CN1287168A - New human hair protein and its sequence - Google Patents

Abstract

The present invention provides the cDNA sequence of a new human hair protein. The cDNA encoded protein is the homologue of high-glycine/tyrosine protein type 1 alpha (HGT 1 alpha) of mouse. The present invention also relates to the nucleotide encoded polypeptide, the application of the polynucleotide and the polypeptide, and the production process of the polynucleotide and the polypeptide.

Description

A kind of new HAIR PROTEIN and encoding sequence thereof

The present invention relates to genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to new HAIR PROTEIN HP (Hair Protein, hair protein) cDNA sequence, the albumen of this cDNA coding is the homologue of mouse HGT 1 α (High-glycine/tyrosine protein type 1 α, the high glycine/tyrosine protein of 1 α type).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.

Hair is a kind of keratinization tissue, and it is by generating in hair follicle, and has outer keratinized sheath (cuticle sheath), internal layer cortex (inner cortex) and this common configuration of central medullary substance (central medulla).The major structural protein that forms hair is Keratin sulfate, thereby be cross-linked with each other between these Keratin sulfate and some other associated protein, forms the trunk of hair.

The albumen that forms hair has more than 50-100 kind according to estimates, these albumen may belong at least 10 different families (Powell BC et al.Ann NY Acad Sci 1991,642:1-20).Keratin sulfate is to consist of several multigene families equally: Keratin sulfate can be divided into intermediate filament (intermediate filaments, IF) and the intermediate filament albumen (intermediate filament-associated proteins, IFAP) that is connected.And the latter can be subdivided into again high-sulfur (high sulfur) albumen, ultra-high-sulfur(UHS) (ultra-high sulfur) albumen and high glycine/tyrosine protein (high-glycine/tyrosine proteins, HGTps).

HGTps is a proteinoid minimum in Keratin sulfate, and most molecular weight are less than 10000Da.According to the amino acid of HGTps, form, they can be further divided into again I type albumen and II type albumen, wherein I type is less containing halfcystine and more containing phenylalanine comparatively speaking, II type is contrary, two types do not contain methionine(Met) (Powell BC et al.Ann NY Acad Sci 1991,642:1-20).

Because hair protein is of a great variety, its name is also because of a little more complicated.Therefore, be necessary that its name is had to a unified regulation.Nineteen ninety, there is document to its name advise (MacKinnon, P.J.J.Cell Biol.1990,111:2587-2600).In this suggestion, the I type in HGTps is subdivided and is called KRTAP7 and 8 (or referred to as KAT7,8), and II type is called as KAP6.

In the hair of different plant species, the content of HGTps (or KAPs) differs very large, from people and Lincoln sheep, be less than 3%, to 13% in merino wool, to in echidna quill up to 30-40% (Gillespie JM, Comp.Biochem.Physiol.1972,41B:723-734).The otherness of this content likely has different explanations, one of them extracts albumen from people's hair is extremely difficult, sometimes extracting to 5% (may by due to experimental error) (the Gillespie JM that only has total protein, et al.1991 in Physiology, Biochemistry, and Molecular Biology of the Skin (Goldsmith LA ed) 2nd Ed, pp.625-659, Oxiford University Press, New York).

Therefore,, for the object for the treatment of and improving looks, R and D people's hair protein is significant.

An object of the present invention is to provide a kind of new polynucleotide, the homologue of this polynucleotide encoding mouse HGT 1 α, cDNA of the present invention is named as people HP gene, and it is a kind of hair protein gene.

Another object of the present invention is to provide a kind of new people's mouse HGT 1 alpha homologues, and this albumen is named as people HP albumen, and it is high glycine/tyrosine protein of people.

A further object of the present invention is to provide a kind of method of utilizing recombinant technology to produce described new people HP polypeptide.

The present invention also provides the application of this people HP nucleotide sequence and polypeptide.

