CN1384114A - Growth promoting factor and its prepn and use - Google Patents

Abstract

The present invention provides DPF protein as one kind of development promoting factor, polynucleotides of coding the DPF protein and the recombination technological process of producing the DPF protein. The DPF protein comes from secretion of oviduct and is the unique factor to the overcome embryo development blockage. The present invention also discloses the application of DPF protein and its coding sequences as well as the application of DPF protein acceptor in diagnosing and treating several diseases. The present invention also provides medicine composition containing DPF protein.

Description

Growth promoting factor, its method for making and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding growth promoting factor (Development Promoting Factor abbreviates " DPF " as), and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind ofly to be derived from fallopian tube secretions, to have the unique factor that overcomes the early embryonic development blocking-up.

Background of invention

The growth of Mammals body early embryo is subjected to the parent form gene regulating, promptly by maternal mRNAs and its deutero-protein regulation of being stored in the ossphere.These parent forms mRNAs and proteinic in good time location, activation, expression and disappearance are determining paotoblastic growth destiny early.

Early embryonic development must realize that to the certain period parent form transcript is degraded in batch when the time comes by the transition of parent form regulation and control to the zygosity regulation and control, and zygosity genetic expression is also dominated further and grown.Mouse, this transition occurs in the 2-cell stage.Yet, if with the mouse fertilized egg isolated culture in the substratum that chemical composition is determined, body early embryo often can not be finished the growth whole process from the zygote to the blastaea, but is parked on the 2-cell stage, this phenomenon is called " blocking-up of 2-cell ".What cause that people pay much attention to is, this class is grown blocking-up and not only is prevalent in the Mammals, and the time of its blocking-up is fitted like a glove by the time of parent form to zygosity regulation and control transition with embryonic development early.On the contrary,, or in uterine tube liquid, cultivate if zygote is placed uterine tube, or in the uterine tubal epithelium co-culture of cells, or in uterine tubal epithelium cell conditioned medium, cultivate, all can overcome this type of and grow blocking-up, continue normal development.In addition, research also shows, is derived from inspiring of uterine tubal epithelium emiocytosis thing and educates the no species specificity of effect.Infer that thus uterine tube is not only " place " of embryonic development early, and secretes the factor that some participation overcomes the growth blocking effect, thereby guarantee that early embryo is realized the transition that its normal development is regulated and control.Be derived from shortage under the condition of " signal " of uterine tubal epithelium cell, mouse early embryonic development just is parked on 2-cell stage G2 phase, and embryonic development blocking-up early takes place.

Up to now, only see to relate to the report that may have such signal or the factor " relevant evidence ", and further conclusive evidence, separation and purification and clone do not appear in the newspapers to this class factor.Therefore whether exist the factor of such regulation and control transition just disputable always.Particularly other body cell (as inoblast) is being cultivated altogether as vitro culture " feeder layer cells " and embryo early, or in conventional substratum, add somatomedin, ion chelating agent or remove free radical reagent, or the interpolation time of change glucose, all work to overcome embryonic development blocking-up early to a certain extent.Bavister was 1992 even proposition, as long as the environmental quality of strict control vitro culture is selected suitable substratum, early embryo can be grown smoothly, need not the existence of uterine tubal epithelium cell.

In contrast, confirmation such as Poueymirou has the catalytic protein phosphorylation reaction of the protein kinase (PKA) that relies on cAMP to participate in the mouse 2-cell stage regulatory mechanism of embryo zygosity gene activation early.Schwartz etc. further confirm the expression of the beta-galactosidase enzymes reporter gene that TPA response element (TRE) drives in the incompetent ovocyte that stimulates tool intact cell nuclear of Buddhist ripple ester " TPA " again, but can sting regular menstruation during early pregnancy aphidicolin and handle and still can check in the expression of the reporter gene in the embryo early of 1-cell stage.These experiment analysis results are all pointed out the possibility of different signal transduction system participating in activation zygosity genes.

Yet, the report of finding the material relevant with the fetal development blocking-up is not also arranged up to now.

Therefore, this area presses for exploitation material relevant with the fetal development blocking-up and preparation method thereof.

