CN1250095A - New human gene series and the polypeptide therewith and its preparation - Google Patents

Abstract

The present invention provides one new member HSyll of secl gene family. The present invention provides the cDNA coding sequence of the human HSycl, the sequence encoded polypeptide and the method of utilizing recombination technology in producing new human HSlyl. The present invention also provides the application of this new kind of human HSlyl gene and polypeptide.

Description

A kind of new people's gene sequence, its encoded polypeptides and preparation method

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new people's gene and albumen.More particularly, the present invention relates to a kind of member HSly1 of new sec1 gene family.The invention provides the cDNA encoding sequence of this people HSly1, the polypeptide of this sequence encoding, and utilize recombinant technology to produce the method for described new people HSly1.The present invention also provides the application of this new person HSly1.

In eukaryotic cell, golgi body is a main transport hub of macromole transportation in the cell., classification processed in golgi body by endoplasmic reticulum synthetic protein, lipid with pack, be transported to the specific position of cell then respectively or be secreted into the extracellular.The Secretory Pathway of golgi body mainly divides two classes: in the inherency Secretory Pathway, by the successive exocytosis protein and phosphatide are transported to cell surface; In the modulability Secretory Pathway, temporarily be stored in after the secretory vacuole maturation in the tenuigenin, under extraneous signal stimulated, the secretory vacuole inclusion discharged (" cytobiology ", (1995), Higher Education Publishing House, Zhai Zhonghe chief editor) to the extracellular.At present find in the transportation of two kinds of approach, all to relate to the transhipment and the fusion of film, and with film on receptor related.By several relevant gene families, instruct jointly vesica sprout, directed move with different compartments between the fusion that takes place.Two gene families that syntaxin (syntaxin) and sec1 come to this.

Acceptor in the syntaxin gene family coding gang vesica transportation on the film, all relevant with the transhipment of film in the two class Secretory Pathways.The member of this family comprises syntaxin 1A in the mammalian cell, 1B, 2,3A-3E, 4,5,6 and yeast in Pep12p, Sso1/2p and Sed5p etc. (Dascher C et al, J.Biol.Chem, 1994,269 (47), 29363-29366).Wherein, syntaxin 1A/B, 2,3,4 is the docking receptor on the cytolemma, discerns and combines with the membranin on the vesica, promotes vesica film and the fusion of cytolemma and the release of inclusion.(Bennett?MK?et?al，Proc.Natl.Acad.Sci.U.S.A，1993，90，2559-2563)。And syntaxin 5 is found in the golgi body zone, it is that endoplasmic reticulum synthetic product is transported to necessary protein (Dascher C et al, J.Biol.Chem, 1994 in the vesica transportation of gorky's somatocyst heap, 269 (47), 29363-29366).Under the steady state, syntaxin 5 is main relevant with the activity of golgi body inboard, quilt recycle promptly (Walch-Solimena C et al. in its compartment between endoplasmic reticulum and preceding golgi body or golgi body inboard, J.Cell.Biol, 1995,128,637).The overexpression meeting of syntaxin 5 causes the preceding accumulation of golgi body intermediate in cell.In addition, syntaxin 5 also may be relevant with maturation with the formation of preceding golgi body intermediate.Syntaxin 5 exists down, and intermediate could generate normally, forms the network of golgi body inboard, for the maturation of golgi body provides basis (Presley JFet al., Nature, 1997,389,81).