In one aspect of the invention, a kind of isolated DNA molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people HP protein-active, in described nucleotide sequence and SEQ ID NO.3, from the nucleotides sequence of Nucleotide 1-246 position, shows at least 70% homology; Or described nucleotide sequence can be under moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 1-246 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in SEQID NO.4.More preferably, this sequence has in SEQID NO.3 the nucleotide sequence from Nucleotide 1-246 position.

In another aspect of this invention, provide a kind of people HP protein polypeptide of separation, it comprises: there is polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is the polypeptide with SEQ ID NO.4 sequence.

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

In another aspect of this invention, the host cell that provides a kind of described carrier to transform.

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

(a) nucleotide sequence that coding is had to a polypeptide of people HP protein-active is operationally connected in expression regulation sequence, form people HP protein expression vector, in described nucleotide sequence and SEQ ID NO.3, from the nucleotides sequence of Nucleotide 1-246 position, show at least 70% homology;

(b) expression vector in step (a) is proceeded to host cell, form the reconstitution cell of people HP albumen;

(c) be applicable to expressing under the condition of people HP protein polypeptide the reconstitution cell in culturing step (b);

(d) isolate the polypeptide with people HP protein-active.

In a specific embodiments of the present invention, the polynucleotide total length of separation of the present invention is 345 Nucleotide, and its detailed sequence is shown in SEQ ID NO.3, and wherein open reading frame is positioned at 1-246 position Nucleotide.

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

In the present invention, term " people HP albumen (or polypeptide) encoding sequence " refers to that coding has the nucleotide sequence of the polypeptide of people HP protein-active, as 1-246 position nucleotide sequence and degenerate sequence thereof in SEQ ID NO.3.This degenerate sequence refers to, is arranged in the encoder block 1-246 position Nucleotide of SEQ ID NO.3 sequence, the sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.Due to the degeneracy of codon, thus with SEQ ID NO.3 in 1-246 position nucleotide sequence homology be low to moderate the sequence that approximately 70% degenerate sequence also can be encoded out described in SEQ ID NO.4.This term also comprises can be under moderate stringent condition, more preferably under height stringent condition, with in SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 1-246 position.Also term also comprise with SEQ ID NO.3 in from the homology at least 70% of the nucleotide sequence of Nucleotide 1-246 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form albumen, SEQ ID NO.4 sequence with people HP identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60,1-20 more preferably, 1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and add several Nucleotide at 5 ' and/or 3 ' end.Encoding sequence of the present invention can be DNA or RNA, can be strand or two strands.

In the present invention, " substantially pure " protein or polypeptide refer to that it at least accounts at least 20% of the total material of sample, 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 by any suitable method, as measured the purity of polypeptide by column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under native state.

In the present invention, term " people HP protein polypeptide " refers to have the polypeptide of the SEQ ID NO.4 sequence of people HP protein-active.This term also comprises having and variant form people HP albumen 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 (being generally in 20 at C-terminal and/or N-terminal, being preferably in 10, is more preferably in 5) amino acid.For example, in the art, while replacing with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, at C-terminal and/or N-terminal, add one or several amino acid and conventionally also can not change the function of protein.This term also comprises active fragments and the reactive derivative of people HP albumen.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, albumen that can be coded with the DNA of people HP DNA hybridization under high or low stringency condition and polypeptide or the albumen that utilizes the antiserum(antisera) acquisition of anti-human HP polypeptide.The present invention also provides other polypeptide, as the fusion rotein that comprises people HP polypeptide or its fragment.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people HP polypeptide.Conventionally, this fragment have people HP peptide sequence at least about 10 continuous amino acids, conventionally 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 HP albumen or polypeptide.The difference of these analogues and natural human HP polypeptide can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.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 the analogue with non-natural amino acid (as β, gamma-amino acid) that exist or synthetic.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide exemplifying.

(conventionally the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as those carry out polypeptide glycosylation modified and that produce in procedure of processing in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and completes by polypeptide is exposed to.Modified forms also comprises the have 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 HP 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 are replaced by the similar or close amino acid of character and form polypeptide at the most best.These conservative property variation polypeptide preferably carry out amino acid substitution according to table 1 and produce.