The purpose of this invention is to provide a kind of new growth promoting factor (Development Promoting Factor, DPF)-DPF albumen with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated DPF polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned DPF polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or 3.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 53-1477 position among the SEQ ID NO:1; (b) has the sequence of 101-1477 position among the SEQ ID NO:1; (c) has the sequence of 1-1906 position among the SEQ ID NO:1.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of DPF protein-active, this method comprises: (a) be fit to cultivate the above-mentioned host cell that is transformed or transduce under the proteic condition of expression DPF; (b) from culture, isolate polypeptide with DPF protein-active.

In a fifth aspect of the present invention, provide and above-mentioned DPF polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains the about 15-1906 of a successive Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism DPF polypeptide active is provided, and the compound that suppresses the DPF polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of DPF polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of DPF in the test sample, it comprises: sample is contacted with the proteic specific antibody of DPF, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DPF albumen.

In a eighth aspect of the present invention, a kind of disease relevant with DPF polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes the DPF polypeptide active, and perhaps screening suppresses the antagonist of DPF polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of DPF of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains DPF polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as women infertility.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is the dose response curve (every group of zygote number is 40-42 piece) that anti-DPF polyvalent antibody and mouse fertilized egg are grown relation.

Fig. 2 is the cDNA sequence of DPF of the present invention and the proteic full length amino acid sequence of DPF of inferring.

Fig. 3 is the dose response curve (every group of zygote is 37-39 piece) that GST-NTP367 DPF and mouse fertilized egg are grown relation.The dosage of GST-NTP367 DPF is meant the mg number that adds GST-NTP DPF in the 50ml nutrient solution, and nutrient solution is made up of F10, M16, foetal calf serum, EGF, transferrin and Regular Insulin.

Fig. 4 is the dose response curve (every group of zygote is 37-39 piece) that GST-CTP92 DPF and mouse fertilized egg are grown relation.The dosage of GST-CTP92 DPF is meant the mg number that adds GST-CTP92 DPF in the 50ml nutrient solution, and nutrient solution is made up of F10, M16, foetal calf serum, EGF, transferrin and Regular Insulin.

Fig. 5 is the dose response curve (every group of zygote is 38-41 piece) that anti-CTP92 DPF polyvalent antibody and mouse fertilized egg are grown relation.The dosage of anti-CTP92 DPF polyvalent antibody is meant the ml number that adds anti-CTP92DPF polyvalent antibody in the 50ml nutrient solution, and nutrient solution is made up of F10, M16, foetal calf serum, EGF, transferrin and Regular Insulin.

Fig. 6 has shown the Northern trace result of the total RNA of different tissues.Wherein with ³²The DPF cDNA fragment of P mark is a probe, and swimming lane 1-8 represents the Northern trace result from total RNA of rabbit muscle, heart, kidney, uterine tube, liver, spleen, lung and small intestine respectively.

Western engram analysis result after the gel electrophoresis of Fig. 7 different tissues protein SDS-PAGE separates makes probe with anti-DPF monoclonal antibody.Swimming lane 1-10 represents the protein immunoblot result from liver, heart, lung, spleen, uterine tube, uterus, ovary, small intestine, skeletal muscle and cerebral tissue respectively.

Western engram analysis result after the protein SDS-PAGE gel electrophoresis of Fig. 8 people oviduct tissue separates makes probe with anti-DPF monoclonal antibody, the protein immunoblot result of people oviduct tissue.

The Western trace density scan collection of illustrative plates of DPF and associated molecule in Fig. 9 different plant species uterine tube albumen.Wherein, make probe with anti-DPF monoclonal antibody.Last figure represents the collection of illustrative plates from the uterine tube Western blot densitometric scan of kunming mice and new zealand white rabbit respectively.

The Northern trace result of the total RNA of Figure 10 people oviduct tissue.Be template synthesizing radioactive label probe wherein with DPF cDNA.Swimming lane 3,4 is represented the Northern trace result of two total RNA of different people oviduct tissue respectively.