The member of sec1 gene family comprises the Sly1 in distinctive Munc-18/n-Sec1/rb-Sec1, the yeast, Vps33 in the neurone, Vps45 and (the William EB etal. such as isotype Munc-18 of wide expression in Mammals, J Biol.Chem. (1996) Vol 271 (27), 15866-15869).This family member concrete mechanism of action in the vesica transportation is still not quite clear, but because they can both form mixture with the member of the known syntaxin of function family, ras family, influence the latter's function, in the vesica transportation, very important function must be arranged so can infer the member of sec1 family.Wherein, yeast Sly1 can form mixture respectively with the different members of syntaxin gene family such as Sed5, syntaxin 1,2,3,4, also can form mixture (Ossig R et al. with the necessary regulatory factor Ypt1 of Secretory Pathway in the eukaryotic cell cell, Mol.Cell.Biol, 1991, Vol11 (6), 2980-2993).Ypt1 is a member of ras gene superfamily, is little gtp binding protein.Because some albumen is in conjunction with GTP or GDP the time, in Secretory Pathway vesica sprout or different compartment between take place to show different activity in the incident such as fusion, the specificity of interior transportation of born of the same parents and the interior albumen sorting of vesica finally depends on the gtp binding protein of many different sites in the different Secretory Pathways, comprises Ypt1, Sec4p, ARF1, ARF2 etc.Lack Ypt1 and will cause the blocking-up of endoplasmic reticulum to the golgi body haulage track.The point mutation of Sly1 can suppress Ypt1 afunction (Dascher MR et al., Mol Cell.Biol, 1991, Vol11,872-885).The product of Sly1 gene plays a role in early days at Secretory Pathway, and experimental observation is arrived, and lacks in the cell of Sly1 to accumulate endoplasmic reticulum, normally secretory protein.People such as nineteen ninety-five Peterson MR at first find the homologous gene of Sly1 in the Mammals, called after rSly1 in the liver of mouse.Its protein product can form mixtures with syntaxin 5, regulation and control syntaxin 5 the function of endoplasmic reticulum in the golgi body transportation (Peterson MR et al, Gene, (1996), 169,293-294).Before the present invention, also there is not to disclose or delivered the homologous gene of the Sly1 in the human body.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding sec1 gene family, the gene of the people sec1 family that the present invention is new is named as HSly1.

Another object of the present invention provides a kind of new sec1 protein family member, and this albumen is named as HSly1 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people HSly1.

The invention still further relates to the application of this people HSly1 gene order and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people HSly1 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 35-1888 position among described nucleotide sequence and the SEQ ID NO.5; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.5 in from the nucleotide sequence hybridization of Nucleotide 35-1888 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.6.More preferably, this sequence has among the SEQ ID NO.5 nucleotide sequence from Nucleotide 35-1888 position.

In another aspect of this invention, provide a kind of isolating HSly1 protein polypeptide, it comprises: have polypeptide or its active fragments of SEQID NO.6 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.6 polypeptide of sequence.

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

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

In another aspect of this invention, the method that provides a kind of generation to have the polypeptide of HSly1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HSly1 protein-active operationally is connected in expression regulation sequence, form the HSly1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 35-1888 position among described nucleotide sequence and the SEQ ID NO.5;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of HSly1;

(c) be fit to express under the condition of HSly1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HSly1 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 583 Nucleotide, and its detailed sequence is seen SEQ ID NO.5, and wherein open reading frame is positioned at 35-1888 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 " HSly1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HSly1 protein-active is as 35-1888 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.5.This degenerate sequence is meant, is arranged in the encoder block 35-1888 position Nucleotide of SEQ ID NO.5 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.5 in 35-1888 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.6 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.5 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 35-1888 position.In addition, this term also comprise with SEQ ID NO.5 in from the homology of nucleotide sequence at least 70% of Nucleotide 35-1888 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.5 with people HSly1 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 " HSly1 protein polypeptide " refers to have the SEQ ID NO.6 polypeptide of sequence of HSly1 protein-active.This term also comprises having and variant form people HSly1 albumen identical function, SEQ IDNO.6 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 HSly1 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of HSly1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-HSly1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises HSly1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of HSly1 polypeptide.Usually, this fragment have the HSly1 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 HSly1 albumen or polypeptide.The difference of these analogues and natural HSly1 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 can also comprise having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

The present invention also comprises the antisense sequences of HSly1 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of HSly1 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer.This probe molecule has 8-100 of HSly1 nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the HSly1 that encodes.

The present invention also comprises the method that detects the HSly1 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 can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of HSly1 polypeptide.Primer length is generally 20-50 Nucleotide.

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

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

On the other hand, the present invention also comprises HSly1 DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into HSly1 gene product or fragment.Preferably, refer to that those can combine with HSly1 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 HSly1, comprise that also those do not influence the antibody of HSly1 protein function.The present invention also comprise those can with modify or without the HSly1 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 HSly1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing HSly1 or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology 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 the HSly1 function and the antibody that does not influence the HSly1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of HSly1 gene product, obtains by immunological technique.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 HSly1 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).