Table 1

Initial residue	Representational replacement	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)	I1e；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises the antisense sequences of people HP polypeptid coding sequence and fragment thereof.This antisense sequences can be used for suppressing the expression of people HP in 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 HP polypeptid coding sequence, preferably 15-50 continuous nucleotide conventionally.This probe can be used for detecting the nucleic acid molecule that whether has encoding human HP in sample.

The present invention also comprises the method that detects people HP nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing, and then whether detection probes combination has occurred.Preferably, this sample is the product after pcr amplification, and wherein pcr amplification primer is corresponding to the encoding sequence of people HP polypeptide, and can be positioned at 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, as commercially available carrier.Such as, select commercially available carrier, then the nucleotide sequence of code book invention polypeptide is operationally connected in to expression regulation sequence, can form protein expression vector.

As used herein, " being operationally connected in " refers to a kind of like this situation, and some part of linear DNA sequence can affect 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 is operationally connected in polypeptid DNA so; If transcribing of promotor control sequence, it is to be operationally connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is to be operationally connected in encoding sequence so.Generally, " being operationally connected in " means adjoining, for secretion leader sequence, means in reading frame adjacent.

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

On the other hand, the present invention also comprises people HP DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into people HP gene product or fragment.Preferably, refer to that those can be combined with people HP gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.In the present invention antibody comprise those can in conjunction with and suppress the molecule of people HP albumen, also comprise that those do not affect the antibody of people HP protein function.The present invention also comprise those can with the antibody of modifying or being combined without the people HP gene product 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 (Tab) 2 fragments; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as there is murine antibody binding specificity but still retain the 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 HP gene product of purifying or its have antigenic fragment, can be applied to animal with the generation of induction polyclonal antibody.Similarly, expression people HP or its cell with antigenic fragment can be used to immune animal and produce antibody.Antibody of the present invention can be also monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see the people such as Kohler, Nature 256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises can block the antibody of people HP function and the antibody that does not affect people HP function.Each antibody-like of the present invention can utilize fragment or the functional zone of people HP gene product, by routine immunization technology, obtains.These fragments or functional zone can utilize recombination method preparation or utilize Peptide synthesizer synthetic.The antibody of being combined with the unmodified form of people HP gene product can for example, carry out immune animal and produce by the gene product of producing in prokaryotic cell prokaryocyte (E.Coli); The antibody of being combined with posttranslational modification form (as the albumen of glycosylation or phosphorylation or polypeptide), can for example, carry out immune animal and obtain by the gene product producing in eukaryotic cell (yeast or insect cell).

People HP Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For 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 need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.

Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This carrier of normally being cloned people, then proceed to cell, then by ordinary method separation from the host cell propagation, obtain relevant sequence.

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

At present, can be completely by chemosynthesis, carry out the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof).Then this DNA sequence dna can be introduced in DNA molecular (as carrier) and cell.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.

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

The encoding sequence of albumen of the present invention also can be used for the assignment of genes gene mapping.For example, by fluorescence in situ hybridization technique (FISH), cDNA clone is hybridized with the karyomit(e) of metaphase, can carry out exactly chromosomal localization.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 ofBasic Techniques, Pergamon Press, New York (1988).

Once sequence is located in certain exact position on karyomit(e), sequence can be associated with genetic map data by the physical location on karyomit(e).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, by linkage analysis, carry out identified gene and be positioned the dependency between the disease of same chromosomal region.

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

Utilize albumen of the present invention, by various conventional screening methods, can filter out with HP interactional material occurs, as acceptor, inhibitor or antagonist etc.

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

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

When people HP protein polypeptide of the present invention is used as medicine (as hair growth or oligotrichosis), this polypeptide for the treatment of effective dose can be applied to Mammals, wherein this treatment effective dose is conventionally at least about 10 micrograms/kg body weight, and be in most of the cases no more than approximately 8 mg/kg body weight, preferably this dosage is the about l mg/kg body weight of 10 micrograms/kg body weight-Yue.Certainly, concrete dosage also should be considered the factors such as route of administration, patient health situation, and these are all within skilled practitioners skill.

