymes of a kind of Dutch's of being more common in familial prevalence mole sample melanoma, 1998), and flavoprotein class oxide-reductase protein encoding gene is enriched in chromosomal 1q No. 1, and the sudden change of this regional karyomit(e) and gene can cause the generation of relative disease.Because humNCB5Rh is positioned karyomit(e) 1q2.3-3.2 No. 1, be positioned at equally this enrichment region near, so humNCB5Rh of the present invention probably with this class disease-related.

Recently, the someone finds, at non-O ₂In the susceptibility reduction reaction, the NADH-Cytb5 reductase enzyme participates in the reduction to toxicant, by detecting NCB5R enzymic activity in the tumour cell, find some hepatoma cell strains (Zoeller.R.A.et al. Lipids 19:488-91,1984) and brain tumor (Rampling.R.et al.Int.J.Radiat.Onco.Biol.Phys.29:427-431,1994) the NCB5R activity is very low in, and prompting NCB5R may have effect in the generation of tumour.Therefore humNCB5Rh of the present invention also may have effect in the generation of tumour.In addition, need NCb5R to participate in (Mikami.K.etal.Gan To Kagaku Ryoho 24:1606-10 owing to there are some researches show the biological reducing of ametycin, 1997) competence exertion antitumor action, prompting can not be treated the very low tumour of this class NCB5R enzymic activity with medicines such as ametycins.That is to say that humNCb5Rh of the present invention is useful on the prospect of the screening of antitumor drug.

People humNCb5Rh of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor humNCb5Rh can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor humNCb5Rh and the N end of mouse NCb5R are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor humNCb5Rh, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor humNCb5Rh nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people humNCb5Rh or the overexpression that suppresses people humNCb5Rh.People humNCb5Rh albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people humNCb5Rh 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 people humNCb5Rh in intestinal bacteria

In this embodiment, with positive colony that contains ORF among the embodiment 1 or the cDNA library of molecules of organizing with people body-centered, brain, placenta, liver, kidney, marrow, skeletal muscle, peripheral blood leucocyte and testis etc. (available from Clontech company) is template, use PCR Oligonucleolide primers to increase, obtain people humNCb5Rh cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end.

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

5 '-TCAGGTCGAC ATGGGGATCC AGACGAGCC-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of SalI restriction enzyme, is 19 Nucleotide of the people humNCb5Rh encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-TTGGAAGCTT TCAGTAGGTG AATCGCATC-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people humNCb5Rh of HindIII restriction enzyme.

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 SalI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, extracting plasmid, PstI enzyme are cut the cDNA fragment of identifier humNCb5Rh and have 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 into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people humNCb5Rh from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people humNCb5Rh from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.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.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 39KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 4

The expression of people humNCb5Rh in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with positive colony that contains ORF among the embodiment 1 or the cDNA library of molecules of organizing with people body-centered, brain, placenta, liver, kidney, marrow, skeletal muscle, peripheral blood leucocyte and testis etc. (available from Clontech company) is template, use PCR Oligonucleolide primers to increase, obtain people humNCb5Rh cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end.

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

5′-TCAGAAGCTT?ATGGGGATCC?AGACGAGCC-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the people humNCb5Rh encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TTGGGAATTC?TCAGTAGGTG?AATCGCATC-3’(SEQ?ID?NO.8)

This primer contains the part encoding sequence of the restriction enzyme site of EcoRI restriction enzyme, translation termination and people humNCb5Rh.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and BamHI 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, the cDNA fragment of PstI sequence verification people humNCb5Rh has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTris HCl (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 50mMTris á HCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mM Tris á HCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 39kDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people humNCb5Rh gene translation product with it.Found that antibody can precipitate with protein-specific of the present invention ground specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information: (i) applicant: Fudan University is denomination of invention (ii): people's reduced form nicotinamide adenine nucleotide-cytochrome b5 reductase