The inventor is through for many years extensive and deep research, obtained to have " afunction evidence " that the fallopian tubal factor that promotes to grow function exists, confirmed that the early comprehensive normal development of embryo in the body must rely on intrinsic natural " signal " that is derived from the synthetic and secretion of body cell.

Particularly, " correlation " and " afunction evidence " that the present invention obtains by means of experimental analysis, confirm first to have in the rabbit fallopian tube secretions the early embryonic development blocking-up of releasing after fertilization and realize that the material that morning, embryo was regulated and control transition by parent form to zygosity is a kind of glycoprotein, called after Development Promoting Factor (DPF). DPF albumen apparent molecular weight is 64KD, and pI is 7.2-8.1, does not have species specificity on the function, and the development of fertilized ova of ox, sheep, pig, rat and mouse is had facilitation. In addition, DPF albumen is different from present estrogen-dependent oviductins that pay close attention in the world and that function it be unclear that.

In one embodiment of the invention, set up the cDNA library of rabbit Mucous Epithelium of Fallopian Tube, and 3 ＇ that arrive about long 0.7kb by antibody screening hold DPF cDNA. DPF distribution tool tissue specificity only is present in fallopian tubal, does not all exist in other main organs. The Northern engram analysis also confirms that this protein mRNA s only is present in fallopian tubal and confirms that its mRNA total length is about 1.9kb. With the end of 3 ＇ about 0.7kb DPF cDNA, and order-checking and sequence analysis have been carried out. Prove that by the gene retrieval resulting gene is a new gene. Further found the DPF cDNA of total length after screening and cloning and the order-checking.

In another embodiment of the present invention, utilize GST fusion expression system, express and purifying DPF, NTP DPF (1-367) (the N terminal peptide of DPF (N-terminal peptide of DPF)) and CTPDPF (368-459) (the C terminal peptide of DPF (C-terminal peptide of DPF)), and the corresponding antiserum that prepared, with GST-Sepharose 4B affinity column and GST-CTP DPF (368-459)-Sepharose 4B affinity column resist carried out purifying more, made up affinity column and be purified into the natural DPF of rabbit with it with how anti-the anti-CTP DPF of the rabbit of purifying (368-459) is.

In another embodiment, carry out the gain-of-function experiment with the fusion of natural DPF and expression and purification, obtained the gain-of-function evidence of DPF. With the many anti-main positions of function enclosed experiment to determine to work among the DPF of having carried out.

In the present invention, term " DPF albumen ", " DPF polypeptide " or " growth promoting factor DPF " are used interchangeably, and (full length sequence is seen SEQ ID NO:2 all to refer to have rabbit growth promoting factor DPF amino acid sequence; The sequence of maturation protein is seen SEQ ID NO:3) albumen or polypeptide. They comprise the growth promoting factor DPF that contains or do not contain initial methionine, and the growth promoting factor DPF that contains or do not contain signal peptide

As used herein, " separation " refers to that material separates (if crude, primal environment namely is natural surroundings) from its primal environment. Do not have separation and purification such as the polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " DPF albumen or the polypeptide of separation " refers to that the DPF polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the purified technology of protein purifying DPF albumen of standard. Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis, or uses recombinant technique to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.

The present invention also comprises fragment, derivative and the analog of DPF albumen. As used herein, term " fragment ", " derivative " refer to basically keep the identical biological function of natural DPF albumen of the present invention or active polypeptide with " analog ". Polypeptide fragment of the present invention, derivative or analog can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide half-life, polyethylene glycol for example) merges formed polypeptide, or (iv) additional amino acid sequence is fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, these fragments, derivative and analog belong to the known scope of those skilled in the art.

In the present invention, term " DPF polypeptide " refers to have the SEQ ID NO.3 of DPF protein active or the polypeptide of 2 sequences. This term also comprises having and variant forms DPF albumen identical function, SEQ ID NO.3 or 2 sequences. These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-is 30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several and (be generally in 20 in that C end and/or N are terminal, preferably being in 10, more preferably is in 5) amino acid. For example, in the art, when replacing with the close or similar amino acid of performance, usually can not change the function of protein. Again such as, add the function that or several amino acid also can not change protein usually in that C end and/or N are terminal. This term also comprises active fragment and the reactive derivative of DPF albumen.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low stringency condition can with the coded albumen of the DNA of DPF DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-DPF polypeptide to obtain. The present invention also provides other polypeptide, as comprises the fusion (such as the fusion of DPF and GST) of DPF polypeptide or its fragment. Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of DPF polypeptide. Usually, this fragment have the DPF 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 analog of DPF albumen or polypeptide. The difference of these analogs and natural DPF polypeptide can be the difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can obtain by various technology, as by radiation or be exposed to mutagens and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars. Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(such as D-amino acid), and the analog with that non-natural exists or synthetic amino acid (such 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.