In one embodiment of the invention, the cDNA nucleotide sequence of people HP is so to obtain, take people's ovary λ gtllcDNA library (purchased from Clontech company) is template, with a pair of oligonucleotide, being primer---A:5 '-ATGAGCTACTACGGCAGCTAC-3 ' is forward primer, oligonucleotide B:5 '-TGCAAATAGCTCCAGCATGATC-3 ＇ is reverse primer, carries out PCR.After being checked order, amplified production obtains the full length cDNA sequence of SEQ ID NO.3.

People HP is form hair-keratin a kind of, and it and some other related angle albumen, as the High-sulphur protein that intermediate filament and intermediate filament are connected in albumen, the trunk of formation hair thereby ultra-high-sulfur(UHS) albumen etc. are cross-linked with each other.

Because people HP of the present invention has the natural acid sequence that is derived from people, therefore, compare with the albumen of the same clan that derives from other species, estimate when being applied to people to there is higher active and/or lower side effect (for example the immunogenicity in human body lower or do not have).

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence (D86422) of people HP of the present invention and mouse HGT 1 α.Wherein, identical amino acid marks with amino acid monocase 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 are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

Clone and the mensuration of the cDNA sequence of people HP

1. primer amplification

Take people's ovary λ gtllcDNA library (purchased from Clontech company) is template, with a pair of oligonucleotide, being primer---A:5 '-ATGAGCTACTACGGCAGCTAC-3 ' (SEQID NO:1) is forward primer, oligonucleotide B:5 '-TGCAAATAGCTCCAGCATGATC-3 ' (SEQ ID NO:2) is reverse primer, carries out PCR.The PCR condition of A/B be 93 ℃ 4 minutes, with 93 ℃ 1 minute, 60 ℃, within 1 minute and 72 ℃, within 1 minute, carry out 35 circulations thereupon, last 72 ℃ are extended 5 minutes.Electrophoresis detection obtains the object fragment of about 350bp.

The order-checking of 2.PCR product

Above-mentioned pcr amplification product A/B is connected with pGEM-T carrier (Promega), transform intestinal bacteria JM103, with QIAprep Plasmid test kit (QIAGEN), extract plasmid, with SequiTherm EXCELTM DNA sequencing kit (Epicentre Technologies), plasmid is checked order, finally, by computer software splicing order, obtain full length cDNA sequence, altogether 345bp, detailed sequence is shown in SEQ ID NO.3, and wherein open reading frame is positioned at 1-246 position Nucleotide.

According to the full length cDNA sequence obtaining, derive the aminoacid sequence of people HP, totally 81 amino-acid residues, its aminoacid sequence refers to SEQ ID NO.4.

Embodiment 2

Homology comparison

CDNA sequence and proteins encoded thereof with people HP of the present invention, in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, with blast program, carry out nucleic acid and albumen homology retrieval.Found that, they and mouse HGT 1 α (D86422) have high homology, and homology relatively finds that their identity on protein level has reached 64% (Fig. 1).In addition, study and also show, the HGTps of some that found in people HP albumen and mouse, rat and sheep also has higher homology, and therefore, HP albumen is considered to a kind of new people HGTp., the analysis of HP albumen is found, its phenylalanine content (7.4%) is higher than the content (6.2%) of halfcystine meanwhile, and this points out it may belong to I type HGTp.According to structures shape function, the Biological Principles that structural similitude function is relevant, can infer according to the function of PARP and 17HSD7 the function of albumen of the present invention.