And (iii) sequence number of encoding sequence: information (i) sequence signature of 10 (2) SEQ ID NO.1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:GTTCGCTGCA GTTGTTCCCC ATC 23 (2) SEQ ID NO.2

(A) length: 27 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2:TGCCATCTCC ATAAGACCAC CTCTG 25 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 1633bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( iii ) ：SEQ ID NO.3：GTCGGCTTGT CAGGTGGTGG AGGAAAAGGC GCTCCGTCAT GGGGATCCAG ACGAGCCCCG 60TCCTGCTGGC CTCCCTGGGG GTGGGGCTGG TCACTCTGCT CGGCCTGGCT GTGGGCTCCT 120ACTTGGTTCG GAGGTCCCGC CGGCCTCAGG TCACTCTCCT GGACCCCAAT GAAAAGTACC 180TGCTACGACT GCTAGACAAG ACGACTGTGA GCCACAACAC CAAGAGGTTC CGCTTTGCCC 240TGCCCACCGC CCACCACACT CTGGGGCTGC CTGTGGGCAA ACATATCTAC CTCTCCACCC 300GAATTGATGG CAGCCTGGTC ATCAGGCCAT ACACTCCTGT CACCAGTGAT GAGGATCAAG 360GCTATGTGGA TCTTGTCATC AAGGTCTACC TGAAGGGTGT GCACCCCAAA TTTCCTGAGG 420GAGGGAAGAT GTCTCAGTAC CTGGATAGCC TGAAGGTTGG GGATGTGGTG GAGTTTCGGG 480GGCCAAGCGG GTTGCTCACT TACACTGGAA AAGGGCATTT TAACATTCAG CCCAACAAGA 540AATCTCCACC AGAACCCCGA GTGGCGAAGA AACTGGGAAT GATTGCCGGC GGGACAGGAA 600TCACCCCAAT GCTACAGCTG ATCCGGGCCA TCCTGAAAGT CCCTGAAGAT CCAACCCAGT 660GCTTTCTGCT TTTTGCCAAC CAGACAGAAA AGGATATCAT CTTGCGGGAG GACTTAGAGG 720AACTGCAGGC CCGCTATCCC AATCGCTTTA AGCTCTGGTT CACTCTGGAT CATCCCCCAA 780AAGATTGGGC CTACAGCAAG GGCTTTGTGA CTGCCGACAT GATCCGGGAA CACCTGCCCG 840CTCCAGGGGA TGATGTGCTG GTACTGCTTT GTGGGCCACC CCCAATGGTG CAGCTGGCCT 900GCCATCCCAA CTTGGACAAA CTGGGCTACT CACAAAAGAT GCGATTCACC TACTGAGCAT 960CCTCCAGCTT CCCTGGTGCT GTTCGCTGCA GTTGTTCCCC ATCAGTACTC AAGCACTATA 1020AGCCTTAGAT TCCTTTCCTC AGAGTTTCAG GTTTTTTCAG TTACATCTAG AGCTGAAATC 1080TGGATAGTAC CTGCAGGAAC AATATTCCTG TAGCCATGGA AGAGGGCCAA GGCTCAGTCA 140CTCCTTGGAT GGCCTCCTAA ATCTCCCCGT GGCAACAGGT CCAGGAGAGG CCCATGGAGC 1200AGTCTCTTCC ATGGAGTAAG AAGGAAGGGA GCATGTACGC TTGGTCCAAG ATTGGCTAGT 1260TCCTTGATAG CATCTTACTC TCACCTTCTT TGTGTCTGTG ATGAAAGGAA CAGTCTGTGC 1320AATGGGTTTT ACTTAAACTT CACTGTTCAA CCTATGAGCA AATCTGTATG TGTGAGTATA 1380AGTTGAGCAT AGCATACTTC CAGAGGTGGT CTTATGGAGA TGGCAAGAAA GGAGGAAATG 1440ATTTCTTCAG ATCTCAAAGG AGTCTGAAAT ATCATATTTC TGTGTGTGTC TCTCTCAGCC 1500CCTGCCCAGG CTAGAGGGAA ACAGCTACTG ATAATCGAAA ACTGCTGTTT GTGGCAGGAA 1560CCCCTGGCTG TGCAAATAAA NTGGCTGAGG CCCCTGTGTG ATATTGAAAA AAAAAAAAAA 1620AGTCGAGCAA AAA 1633 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 305 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4：Met Gly Ile Gln Thr Ser Pro Val Leu Leu Ala Ser Leu Gly Val 15Gly Leu Val Thr Leu Leu Gly Leu Ala Val Gly Ser Tyr Leu Val 30Arg Arg Ser Arg Arg Pro Gln Val Thr Leu Leu Asp Pro Asn Glu 45Lys Tyr Leu Leu Arg Leu Leu Asp Lys Thr Thr Val Ser His Asn 60Thr Lys Arg Phe Arg Phe Ala Leu Pro Thr Ala His His Thr Leu 75Gly Leu Pro Val Gly Lys His Ile Tyr Leu Ser Thr Arg