CN1278556A - Human montage factor II and its code sequence, preparation and use - Google Patents

Abstract

The present invention provides a cDNA sequence of new coded human splicing factor 2 (called SF2 for short), and said c DAN coded protein is member of RNP motif family. Said invention also relates to polypeptide of said nucleotide sequence code, application of these polynucleotides and polypeptide and production method of them.

Description

Human montage factor II and encoding sequence thereof, and method for making and purposes

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of human montage factor II (Splicing Factor 2 abbreviates " SF2 " as), this cDNA encoded protein is RNP motif family member.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.

The RNA splicing factor is to contain one or more ribonucleoproteins (ribonucleoprotein, RNP) albumen of motif.The RNP motif, have another name called " total type RNA is in conjunction with the territory " (consensus type RNA binding domain, CS-RBD) or " RNA discerns motif " (RNA recognition motif, RRM) be that (The New Biologist 1992,4 (5): 421-429 for about 80 the amino acid whose conserved sequences of a segment length; Nucleic Acids Res.1993,21 (25): 5803-5816).The albumen that contains the RNP motif has been formed a big and miscellaneous family---and " RNP motif protein family ", comprising various and heterogeneous nuclear RNA (hnRNA), microRNA (snRNA), rRNA, mRNA and RNA polymerase III transcript bonded protein molecular.To 1992, had been found that this family more than 30 the member and more than 130 independently RNP motif order (The NewBiologist 1992,4 (5): 421-429).The metabolism of many RNP motif albumen and mRNA is relevant, as with the Hela nucleus in 6 albumen to be arranged in 20 kinds of protein of newborn hnRNA bonded be RNP motif albumen.

RNP motif albumen all has vital role in composing type or adjustment type montage, some albumen participates in regulating the selection of splice site, and some protein binding is in the Special Areas of transcript, causes or suppresses specific montage approach.In other course of processing of mRNA, as the polyA polymerase that adds in the polyA tail process also is this family member.In the mRNA translation initiation stage, the albumen that is similar to eukaryotic initiation factor eIF-4B etc. all is RNP motif albumen in addition.

1993, one of discovery such as Imai was developed in the liver cell cancer patient body by liver cirrhosis and has produced new antinuclear antibody.They separate the nuclear autoantigen HCC1 that makes new advances from serum, it has three RNP motifs and a RS zone of being rich in arginine, Serine.The albumen that contains RNP and RS territory belongs to non-snRNA splicing factor subfamily, their discord snRNA combinations (J.Clin.Invest.1993,92:2419-2426).

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding RNP motif family, RNP motif family member of the present invention are named as human montage factor II (Splicing Factor2 abbreviates " SF2 " as).

Another object of the present invention provides a kind of new RNP motif family member, and this albumen is named as people SF2 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's SF2 polypeptide.

The present invention also provides this people's the SF2 nucleotide sequence and the application of polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people SF2 protein-active, shows at least 70% homology from the nucleotides sequence of 16-1293 in Nucleotide among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of 16-1293 in Nucleotide.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.7.More preferably, this sequence has among the SEQ ID NO.6 nucleotide sequence from 16-1293 in Nucleotide.

In another aspect of this invention, provide a kind of isolating people SF2 protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.7 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

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

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

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people SF2 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people SF2 protein-active operationally is connected in expression regulation sequence, form people SF2 protein expression vector, show at least 70% homology from the nucleotides sequence of 16-1293 in Nucleotide among described nucleotide sequence and the SEQ ID NO.6;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people SF2;

(c) under the condition that is fit to expressing human SF2 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people SF2 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1803 Nucleotide, and its detailed sequence is seen SEQ ID NO.6, and wherein open reading frame is positioned at 16-1293 Nucleotide.