The major structural protein that forms hair is Keratin sulfate, and Keratin sulfate can be divided into intermediate filament and the intermediate filament albumen that is connected, and the latter, can be subdivided into High-sulphur protein again, ultra-high-sulfur(UHS) albumen and high glycine/tyrosine (HGTps).Thereby be cross-linked with each other between these Keratin sulfate and some other associated protein, form the trunk of hair.Each proteinoid is all by a plurality of similar member composition family.So numerous similar members, may be that biology produces by gene redundancy during evolution in order to adapt to the needs of concentrated great expression hair protein in growth course.At present, the concrete effect for each hair protein does not still understand.But an interesting discovery is that the wool that has a kind of transgenation to produce, hardly containing HGTps, consequently this wool is malthoid sample gloss (felting lustermutant) (Gillespie JM and Darskus RL, Aust, J.Biol.Sci.1971,24:1189-1197)

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

For the antibody of inventor HP, for screening other members of this family, or for affinity purification associated protein (other members of Ru Gai family).

In addition, inventor HP nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, to improve the overexpression of expression level or the inhibition people HP of people HP.People HP albumen of the present invention or its active polypeptide fragment can be applied to patient, to treat or to alleviate because of people HP disappearance, nonfunctional or the abnormal related disorders causing.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 HP in intestinal bacteria

In this embodiment, the pcr amplification product A/B in embodiment 1 of take is template, use the PCR Oligonucleolide primers (SEQ ID NO.5 and 6) corresponding to 5 of this DNA sequence dna ' and 3 ' end to increase the cDNA sequence of encoding human HP, obtain people HP cDNA as Insert Fragment.

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

5 '-TCAGGGATCCATGAGCTACTACGGCAGCT-3 ＇ (SEQ ID NO.5), the restriction enzyme site that this primer contains BamHI restriction enzyme is 19 Nucleotide of the people HP encoding sequence that started by initiator codon after this restriction enzyme site;

3 ' end primer sequence is

5 '-TTGGAAGCTTTTAATAGAATCCAGAGAAT-3 ＇ (SEQ ID NO.6), this primer contains the restricted part encoding sequence of cutting restriction enzyme site, translation termination and the people HP of enzyme of Hind III.

The restriction enzyme site of the restriction enzyme on primer is corresponding to the restriction enzyme digestion sites on bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA), 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 BamH I and Hind III digestion pQE-9 carrier and Insert Fragment, subsequently Insert Fragment be connected to pQE-9 carrier and keep open reading frame initial at bacterium RBS.With connecting mixture, transform purchased from Qiagen subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, the plasmid pREP4 that M15/rep4 contains multiple copied, it is expressed lac I repressor and carries kalamycin resistance (Kan ^r).On the LB culture dish that contains Amp and Kan, screen transformant, extracting plasmid, the cDNA fragment of sequence verification people HP has correctly been inserted carrier.

In the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml), incubated overnight (O/N) is containing the positive transformant clone of required construction.Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be then inoculated in large volume culture base, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) while being 0.4-0.6, add IPTG (" isopropylthio-β-D-galactoside ") to final concentration be lmM.By making lac I repressor inactivation, IPTG induction starts P/O and causes gene expression dose to improve.Continue culturing cell 3-4 hour, centrifugal (6000 * g, 20 minutes) subsequently.Ultrasonic degradation culture, collecting cell lysate is also diluted in 6M Guanidinium hydrochloride.After clarification, by making under the condition of combining closely containing 6-His marker albumen, with nickel-chelate column chromatography people HP that purifying dissolves from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people HP from post.Available several method is sex change protein precipitation from Guanidinium hydrochloride.Or, use dialysis step to remove Guanidinium hydrochloride, or from nickel-chelate column isolated purifying protein.Protein after 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, uses Guanidinium hydrochloride (pH5.0) wash-out subsequently.Finally, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

With 12% SDS-PAGE glue, carry out electrophoresis, the molecular size range of identifying expressing protein is about 8.2KDa.

In addition, by ordinary method, to reaching N end and the C of albumen, hold the amino acid of each 10 amino acid lengths to check order, find consistent with the sequence of SEQ ID NO.4.

Embodiment 4

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

In this embodiment, the pcr amplification product A/B in embodiment 1 of take is template, use the PCR Oligonucleolide primers (SEQ ID NO.7 and 8) corresponding to 5 of this DNA sequence dna ' and 3 ' end to increase the cDNA sequence of encoding human HP, obtain people HP cDNA as Insert Fragment.