Ile Asp 90Gly Ser Leu Val Ile Arg Pro Tyr Thr Pro Val Thr Ser Asp Glu 105Asp Gln Gly Tyr Val Asp Leu Val Ile Lys Val Tyr Leu Lys Gly 120Val His Pro Lys Phe Pro Glu Gly Gly Lys Met Ser Gln Tyr Leu 135Asp Ser Leu Lys Val Gly Asp Val Val Glu Phe Arg Gly Pro Ser 150Gly Leu Leu Thr Tyr Thr Gly Lys Gly His Phe Ash Ile Gln Pro 165Asn Lys Lys Ser Pro Pro Glu Pro Arg Val Ala Lys Lys Leu Gly 180Met Ile Ala Gly Gly Thr Gly Ile Thr Pro Met Leu Gln Leu Ile 195Arg Ala Ile Leu Lys Val Pro Glu Asp Pro Thr Gln Cys Phe Leu 210Leu Phe Ala Asn Gln Thr Glu Lys Asp Ile Ile Leu Arg Glu Asp 225Leu Glu Glu Leu Gln Ala Arg Tyr Pro Asn Arg Phe Lys Leu Trp 240Phe Thr Leu Asp His Pro Pro Lys Asp Trp Ala Tyr Ser Lys Gly 255Phe Val Thr Ala Asp Met Ile Arg Glu His Leu Pro Ala Pro Gly 270Asp Asp Val Leu Val Leu Leu Cys Gly Pro Pro Pro Met Val Gln 285Leu Ala Cys His Pro Asn Leu Asp Lys Leu Gly Tyr Ser Gln Lys 300Met Arg Phe Thr Tyr 305 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5:TCAGGTCGAC ATGGGGATCC AGACGAGCC 29 (2) SEQ ID NO.6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.6:TTGGAAGCTT TCAGTAGGTG AATCGCATC 29 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TCAGAAGCTT ATGGGGATCC AGACGAGCC 29 (2) SEQ ID NO.8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.8:TTGGGAATTC TCAGTAGGTG AATCGCATC 29 (2) SEQ ID NO.9

(A) length: 19 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.9:ATGGGGATCC AGACGAGCC 19 (2) SEQ ID NO.10

(A) length: 19 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.10:TCAGTAGGTG AATCGCATC 19

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people humNCb5Rh protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 39-956 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 39-956 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 39-956 position.

4. isolating people humNCb5Rh protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ IDNO.4 aminoacid sequence, or its reactive derivative.

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

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

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

8. a generation has the method for the polypeptide of people humNCb5Rh protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people humNCb5Rh protein-active operationally is connected in expression regulation sequence, form people humNCb5Rh protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 39-956 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people humNCb5Rh;

(c) under the condition that is fit to expressing human humNCb5Rh protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people humNCb5Rh protein-active.

9. energy and the described people humNCb5Rh of claim 4 protein polypeptide specificity bonded antibody.

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