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

In the present invention, term " people SF2 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people SF2 protein-active is as 16-1293 nucleotide sequences among the SEQ ID NO.6 and degenerate sequence thereof.This degenerate sequence is meant, is arranged in 16-1293 Nucleotide of encoder block of SEQ ID NO.6 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.6 in 16-1293 nucleotide sequence homologies be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.7 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.6 from the nucleotide sequence of the nucleotide sequence hybridization of 16-1293 in Nucleotide.Also term also comprise with SEQ ID NO.6 in from the homology of nucleotide sequence at least 70% of 16-1293 in Nucleotide, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.7 sequence of people SF2 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 add several at 5 and/or 3 ends and (be generally in 60, preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

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

In the present invention, term " people SF2 protein polypeptide " refers to have the SEQ ID NO.7 polypeptide of sequence of people SF2 protein-active.This term also comprises having and variant form people SF2 albumen identical function, SEQ ID NO.7 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people SF2 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people SF2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people SF2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people SF2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people SF2 polypeptide.Usually, this fragment have people SF2 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people SF2 albumen or polypeptide.The difference of these analogues and natural human SF2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from natural L-amino acid whose residue (as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, γ-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

In the present invention, " people SF2 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.7, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp

Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises people SF2 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people SF2 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of people SF2 polypeptid coding sequence, preferably 15-50 continuous nucleotides usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people SF2.

The present invention also comprises the method that detects people SF2 nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of people SF2 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

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

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

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

On the other hand, the present invention also comprises people SF2 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people SF2 gene product or fragment.Preferably, refer to that those can combine with people SF2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people SF2, comprise that also those do not influence the antibody of people SF2 protein function.The present invention also comprise those can with modify or without the people SF2 gene product bonded antibody of modified forms.

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

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people SF2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human SF2 or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people SF2 function and the antibody that does not influence people SF2 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people SF2 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people SF2 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People SF2 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

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

In addition, also the method for available synthetic is synthesized relevant sequence.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the cDNA nucleotide sequence of people SF2 is so to obtain, with people's kidney λ gtllcDNA library (available from Clontech company) is template, with three oligonucleotide is primer---A1:5-CACAACATACTCAAAGGAACGGAG-3 ' is that outside forward primer, A2:5-AACTGATCTGACAGGATGGCATC-3 are inboard forward primer, oligonucleotide B:5 '-AGTAGATGTGAAGTGGCAGGATC-3 is a reverse primer, carries out nested PCR.With people's kidney λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---C:5-CACAGTATACTGCCTCCTTGTGC-3 ' is a forward primer, oligonucleotide D:5 '-TCAATGCACGTCCATGCTATCTC-3 is a reverse primer, carries out PCR.To amplified production check order and splice after obtain the full length cDNA sequence of SEQ ID NO.6.

The RNA splicing factor contains RNP (ribonucleoprotein) motif.Two highly conserved sequences are arranged in the RNP motif, and one section eight body sequence is called RNP common sequences or RNP1 (Mol.Cell.Biol.1986,6:2932-2943), and they may also have a RNP2 who is made up of six amino acid with relevant with the RNA combination.(Nature?1990，348：515—519；Trends?Biochem．Sci．1991，16：214—220；Proc．Natl．Acad．Sci．U．S．A．1991，88：2495—2499；The?New?Biologist?1992，4(5)：421—429)。RNP motif albumen has more than and contains the RNP motif, and they also have some other auxiliary area, as is rich in glycine/fragrance group amino acid region, the RS zone of being rich in arginine, Serine, the auxiliary area of altitudinal belt electric charge.The various mRNA metabolic reactions of RNP motif albumen wide participation, and therefore with the part disease-related.

The SF2 albumen that the present invention cloned contains two and HCC1 albumen height homologous RNP motif, and RNP1 wherein and RNP2 conserved sequence are in full accord, so people SF2 also is the splicing factor of a mRNA.People SF2 albumen of the present invention helps people to study the proteic function of RNP motif.In addition, because SF2 of the present invention has the natural acid sequence that is derived from the people, therefore, compare, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour with the albumen of the same clan that derives from other species.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people SF2 of the present invention and HCC1.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

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

Embodiment 1

The clone and the mensuration of the cDNA sequence of people SF2

1. primer amplification

With people's kidney λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5-CACAACATACTCAAAGGAACGGAG-3 (SEQ ID NO:1) is a forward primer, oligonucleotide B:5-AGTAGATGTGAAGTGGCAGGATC-3 (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A1/B be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 94 ℃ 30 seconds, 60 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 10 minutes.With the PCR product is template, using a pair of oligonucleotide again---A2:5-AACTGATCTGACAGGATGGCATC-3 (SEQ ID NO:3) is a forward primer, B:5-AGTAGATGTGAAGTGGCAGGATC-3 (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A2/B be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 94 ℃ 30 seconds, 65 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 10 minutes.Electrophoresis detection obtains the purpose Segment A 2/B of about 1.2kb.With people's kidney λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---C:5-CACAGTATACTGCCTCCTTGTGC-3 (SEQ ID NO:4) is a forward primer, oligonucleotide D:5-TCAATGCACGTCCATGCTATCTC-3 (SEQ ID NO:5) is a reverse primer, carries out PCR.The PCR condition of C/D be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 60 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment C/D of about 500bp.