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

5′-TCAGAAGCTTATGAGCTACTACGGCAGCT-3′(SEQ?ID?NO.7)

The restriction enzyme site that this primer contains Hind III restriction enzyme is 19 Nucleotide of the people HP encoding sequence that started by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5-TTGGGGATCCTTAATAGAATCCAGAGAAT-3i(SEQ?ID?NO.8)

The part encoding sequence of the restriction enzyme site that this primer contains BamH I restriction enzyme, translation termination and people HP.

The restriction enzyme site of the restriction enzyme on primer is corresponding to the restriction enzyme digestion sites on expressing cho cell carrier pcDNA3, 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 Hind III and BamH I digestion pcDNA3 carrier and Insert Fragment, subsequently Insert Fragment is connected to pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.On the LB culture dish that contains Amp, screen transformant, in the LB liquid nutrient medium of adding Amp (100 μ g/ml), incubated overnight (O/N) is containing the clone of required construction.Extracting plasmid, cuts and the cDNA fragment of sequence verification people HP has correctly been inserted carrier with EcoR I enzyme.

Plasmid transfection Chinese hamster ovary celI is to adopt lipofection, with Lipofectin (GiBco Life), carries out.After transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing clone cell Method of Limited Dilution, select to have the cell subclone compared with high protein activity.Cultivate in a large number according to a conventional method above-mentioned positive subclone.After 48 hours, start collecting cell and supernatant, with ultrasonic degradation method smudge cells.50mMTrisHCl (pH7.6) solution of take containing 0.05%Triton is balance liquid and elutriant, uses the Peak Activity of collecting above-mentioned albumen through the Superdex of pre-equilibration G-75 post.Use again the DEAE-Sepharose post of 50mMTrisHCl (pH8.0) balance, take and carry out gradient elution containing 50mMTrisHCl (pH8.0) solution of 0-1M NaCl as elutriant, collect the Peak Activity of above-mentioned albumen.Then the PBS (pH7.4) of take dialyses to expressing protein solution as dialyzate.Last freeze-drying is preserved.

With 12% SDS-PAGE glue, carry out electrophoresis, identify that the molecular size range of expressing protein is about 8.2KDa.

In addition, by ordinary method, to reaching N end and the C of albumen, hold the amino acid of each 10 amino acid lengths to check order, find consistent with the sequence of SEQ ID NO.4.

Embodiment 5

Dispersal risk

The recombinant protein obtaining in embodiment 3 and 4 is used for to immune animal to produce antibody, and concrete grammar is as follows.It is rear standby that recombinant molecule carries out separation by chromatography.Also available SDS-PAGE gel electrophoresis carries out separation, electrophoretic band is cut 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, with the same antigen of non-complete Freundis adjuvant emulsion, mouse is carried out to peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml.Every 14 days, carry out one time booster immunization, at least carry out three times.The sero-fast specific reaction activity obtaining is assessed by the ability that it precipitates people HP gene translation product in vitro.Found that, antibody can precipitate with albumen of the present invention specifically.

All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that 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 limited range equally.

Sequence table (1) general information:

(I) denomination of invention: a kind of new HAIR PROTEIN and encoding sequence thereof

(II) sequence number: the information of 8 (2) SEQ ID NO.1

(I) sequence signature

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: oligonucleotide

(X I) sequence description: the information of SEQ ID NO.1:ATGAGCTACT ACGGCAGCTAC 21 (2) SEQ ID NO.2

(I) sequence signature

(A) length: 22 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: oligonucleotide

(X I) sequence description: the information of SEQ ID NO:2:TGCAAATAGC TCCAGCATGA TC 22 (2) SEQ ID NO.3:

(I) sequence signature:

(A) length: 345bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(II) molecule type: cDNA