HSly1 nucleotide sequence 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

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

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

In one embodiment of the invention, polynucleotide total length of the present invention is 1992 Nucleotide, and its detailed sequence is seen SEQ ID NO.5, and wherein open reading frame is positioned at 35-1888 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A:5 '-TATTCGGGAAAGGCAGACAGTGG-3 ' (SEQ ID NO.1), forward primer C:5 '-TTGGACATATCAAGCATTGGTGC-3 ' (SEQ ID NO.3) and reverse primer B:5 '-AACAGCTGATG TTAGCTTAGCGG-3 ' (SEQ ID NO.2), reverse primer D:5 '-TTAGGACACTGTTACAG AGAAGG-3 ' (SEQ ID NO.4) carry out twice PCR, obtain the purpose fragment of 1992bp.Obtain the full length cDNA sequence of SEQ ID NO.5 after the order-checking.

According to homology result relatively, the sec1 family member of nucleotide sequence of the present invention and encoded protein matter sequence and different sources has shown significant homology, therefore, this shows that it is a newcomer of sec1 family, and has some critical functions of sec1 family protein.

HSly1 is the important composition of protein excretion mechanism in the human body cell, in vesica transportation (the especially transportation of the vesica from the endoplasmic reticulum to the golgi body) important function is arranged, also may be relevant with the maturation and the normal enforcement of function of golgi body.When cell was subjected to extraneous pressure, HSly1 can change biochemical synthetic direction, significantly improved the output of specific protein and promoted proteic release.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the nucleotide sequence (RSLY1N) of people HSly1 nucleotide sequence of the present invention (HSLY1N) and mouse RSLY1.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people HSly1 albumen of the present invention (HSLY1P) and mouse RSLY1 albumen (RSLY1P).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 HSly1

1. primer amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with two pairs of oligonucleotide is primer---A:5 '-TATTCGGGAAAGGCAGACAGTGG-3 ' (SEQ ID NO.1) and C:5 '-TTGGACATATCAAGCATTGGTGC-3 ' (SEQ ID NO.3) they are forward primer, oligonucleotide B:5 '-AACAG CTGATGTTAGCTTAGCGG-3 ' (SEQ ID NO.2) and D:5 '-TTAGGACACTGTTACAGAGAAGG-3 ' (SEQ ID NO.4) are reverse primer, carry out PCR.The A/B primer to the right PCR condition of C/D primer be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 67 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is respectively 1117, the purpose fragment of 1211bp.

2.PCR the order-checking of product

With two sections pcr amplification products as above obtaining respectively with pGEM-T ^Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1992bp, detailed sequence is seen SEQ ID NO.5, and wherein open reading frame is positioned at 35-1888 position Nucleotide.

Derive the aminoacid sequence of HSly1 according to the full length cDNA sequence that obtains, totally 617 amino-acid residues, its aminoacid sequence sees SEQ ID NO.6 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with HSly1 carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database.The member who finds they and sec1 gene family has higher homology, wherein reaches 85.5% identical (Fig. 1) with the rSly1 gene of mouse on nucleic acid level, reaches 96% identical on protein level, 98% similar (Fig. 2).Thereby can think that the gene that we found is the homologous gene of rSly1, be a newcomer of sec1 gene family.

RSly1 and zymic Sly1 are homologous gene (homology of nucleic acid are 30%), its open reading frame hydrophilic albumen that molecular weight is 72～73kD of having encoded.This albumen can form mixture with syntaxin 5, is just regulating and control the function of syntaxin 5 in endoplasmic reticulum is transported to golgi body.Possible regulatory mechanism is: the formation that can promote to stop-merge mixture that combines of rSly1 and syntaxin 5.The overexpression of syntaxin 5 will be with exhausting endogenic rSly1, and the syntaxin 5 that does not form mixture will hinder the formation of normal protein complex in vesica stop and the fusion process.Only when the rSly1 expression was equivalent to or is in excess in syntaxin 5, syntaxin 5 could normal functionating.And the rSly1 of overexpression itself does not have remarkable negative effect to endoplasmic reticulum to the transportation of golgi body.(William?EB?et?al.，J?Biol.Chem.(1996)Vol?271(27)，15866-15869)。RSly1 is the same with other members of sec1 family, does not have transmembrane domains.In eight different tissues of mouse, found the expression of rSly1, wherein maximum in brain and in the lung.The wide expression of rSly1 hint rSly1 may the endoplasmic reticulum in a organized way play a role in the transportation of golgi body (Peterson MR et al, Gene (1996), 169,293-294).