2.PCR the order-checking of product

Above-mentioned pcr amplification product A2/B is connected with pGEM-T carrier (Promega), and transformed into escherichia coli JM103 extracts plasmid with QIAprep Plasmid test kit (QIAGEN), carries out Rapid identification and ordering with PCR then.Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively.With above-mentioned pcr amplification product C/D and pGEM-T ^Carrier (Promega) connects, and transformed into escherichia coli JM103 extracts plasmid with QIAprep Plasmid test kit (QIAGEN), uses SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) inserts fragment to plasmid and checks order.With the computer software splicing row that check order, obtain full length cDNA sequence at last, be total to 1803bp, detailed sequence is seen SEQ ID NO.6, and wherein open reading frame is positioned at 16-1293 Nucleotide.

Derive the aminoacid sequence of people SF2 according to the full length cDNA sequence that obtains, totally 425 amino-acid residues, its aminoacid sequence sees SEQ ID NO.7 for details.

Buy and execute example 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people SF2 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that, the splicing factor gene and the proteins encoded thereof of they and different sources have homology widely, if find relatively that with PCGENE software it and people HCC1 (L10911) identity and the similarity on protein level reaches 54.1% and 64.7% (Fig. 1) respectively.What is more important, albumen of the present invention have two groups of typical R NA in conjunction with the territory, are respectively RNP1-191KGIAYVEF198,288KGYGFITF295 and RNP2-152VFCMQL157,249LYVGSL254.Therefore, people SF2 albumen of the present invention can be included into splicing factor albumen homologue, and can infer that it has the general utility functions of splicing factor albumen homologue.

The RNA splicing factor is the same with some other rna binding protein, all contains about 80 the amino acid whose conserved sequences of a segment length of one or more copies, is called RNP (ribonucleoprotein) motif (The New Biologist1992,4 (5): 421-429; Nucleic Acids Res.1993,21 (25): 5803-5816), thereby belong to RNP motif protein family.The metabolism of many RNP motif albumen and mRNA is relevant.The sequence that two high conservatives are arranged in the RNP motif, one section eight amino acid whose sequences-[RK] G{EDRKHPCG}[AGSCI] [FY] [LIVA] X[FYLM] (any one amino acid that expression is wherein drawn together in the square brackets wherein, any one that the amino acid that braces is represented wherein to be drawn together is outer, X represents any one amino acid), called after RNP common sequences or RNP1 (Mol.Cell.Biol.1986,6:2932-2943), they may be with relevant with the RNA combination.The RNP2 that another highly conserved sequence is made up of six amino acid.Use X-ray crystalline diffraction technology and nuclear magnetic resonance, technology, people find in the research to RNP motif albumen U1A, the secondary structure of RNP motif is made up of 4 antiparallel β chains and 2 α spirals that are positioned at the βZhe Die homonymy, RNP1 and RNP2 are positioned on adjacent β 1 and β 3 chains, aromatic residue is positioned at folding surface (Nature1990,348:515-519; Trends Biochem.Sci.1991,16:214-220; Proc.Natl.Acad.Sci.U.S.A.1991,88:2495-2499; The New Biologist 1992,4 (5): 421-429).SF2 of the present invention has similar secondary structure.

RNP motif albumen has more than and contains the RNP motif, and they also have some other auxiliary area, as is rich in glycine/die aromatischen Aminosaeuren zone, is rich in the RS zone of arginine, Serine, the auxiliary area of altitudinal belt electric charge.Albumen of the present invention contains the RS zone, and proteic arginine, serine content are obviously higher, and occurs continuously.