(usually the not changing primary structure) form of modification comprises: chemically derived form such as acetylation or the 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 such as those in the synthetic and processing of polypeptide or further. This modification can be carried out glycosylated enzyme (such 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 (such as phosphotyrosine, 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, " DPF albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2 or 3, 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

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ IDNO:2 or 3, but with the differentiated nucleotide sequence of corresponding encoded region sequence shown in the SEQ ID NO:1.

The polynucleotide of encoding D PF mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation encoding D PF.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

DPF 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, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or DPF albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the DPF polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of encoding D PF polypeptide of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, the DPF polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: the expression vector based on T7 of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains DPF DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS cell etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The DPF albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease (as some women infertility) due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism DPF protein function as pharmacological agent DPF protein function.The peptide molecule that can suppress or stimulate the DPF protein function that can be used for seeking therapeutic value with the reorganization DPF protein screening peptide library of expressing.

On the other hand, the present invention also comprises DPF 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 DPF gene product or fragment.Preferably, refer to that those can combine with DPF 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 DPF, comprise that also those do not influence the antibody of DPF protein function.The present invention also comprise those can with modify or without the DPF 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; 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 DPF gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, the DPF albumen of expression 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.Antibody of the present invention comprises the antibody that can block the DPF protein function and the antibody that does not influence the DPF protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of DPF 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 DPF 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).

The proteic antibody of anti-DPF can be used in the immunohistochemistry technology, detects the DPF albumen in the biopsy specimen.

The disease that antibody among the present invention can be used for treating or prevention is relevant with DPF albumen.The antibody that gives suitable dosage can be blocked proteic generation of DPF or activity.Another important use of antibody of the present invention is the medicine as the reversibility contraception.

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

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, these materials can be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of women infertility aspect.When using DPF albumen of the present invention, also can use the other treatment agent simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains DPF polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the DPF albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of DPF also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of DPF of the proteic nothing expression of DPF or unusual/non-activity.The DPF albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic DPF protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the DPF transgenosis to cell.The method that structure carries the recombinant viral vector of DPF gene is found in existing document (Sambrook, et al.).The DPF gene of recombinating in addition can be packaged in the liposome, and then is transferred in the cell.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization DPF protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The DPF protein level that is detected in the test can be with laying down a definition the importance of DPF albumen in various diseases and be used to the disease of diagnosing DPF albumen to work.

Whether having the proteic method of DPF in a kind of detection test sample is to utilize the proteic specific antibody of DPF to detect, and it comprises: sample is contacted with the DPF protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DPF albumen.

The proteic polynucleotide of DPF can be used for the diagnosis and the treatment of DPF protein related diseases.Aspect diagnosis, the proteic polynucleotide of DPF can be used for detecting the proteic expression of DPF DPF abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of DPF as the DPF dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of DPF albumen and also can detect the proteic transcription product of DPF.

The sudden change that detects the DPF gene also can be used for the disease of diagnosing DPF albumen relevant.The form of DPF protein mutation comprises that the point mutation compared with normal wild type DPF dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of DPF prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from rabbit uterine tube cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1906 bases, and its open reading frame is positioned at the 53-1477 position, and the coding total length is 475 amino acid whose DPF albumen (SEQ ID NO:2).Remove the ripe DPF behind 19 amino acid signal peptides, had 459 amino acid (SEQ ID NO:3).