The research of being carried out so far shows that all it is high conservative that the necessity in the protein excretion mechanism is formed in evolution.By sequential analysis and homology result relatively to HSly1, can infer, HSly1 is the important composition of protein excretion mechanism in the human body cell, in vesica transportation (the especially transportation of the vesica from the endoplasmic reticulum to the golgi body), important function is arranged, also may be relevant with the maturation and the normal enforcement of function of golgi body.Because golgi body is in the intermediary status in endomembrane system, many important macromolecular transportations and secretion all will be passed through golgi body, as the secretory granules of gland cell, the cytoplasmic granule of hemocyte and the acrosome of spermoblast.Golgi body is also relevant with proteinic transformation with the formation of new cytoplasmic membrane, cell walls.Pathologic variation takes place in the cell that will make unusually of golgi body and protein excretion approach, or the change of differentiation degree takes place, and causes illnesss such as joint inflammation.

In another research, find, (protein product of this gene is named as RA410 again to rSly1 in this research, Matsuo N et al., J.Biol.Chem., 1997, Vol272 (26), 16438) with the albumen processing of back golgi body with transport relevant, again during oxygen supply, the expression amount of RA410 in cell can obviously increase after astroglia cell is because of the local asphyxia anoxic.This means that RA410 is likely the major function composition of the protein transportation of carrying out when astroglia cell is subjected to extraneous pressure.Astroglia cell has very important function in central nervous system, with keeping of hemato encephalic barrier and moving of excitatory neurotransmitter relation is arranged all.Astroglia cell can the founder cell factor, as interleukin I L-1, and IL-6 and Interferon, rabbit etc.Verified, again during oxygen supply, can generate IL-6 after the astroglia cell local asphyxia.If but the expression of RA410 is suppressed, IL-6 can't normally generate.As seen, RA410 after anoxic again provide optimized transportation for IL-6 in the astroglia cell of oxygen.Can infer that the homologous gene product of the Sly1 that finds also may have similar function with RA410 in human body, promptly when cell is subjected to extraneous pressure, can change biochemical synthetic direction, significantly improve the output of specific protein and promote proteic release.

Understanding to structure and the sequence of HSly1 not only helps the further research to its function, more helps further to illustrate the molecular mechanism of intracellular matter transportation, for the treatment of relative disease provides theoretical foundation.

People HSly1 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 HSly1 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor HSly1 and the N end of mouse RSly1 are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor HSly1, 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 HSLY1 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people HSLY1 or the overexpression that suppresses people HSLY1.People HSLY1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people HSLY1 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 HSly1 in intestinal bacteria

In this embodiment, two fragments that will obtain in embodiment 1 are cut with Xmn I enzyme respectively, mix and add ligase enzyme and connect, and walk electrophoresis after connection is finished, and identify and also reclaim the correct full length fragment that connects.As template, use the PCR Oligonucleolide primers corresponding to 5 ', 3 ' end of this dna sequence dna to increase the dna sequence dna of coding HSly1, the cDNA that obtains HSly1 is as inserting fragment.

5 ' Oligonucleolide primers sequence is

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

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 19 Nucleotide of the HSly1 encoding sequence that begun by initiator codon;

3 ' Oligonucleolide primers sequence is

5’-ACAGGTCGACTTACTTTTGTCCAAGTTGT-3’(SEQ?ID?NO.8)，

This primer contains the restriction enzyme site of SalI restriction enzyme, the encoding sequence of translation termination and HSly1.

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

With BamHI and SalI 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, 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).The extracting plasmid is cut with the HindIII enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the cDNA of HSly1 inserts the fragment carrier of correctly packing into.

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 HSly1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out HSly1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, 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 70KDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.6 with ordinary method.