The neoantigen HCC1 that Imai etc. find has three RNP motifs and a RS zone of being rich in arginine, Serine.The albumen that contains RNP and RS territory belongs to non-snRNA splicing factor subfamily, and they do not combine (J.Clin.Invest.1993,92:2419-2426) with snRNA.The RS territory can be used as little of the special subnucleus of signal for locating and pilot protein in the nuclear, is called as little of spottiness.Immunofluorescence experiment shows, the distribution of HCC1 albumen in nuclear is that the position of a spottiness mesh mode and the snRNP that is rich in uridine, non-snRNP spliceosome moiety is suitable.The constructional feature of HCC1 holds itself out to be a nuclear protein matter, and is relevant with the montage of mRNA precursor.The antibody of HCC1 worsens the earlier month of being diagnosed out up to liver and just can be detected, and is one of relevant new antibody response of several and pernicious transfer but be proved it.We clone's SF2 albumen contains two and HCC1 albumen height homologous RNP motif, and RNP1 wherein and RNP2 conserved sequence are in full accord, infers that SF2 also is the splicing factor of a mRNA.In addition, SF2 has the RS territory that is different from HCC1, points out it may be positioned at the different zones of nuclear.

People SF2 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 SF2 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor SF2 and the N end of HCC1 are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor SF2, 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 SF2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people SF2 or the overexpression that suppresses people SF2.People SF2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of what people SF2 caused unusually related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people SF2 in intestinal bacteria

In this embodiment, with pcr amplification product A2/B among the embodiment 1 is template, with the cDNA sequence of coding people SF2 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.8 and 9) of 3 ' end increase, with the people SF2 cDNA that obtains as inserting fragment.

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

5 '-TCAGCTGCAGATGGCATCTGATGACTTTG-3 ' (SEQ ID NO.8), this primer contains the restriction enzyme site of PstI restriction enzyme, is 19 Nucleotide of the people SF2 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5-TTGGAAGCTTTTACATGGTCTGGGGGGTA-3 (SEQ ID NO.9), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people SF2 of Hind III restriction enzyme.

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

With Pst I and Hind III digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to 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 lac I repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people SF2 has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spent 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-β-D-galactoside ") to final concentration be 1mM.By making lac I repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hours, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people SF2 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people SF2 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS with 12%-PAGE glue carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 47KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.7 with ordinary method.

Embodiment 4

The expression of people SF2 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with pcr amplification product A2/B among the embodiment 1 is template, with the cDNA sequence of coding people SF2 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.10 and 11) of 3 ' end increase, obtain people SF2 cDNA as inserting fragment.

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

5′—TCAGAAGCTTATGGCATCTGATGACTTTG—3′(SEQ?ID?NO．10)

This primer contains the restriction enzyme site of Hind III restriction enzyme, is 19 Nucleotide of the people SF2 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5—TTGGGATATCTTACATGGTCTGGGGGGTA—3(SEQ?ID?NO．11)

This primer contains the part encoding sequence of the restriction enzyme site of EcoRV restriction enzyme, translation termination and people SF2.

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 (fl ori), a virus replication starting point (SV40 ori), T7 promotor, viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and corresponding polyA order.

With Hind III and EcoRV 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 is cut and the cDNA fragment of sequence verification people SF2 has correctly been inserted carrier with Xba I enzyme.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 weeks, 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 50mMTrisHCl (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 50mMTrisHCl (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 with 12%-PAGE glue carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 47KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.7 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsifications carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people SF2 gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.