The Western engram analysis confirms, all contains the DPF associated molecule in the fallopian tube secretions of other detected species (people, mouse, golden hamster).Therefore, utilize content disclosed by the invention (the especially nucleotide sequence of DPF and amino acid), can from people's uterine tube cDNA library, screen, clone the DPE-1 homolgous molecule, and carry out the in-depth analysis of 26S Proteasome Structure and Function aspect.People's uterine tubal epithelium cell cdna library of the better quality that makes up with the inventor shows that by the Northern engram analysis the relevant DPFcDNA of people is about 3.5kb.The research of the screening of this relevant DPFcDNA that behaves and the relevant DPF of people is laid a good foundation.

By further analysis to " DPF " 26S Proteasome Structure and Function, by means of the research of DPF to protoblast signal transducting system influence morning, and to causing the driving mechanism (p34 that G2 → transition of M phase is relied on ^Cdc2/ Cyclin B) research of formation and reactivation process influence, and observe the chromatin Structure variation that may bring thus, help to illustrate the mechanism of after fertilization embryonic development morning and growth blocking-up fully.At aspects such as medical science, husbandry very important using value is arranged also in addition, by transforming, select suitable carriers and immunization route, therefore thereby have a great application prospect DPF also is expected to become and a kind ofly disturbs local microenvironment and block the novel contraceptive vaccine of embryonic development early.

Particularly, the present invention has following potential application prospect:

(1) artificial Assisted Reproductive Technology ART:

Determining to help the further quality of growth of pregnant success ratio and embryo for the human embryos quality of transplanting.Before the implantation early one of key link of embryonic development be to overcome embryonic development blocking-up early, make the embryo can be according to the normal development of inherent program.DPF is exactly the unique factor that overcomes embryonic development blocking-up early that the Mucous Epithelium of Fallopian Tube cell provides.

(2) immunological contraception

The antigen of immunological contraception research at present mainly concentrates on three class target antigens, i.e. the zona pellucida antigen of ovum periphery, sperm antigen and hormone antigen.DPF is expected to become the 4th class antigen that immunological contraception is used.Based on DPF is the distinctive secretory protein of uterine tubal epithelium cell, and the contraception security meeting of its antibody obviously improves.

(3) diagnosis of the women infertility of part unknown cause and treatment

The cause of disease of women infertility is a lot, produces reasons such as anti-DPF antibody and also can cause sterile possibility but still reckon without uterine tubal epithelium cell DPF abnormal gene expression or women so far.DPF that this laboratory provides and antibody thereof can be used for the differential diagnosis of this type of cause of disease.

(4) breeding of domestic animal

The function of DPF does not have species specificity, so this laboratory the rabbit DPF or the people DPF that provide, all can improve the conceptual quotient of domestic animal.

(5) fundamental research of embryonic development early

The quality of embryonic development early behind the implantation of the quality of embryonic development decision early before the implantation.By means of DPF, people might be before the in vitro study implantation the intrinsic program of embryonic development early, the identification beacon and the technology of idioplasm amount lay the foundation in order to set up early comprehensively.

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 SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the proteic antibody of anti-64kDa has blocking effect to early embryonic development

External, in the nutrient solution that composition is determined, embryonic development morning of after fertilization can be blocked.This embryonic development blocking-up morning is ubiquitous phenomenon.Morning, embryonic development was regulated and control period from transition to zygosity (being derived from the developmental regulation element that the zygotic gene group is expressed) that regulate and control by parent form (being derived from the developmental regulation element of ovum) just to grow the time of blocking generation.And different, for example mouse is a 2-cell stage embryo early to parent form to the time follower of zygosity regulation and control transition, and the people be 8-cell stage embryo morning.And fallopian tube secretions (or Mucous Epithelium of Fallopian Tube cell condition training liquid) has the effect that overcomes embryonic development blocking-up early.

For this reason, in this embodiment, collect the rabbit fallopian tube secretions, precipitating proteins.And by preparation property polyacrylamide gel electrophoresis isolated protein each component, immune BALB/c mouse obtains at each component proteinic how anti-.

The result shows, only at molecular weight about 64, the mouse effect of embryonic development early in Mucous Epithelium of Fallopian Tube cell condition training liquid is cultivated in antibody tool 100% inhibition of 000 daltonian protein (DPF), and making it can't be by 2-cell stage embryonic development morning to 4-cell stage embryo (1) morning.