Embodiment 4

The expression of HSly1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, two fragments that will obtain in embodiment 1 are cut with Xmn I enzyme respectively, mix and add ligase enzyme and connect, and walk electrophoresis after connection is finished, and identify and also reclaim the correct full length fragment that connects.As template, use the PCR Oligonucleolide primers corresponding to 5 ', 3 ' end of this dna sequence dna to increase the dna sequence dna of coding HSly1, the cDNA that obtains HSly1 is as inserting fragment.

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

5′-GCATGGATCCATGTTGAATTTCAATGTGC-3′(SEQ?ID?NO.9)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 19 Nucleotide of the HSly1 encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

5’-ACAGGAATTCTTACTTTTGTCCAAGTTGT-3’(SEQ?ID?NO.10)

This primer contains the encoding sequence of the restriction enzyme site of EcoRI restriction enzyme, translation termination and HSly1.

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 BamHI and EcoRI 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, sequence verification result shows that the cDNA of HSly1 inserts the fragment carrier of correctly packing into.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured 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 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (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 70KDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.6 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, specific as follows.Recombinant protein is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation HSly1 gene translation product with it.

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: Xinhuangpu-Fudan Gene Engineering Co., Ltd., Shanghai is denomination of invention (ii): a kind of new people's gene sequence, its encoded polypeptides and preparation method be the sequence number (iii): 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:TATTCGGGAA AGGCAGACAG TGG 23 (2) SEQ ID NO.2

(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.2AACAGCTGAT GTTAGCTTAG CGG 23 (2) SEQ ID NO.3

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO.3

TTGGACATAT?CAAGCATTGG?TGC 23

(2) information of SEQ ID NO.4

(i) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO.4

TTAGGACACT?GTTACAGAGA?AGG 23

(2) information of SEQ ID NO.5:

(i) sequence signature:

(A) length: 1992bp

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO.5 1 TATTCGGGAA AGGCAGACAG TGGCTTTGAA GCGTATGTTG AATTTCAATG TGCCTCATAT 61 TAAAAACAGC ACAGGAGAAC CAGTATGGAA GGTACTCATT TATGACAGAT TTGGCCAAGA121 TATAATCTCT CCTCTGCTAT CTGTGAAGGA GCTAAGAGAC ATGGGAATCA CTCTGCATCT181 GCTTTTACAC TCTGATCGAG ATCCTATTCC AGATGTTCCT GCAGTATACT TTGTAATGCC241 AACTGAAGAA AATATTGACA GAATGTGCCA GGATCTTCGA AATCAACTAT ATGAATCATA301 TTATTTAAAT TTTATTTCTG CTATTTCAAG AAGTAAACTG GAAGATATTG CAAATGCAGC361 GTTAGCAGCT AGTGCAGTAA CACAAGTAGC CAAGGTTTTT GACCAATATC TCAATTTTAT421 TACTTTGGAA GATGATATGT TTGTATTATG TAATCAAAAT AAGGAGCTTG TTTCATATCG481 TGCCATTAAC AGGCCAGATA TCACAGACAC GGAAATGGAA ACTGTTATGG ACACTATAGT541 TGACAGCCTC TTCTGCTTTT ATGGTACTCT GGGTGATGTT CCTATAATCA GATGTTCAAG601 AGGAACAGCA GCAGAAATGG TAGCAGTGAA ACTAGACAAG AAACTTCGAG AAAATCTAAG661 AGATGCAAGA AACAGTCTTT TTACAGGTGA TACACTTGGA GCTGGCCAAT TCAGCTTCCA721 GAGGCCCTTA TTAGTCCTTG TTGACAGAAA CATAGATTTG GCAACTCCTT TACATCATAC781 TTGGACATAT CAAGCATTGG TGCACGATGT ACTGGATTTC CATTTAAACA GGGTTAATTT841 GGAAGAATCT TCAGGAGTGG AAAACTCTCC AGCTGGTGCT AGACCAAAGA GAAAAAACAA901 GAAGTCTTAT GATTTAACTC CGGTTGATAA ATTTTGGCAA AAACATAAAG GAAGTCCATT961 CCCAGAAGTT GCAGAATCAG TTCAGCAAGA ACTAGAATCT TACAGAGCAC AGGAAGATGA1021 GGTCAAACGA CTTAAAAGCA TTATGGGACT AGAAGGGGAA GATGAAGGAG CCATAAGTAT1081 GCTTTCTGAC AATACCGCTA AGCTAACATC AGCTGTTAGT TCTTTGCCAG AACTCCTTGA1141 GAAAAAAAGA CTTATTGATC TCCATACAAA TGTTGCCACT GCTGTTTTAG AACATATAAA1201 GGCAAGAAAA TTGGATGTAT ATTTTGAATA TGAAGAAAAA ATAATGAGCA AAACTACTCT1261 GGATAAATCT CTTCTAGATA TAATATCAGA CCCTGATGCA GGAACTCCAG AAGATAAAAT1321 GAGGTTGTTT CTTATCTATT ATATAAGCAC ACAGCAAGCA CCTTCTGAGG CTGATTTGGA1381 GCAATATAAA AAAGCTTTAA CTGATGCAGG ATGCAACCTT AATCCTTTAC AATATATCAA1441 ACAGTGGAAG GCTTTTACCA AGATGGCCTC AGCTCCGGCC AGCTATGGCA GCACTACCAC1501 TAAACCAATG GGTCTTTTAT CACGAGTCAT GAATACAGGA TCACAGTTTG TGATGGAAGG1561 AGTGAAGAAC CTGGTTTTGA AACAGCAAAA TCTACCTGTT ACTCGTATTT TGGACAATCT1621 TATGGAGATG AAGTCAAACC CCGAAACTGA TGACTATAGA TATTTTGATC CCAAAATGCT1681 GCGGGGCAAT GACAGCTCAG TTCCCAGAAA TAAAAATCCA TTCCAAGAGG CCATTGTTTT1741 TGTGGTGGGA GGAGGCAACT ACATTGAATA TCAGAATCTT GTTGACTACA TAAAGGGGAA1801 ACAAGGCAAA CACATTTTAT ATGGCTGCAG TGAGCTTTTT AATGCTACAC AGTTCATAAA1861 ACAGTTGTCA CAACTTGGAC AAAAGTAACA CAGAAGAACC TTACTATGAT AATCTACTTG1921 GAATGTGGAT AAATGTAAAA AGAAGAAAAG TTAGAAGAGC AATATGTTTC CTTCTCTGTA1981 ACAGTGTCCT AA ( 2 ) SEQ ID NO.6： ( i ) ：