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

Sequence Table (A) General information: (Ⅰ) Applicant: Fudan University (Ⅱ) Invention: human splicing factor Ⅱ and its coding sequence, and the preparation method and uses (Ⅲ) Serial Number: 11 (2) SEQ ID NO. An information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 24 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO. 1: CACAACATACTCAAAGGAACGGAG 24 (2) SEQ ID NO. 2 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 23 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 2 AGTAGATGTG AAGTGGCAGG ATC 23 (2) SEQ ID NO. 3 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 23 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 3 AACTGATCTG ACAGGATGGC ATC 23 (2) SEQ ID NO. 4 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 23 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 4 CACAGTATAC TGCCTCCTTG TGC 23 (2) SEQ ID NO. 5 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 23 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 5 TCAATGCACG TCCATGCTAT CTC 23 (2) SEQ ID NO. 6 Information: (Ⅰ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1803bp (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: cDNA (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO. 6 AACTGATCTG ACAGGATGGC ATCTGATGAC TTTGACATAG TGATTGAGGC CATGCTGGAA 60 GCTCCCTATA AAAAAGAAGA GGATGAGCAA CAAAGGAAAG AAGTTAAAAA GGATTATCCT 120 AGCAATACCA CCAGCAGCAC CAGCAACAGT GGCAATGAGA CCAGTGGAAG CAGCACCATC 180 GGGGAGACAA GCAATCGTAG TCGAGATCGG GATCGGTATA GACGGAGAAA TAGTCGGAGC 240 CGAAGTCCAG GTCGGCAGTG TCGTCACCGT AGCCGTAGCT GGGATCGTCG ACATGGTAGT 300 GAGTCGCGAA GTCGGGACCA TCGTCGTGAG GATCGTGTGC ATTACAGGAG TCCTCCACTT 360 GCCACTGGTT ATAGGTATGG ACACAGTAAG AGTCCTCATT TCAGAGAGAA GAGCCCAGTC 420 AGGGAGCCAG TTGATAATCT GAGTCCTGAG GAGCGTGATG CCCGCACAGT TTTCTGTATG 480 CAGTTAGCTG CCCGAATTCG GCCTCGAGAT CTGGAGGACT TTTTCTCTGC TGTAGGCAAG 540 GTTCGCGATG TACGTATCAT CTCAGATCGG AACTCACGTC GTTCTAAGGG CATTGCCTAC 600 GTGGAATTCT GTGAAATCCA GTCTGTGCCA CTGGCCATTG GGCTGACTGG GCAGCGGTTG 660 CTGGGAGTGC CTATCATTGT ACAGGCTTCA CAGGCAGAGA AAAACCGACT GGCAGCCATG 720 GCCAACAACC TGCAAAAGGG CAATGGTGGA CCAATGCGCC TCTATGTGGG TTCCCTGCAC 780 TTCAATATCA CTGAAGACAT GCTCCGGGGC ATCTTTGAGC CCTTTGGTAA AATTGATAAT 840 ATTGTCCTGA TGAAGGACTC AGATACAGGC CGCTCTAAAG GTTATGGTTT CATCACGTTC 900 TCTGATTCTG AGTGTGCCCG GCGGGCCTGT GGAACAGTTG AATGGGGTTT GAGCTTGCTG 960 GGTCGACCTA TGAGGGTTGG CCATGTGACT GAGCGACTGG ATGGTGGCAC AGACATCACT 1020 TTTCCTGATG GGGACCAGGA GCTGGATCTG GGATCAGCAG GTGGACGTTT TCAGCTCATG 1080 GCAAAACTGG CAGAAGGCGC TGGAATCCAA CTGCCAAGCA CTGCTGCTGC TGCTGCTGCC 1140 GCCGCCGCCG CCCAGGCTGC TGCCTTGCAA CTGAATGGAG CAGTTCCCTT GGGGGCCCTG 1200 AATCCAGCAG CTCTGACTGC TCTGAGTCCA GCCCTGAACC TTGCCTCCCA GTGTTTCCAG 1260 CTCTCCAGCC TCTTTACCCC CCAGACCATG TAAATCAGTG GCACAGTATA CTGCCTCCTT 1320 GTGCCTCTGG ATCCTGCCAC TTCACATCTA CTCTTCCATG GCCCCATTTC TCCATTTTGT 1380 GGACCAAGCC ATCCTGAGGG CATGGACATT GTCTCTGAGG AAATTGGGGC CACCCTTAAG 1440 ATACCAAGAA AAGCTCCTGC CCATGGTCCC ACTGGAAATG GACTCTGCTG AGCAAAGCCA 1500 CCAGTTGAAG AGAACAGAAT CCACACCTGC ATTGAATACC TGTTTCTCCA TGTGTATCGT 1560 CTCTGAGATT ACCTTCTTGC CCTTTCCAAC ACCTTAGTGA TTCCTCAATT TCTCCCCCAT 1620 TGGGAAGGCC ATAGGGCATT AACTGAAGGA ACTGACCTCT CTCCTTTTCC TGTACCTTTA 1680 ACCTTTAGTC TGTCAAGGAA AACCCTTAGG ACCTCTGAAT CAAGAGGACT GAGTTTGTGG 1740 GTGAACCTTG AAGGTGCTCT TTCTGCTACA AGGGCCCTGG GAGATAGCAT GGACGTGCAT 1800 TGA 1803 (2) SEQ ID NO. 7 Information: (Ⅰ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 425 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: peptide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO. 7 Met Ala Ser Asp Asp Phe Asp Ile Val Ile Glu Ala Met Leu