( ⅲ ) 序列描述：SEQ ID NO.3： 1 ATGAGCTACT ACGGCAGCTA CTATGGAGGC CTGGGCTATG GCTGTGGAGG CTTTGGTGGC 61 CTGGGCTATG GCTATGGCTG TGGATGTGGC AGCTTCCGCA GACTGGGTTC TGGCTGTGGC121 TATGGAGGCT ACGGATATGG CTCTGGCTTT GGAGGCTATG GATATGGCTC TGGCTTCGGA181 GGCTACGGAT ATGGCTGCTA CCGCCCATCA TACTATGGAG GATATGGATT CTCTGGATTC241 TATTAAACTA CTGCCCCAGC AACACAATGT GTGAAATTAT AAGAGGACTT TCCCAGAGCT301 GACTTCAATC ATTGGACAAC AAAGATCATG CTGGAGCTAT TTGCA ( 2 ) SEQ IDNO.4的信息：

(I) sequence signature:

(A) length: 81 amino acid

(B) type: amino acid

(D) topological framework: linearity

(II) molecule type: polypeptide

(X I) sequence description: the information of SEQ ID NO.4:1 Met Ser Tyr Tyr Gly Ser Tyr Tyr Gly Gly Leu Gly Tyr Gly Cys16 Gly Gly Phe Gly Gly Leu Gly Tyr Gly Tyr Gly Cys Gly Cys Gly31 Ser Phe Arg Arg Leu Gly Ser Gly Cys Gly Tyr Gly Gly Tyr Gly46 Tyr Gly Ser Gly Phe Gly Gly Tyr Gly Tyr Gly Ser Gly Phe Gly61 Gly Tyr Gly Tyr Gly Cys Tyr Arg Pro Ser Tyr Tyr Gly Gly Tyr76 Gly Phe Ser Gly Phe Tyr (2) SEQ ID NO.5

(I) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: oligonucleotide

(X I) sequence description: SEQ ID NO.5:

TCAGGGATCC?ATGAGCTACT?ACGGCAGCT????29

(2) information of SEQ ID NO.6

(I) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: oligonucleotide

(X I) sequence description: SEQ ID NO.6:

TTGGAAGCTT?TTAATAGAAT?CCAGAGAAT????29

(2) information of SEQ ID NO.7

(I) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: oligonucleotide

(X I) sequence description: SEQ ID NO.7:

TCAGAAGCTT?ATGAGCTACT?ACGGCAGCT????29

(2) information of SEQ ID NO.8

(I) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: oligonucleotide

(X I) sequence description: SEQ ID NO.8:

TTGGGGATCC?TTAATAGAAT?CCAGAGAAT????29

Claims (10)

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

In described nucleotide sequence and SEQ ID NO.3, from the nucleotides sequence of Nucleotide 1-246 position, show at least 70% homology; Or

Described nucleotide sequence can be under moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 1-246 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 SEQ ID NO.4.

3. DNA molecular as claimed in claim 1, is characterized in that, this sequence has in SEQ ID NO.3 the nucleotide sequence from Nucleotide 1-246 position.

4. a separated people HP protein polypeptide, is characterized in that, it comprises: there is polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4, is characterized in that, this polypeptide is the polypeptide with SEQ ID NO.4 sequence.

6. a carrier, is characterized in that, it contains DNA claimed in claim 1.

7. the host cell transforming with carrier described in claim 6.

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

(a) nucleotide sequence that coding is had to a polypeptide of people HP protein-active is operationally connected in expression regulation sequence, form people HP protein expression vector, in described nucleotide sequence and SEQ ID NO.3, from the nucleotides sequence of Nucleotide 1-246 position, show at least 70% homology;

(b) expression vector in step (a) is proceeded to host cell, form the reconstitution cell of people HP albumen;

(c) be applicable to expressing under the condition of people HP protein polypeptide the reconstitution cell in culturing step (b);

(d) isolate the polypeptide with people HP protein-active.

9. the antibody of an energy and people HP protein polypeptide specific binding claimed in claim 4.

10. a probe molecule, is characterized in that, it contains 8-100 continuous nucleotide in DNA molecular claimed in claim 1.