(A) length: 617 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.6 1 Met Leu Asn Phe Asn Val Pro His Ile Lys Asn Ser Thr Gly Glu 16 Pro Val Trp Lys Val Leu Ile Tyr Asp Arg Phe Gly Gln Asp Ile 31 Ile Ser Pro Leu Leu Ser Val Lys Glu Leu Arg Asp Met Gly Ile 46 Thr Leu His Leu Leu Leu His Ser Asp Arg Asp Pro Ile Pro Asp 61 Val Pro Ala Val Tyr Phe Val Met Pro Thr Glu Glu Asn Ile Asp 76 Arg Met Cys Gln Asp Leu Arg Asn Gln Leu Tyr Glu Ser Tyr Tyr 91 Leu Asn Phe Ile Ser Ala Ile Ser Arg Ser Lys Leu Glu Asp Ile106 Ala Asn Ala Ala Leu Ala Ala Ser Ala Val Thr Gln Val Ala Lys121 Val Phe Asp Gln Tyr Leu Asn Phe Ile Thr Leu Glu Asp Asp Met136 Phe Val Leu Cys Asn Gln Asn Lys Glu Leu Val Ser Tyr Arg Ala151 Ile Asn Arg Pro Asp Ile Thr Asp Thr Glu Met Glu Thr Val Met166 Asp Thr Ile Val Asp Ser Leu Phe Cys Phe Tyr Gly Thr Leu Gly181 Asp Val Pro Ile Ile Arg Cys Ser Arg Gly Thr Ala Ala Glu Met196 Val Ala Val Lys Leu Asp Lys Lys Leu Arg Glu Asn Leu Arg Asp211 Ala Arg Asn Ser Leu Phe Thr Gly Asp Thr Leu Gly Ala Gly Gln226 Phe Ser Phe Gln Arg Pro Leu Leu Val Leu Val Asp Arg Asn Ile241 Asp Leu Ala Thr Pro Leu His His Thr Trp Thr Tyr Gln Ala Leu256 Val His Asp Val Leu Asp Phe His Leu Asn Arg Val Asn Leu Glu271 Glu Ser Ser Gly Val Glu Asn Ser Pro Ala Gly Ala Arg Pro Lys286 Arg Lys Asn Lys Lys Ser Tyr Asp Leu Thr Pro Val Asp Lys Phe301 Trp Gln Lys His Lys Gly Ser Pro Phe Pro Glu Val Ala Glu Ser316 Val Gln Gln Glu Leu Glu Ser Tyr Arg Ala Gln Glu Asp Glu Val331 Lys Arg Leu Lys Ser Ile Met Gly Leu Glu Gly Glu Asp Glu Gly346 Ala Ile Ser Met Leu Ser Asp Asn Thr Ala Lys Leu Thr Ser Ala361 Val Ser Ser Leu Pro Glu Leu Leu Glu Lys Lys Arg Leu Ile Asp376 Leu His Thr Asn Val Ala Thr Ala Val Leu Glu His Ile Lys Ala391 Arg Lys Leu Asp Val Tyr Phe Glu Tyr Glu Glu Lys Ile Met Ser406 Lys Thr Thr Leu Asp Lys Ser Leu Leu Asp Ile Ile Ser Asp Pro421 Asp Ala Gly Thr Pro Glu Asp Lys Met Arg Leu Phe Leu Ile Tyr436 Tyr Ile Ser Thr Gln Gln Ala Pro Ser Glu Ala Asp Leu Glu Gln451 Tyr Lys Lys Ala Leu Thr Asp Ala Gly Cys Asn Leu Asn Pro Leu466 Gln Tyr Ile Lys Gln Trp Lys Ala Phe Thr Lys Met Ala Ser Ala481 Pro Ala Ser Tyr Gly Ser Thr Thr Thr Lys Pro Met Gly Leu Leu496 Ser Arg Val Met Asn Thr Gly Ser Gln Phe Val Met Glu Gly Val511 Lys Asn Leu Val Leu Lys Gln Gln Asn Leu Pro Val Thr Arg Ile526 Leu Asp Asn Leu Met Glu Met Lys Ser Asn Pro Glu Thr Asp Asp541 Tyr Arg Tyr Phe Asp Pro Lys Met Leu Arg Gly Asn Asp Ser Ser556 Val Pro Arg Asn Lys Asn Pro Phe Gln Glu Ala Ile Val Phe Val571 Val Gly Gly Gly Asn Tyr Ile Glu Tyr Gln Asn Leu Val Asp Tyr586 Ile Lys Gly Lys Gln Gly Lys His Ile Leu Tyr Gly Cys Ser Glu601 Leu Phe Asn Ala Thr Gln Phe Ile Lys Gln Leu Ser Gln Leu Gly616 Gln Lys ( 2 ) SEQ ID NO.7 ( 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.7:GCATGGATCC ATGTTGAATT TCAATGTGC 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:ACAGGTCGAC TTACTTTTGT CCAAGTTGT 29 (2) SEQ ID NO.9

(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.9:GCATGGATCC ATGTTGAATT TCAATGTGC C 29 (2) SEQ ID NO.10

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.10:ACAGGAATTC TTACTTTTGT CCAAGTTGT 29

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people HSly1 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 35-1888 position among described nucleotide sequence and the SEQ ID NO.5; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.5 in from the nucleotide sequence hybridization of Nucleotide 35-1888 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.6.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.5 nucleotide sequence from Nucleotide 35-1888 position.

4. isolating HSly1 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.6 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.6 polypeptide of sequence.

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

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

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

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

10. a generation has the method for the polypeptide of HSly1 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HSly1 protein-active operationally is connected in expression regulation sequence, form the HSly1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 35-1888 position among described nucleotide sequence and the SEQ ID NO.5;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of HSly1;

(c) be fit to express under the condition of HSly1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HSly1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 35-1888 position among the SEQ ID NO.5.

12. energy and the described HSly1 protein polypeptide of claim 4 specificity bonded antibody.

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

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

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