Glu 15 Ala Pro Tyr Lys Lys Glu Glu Asp Glu Gln Gln Arg Lys Glu Val 30 Lys Lys Asp Tyr Pro Ser Asn Thr Thr Ser Ser Thr Ser Asn Ser 45 Gly Asn Glu Thr Ser Gly Ser Ser Thr Ile Gly Glu Thr Ser Asn 60 Arg Ser Arg Asp Arg Asp Arg Tyr Arg Arg Arg Asn Ser Arg Ser 75 Arg Ser Pro Gly Arg Gln Cys Arg His Arg Ser Arg Ser Trp Asp 90 Arg Arg His Gly Ser Glu Ser Arg Ser Arg Asp His Arg Arg Glu 105 Asp Arg Val His Tyr Arg Ser Pro Pro Leu Ala Thr Gly Tyr Arg 120 Tyr Gly His Ser Lys Ser Pro His Phe Arg Glu Lys Ser Pro Val 135 Arg Glu Pro Val Asp Asn Leu Ser Pro Glu Glu Arg Asp Ala Arg 150 Thr Val Phe Cys Met Gln Leu Ala Ala Arg Ile Arg Pro Arg Asp 165 Leu Glu Asp Phe Phe Ser Ala Val Gly Lys Val Arg Asp Val Arg 180 Ile Ile Ser Asp Arg Asn Ser Arg Arg Ser Lys Gly Ile Ala Tyr 195 Val Glu Phe Cys Glu Ile Gln Ser Val Pro Leu Ala Ile Gly Leu 210 Thr Gly Gln Arg Leu Leu Gly Val Pro Ile Ile Val Gln Ala Ser 225 Gln Ala Glu Lys Asn Arg Leu Ala Ala Met Ala Asn Asn Leu Gln 240 Lys Gly Asn Gly Gly Pro Met Arg Leu Tyr Val Gly Ser Leu His 255 Phe Asn Ile Thr Glu Asp Met Leu Arg Gly Ile Phe Glu Pro Phe 270 Gly Lys Ile Asp Asn Ile Val Leu Met Lys Asp Ser Asp Thr Gly 285 Arg Ser Lys Gly Tyr Gly Phe Ile Thr Phe Ser Asp Ser Glu Cys 300 Ala Arg Arg Ala Cys Gly Thr Val Glu Trp Gly Leu Ser Leu Leu 315 Gly Arg Pro Met Arg Val Gly His Val Thr Glu Arg Leu Asp Gly 330 Gly Thr Asp Ile Thr Phe Pro Asp Gly Asp Gln Glu Leu Asp Leu 345 Gly Ser Ala Gly Gly Arg Phe Gln Leu Met Ala Lys Leu Ala Glu 360 Gly Ala Gly Ile Gln Leu Pro Ser Thr Ala Ala Ala Ala Ala Ala 375 Ala Ala Ala Ala Gln Ala Ala Ala Leu Gln Leu Asn Gly Ala Val 390 Pro Leu Gly Ala Leu Asn Pro Ala Ala Leu Thr Ala Leu Ser Pro 405 Ala Leu Asn Leu Ala Ser Gln Cys Phe Gln Leu Ser Ser Leu Phe 420 Thr Pro Gln Thr Met 425 (2) SEQ ID NO. 8 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO. 8: TCAGCTGCAG ATGGCATCTG ATGACTTTG 29 (2) SEQ ID NO. 9 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO. 9: TTGGAAGCTT TTACATGGTC TGGGGGGTA 29 (2) SEQ ID NO. 10 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO 10: TCAGAAGCTT ATGGCATCTG ATGACTTTG 29 (2) SEQ ID NO. 11 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO. 11: TTGGGATATC TTACATGGTC TGGGGGGTA 29 ...

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people SF2 protein-active,

Show at least 70% homology from the nucleotides sequence of 16-1293 in Nucleotide among described nucleotide sequence and the SEQ ID NO.6; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of 16-1293 in Nucleotide.

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.7.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.6 nucleotide sequence from 16-1293 in Nucleotide.

4. isolating people SF2 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

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

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

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

8. a generation has the method for the polypeptide of people SF2 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people SF2 protein-active operationally is connected in expression regulation sequence, form people SF2 protein expression vector, show at least 70% homology from the nucleotides sequence of 16-1293 in Nucleotide among described nucleotide sequence and the SEQ ID NO.6;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people SF2;

(c) under the condition that is fit to expressing human SF2 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people SF2 protein-active.

9. energy and the described people SF2 of claim 4 protein polypeptide specificity bonded antibody.

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