The clone of embodiment 2 DPF cDNA

Make up the cDNA library of rabbit Mucous Epithelium of Fallopian Tube with ordinary method, and pass through the 3 ＇ end DPF cDNA about the long 0.7kb of antibody screening to of embodiment 1.With the end of 3 ＇ about 0.7kb DPF cDNA, and order-checking and sequential analysis have been carried out.By the resulting gene of gene retrieval proof is a new gene.

Further screening is found, it is new full-length cDNA that a cDNA clone's dna sequence dna is arranged.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as DPF albumen, its encoding gene called after DPF protein gene.

The full length cDNA sequence of DPF is 1906bp (Fig. 2 and SEQ ID NO:1), contains complete open frame (53-1477), and coding contains the polypeptide (Fig. 2 and SEQ ID NO:2) of 475 amino-acid residues.According to analysis, shown that DPF albumen contains the signal peptide that 16 amino-acid residues are formed, sophisticated peptide molecule is formed (SEQ ID NO:3) by 459 amino-acid residues.

Embodiment 3: with the proteic encoding sequence of RT-PCR method clone DPF

Extract rabbit Mucous Epithelium of Fallopian Tube cell total rna with ordinary method, get 6mg cell total rna and 0.5 μ gOligo-dT ₁₂- ₁₈Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing ₂O dilutes.The used primer of pcr amplification is as follows: adopted primer: 5 ＇-GCTGGTTCTTGTGCTGGATT-3 ＇ (SEQ ID NO:4) is arranged, antisense primer 5 ＇-GAAAAGCAACTGATGCTCCG-3 ＇ (SEQ ID NO:5).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaqDNA polysaccharase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.The dna sequence analysis result shows the dna encoding sequence of this PCR product shown in 28-1866 among the SEQ ID NO:1, is the DPF cDNA that comprises the coding total length reading frame of signal peptide.

The Northern blot hybridization of embodiment 4 DPF is analyzed

With ³²The DPF cDNA fragment of P mark is a probe, carries out the Northern trace according to a conventional method.The result as shown in Figure 6.

The result shows that DPF only is present in the rabbit Mucous Epithelium of Fallopian Tube cell, and other tissue of rabbit does not all detect in (brain, lung, the heart, liver,spleen,kidney, small intestine, large intestine, ovary, pancreas and uterus).

Recombinant expressed and the purifying of embodiment 5 DPF polypeptide

In the present embodiment, utilize GST fusion gene expression system, express and purifying DPF, NTPDPF (1-367) (the N-terminal peptide of DPF (N-terminal peptide of DPF)) and CTP DPF (368-459) (the C-terminal peptide of DPF (C-terminal peptide of DPF)), and the corresponding antiserum(antisera) that prepared, with GST-Sepharose 4B affinity column and GST-CTP DPF (368-459)-Sepharose 4B affinity column resist carried out purifying more.

In this embodiment, be template with the pcr amplification product among the embodiment 3, increase with the PCR Oligonucleolide primers of following 5 ＇ of sequence and 3 ＇ end, obtain DPF DNA as inserting fragment.The 5 ＇ end Oligonucleolide primers sequence of using in the PCR reaction is as follows:

DPF：

5 ' end primer: 5 '-TAGAATTCTTACCAACTGGCACATAGTCG-3 ' (SEQ ID NO:6)

3 ' end primer: 5 '-TTGCGGCCGCGAAAAGCAACTGATGCTCCG-3 ' (SEQ ID NO:7)

DPF-NTP：

5 ' end primer: 5 '-TAGAATTCTTACCAACTGGCACATAGTCG-3 ' (SEQ ID NO:8)

3 ' end primer: 5 '-TTGCGGCCGCAGGTGAGTTCTTGAAAAATTC-3 ' (SEQ ID NO:9)

DPF-CTP：

5 ' end primer: 5 '-CGGAATTCTTCAAGAACTCACCTGGCCGTG-3 ' (SEQ ID NO:10)

3 ' end primer: 5 '-TTGCGGCCGCGAAAAGCAACTGATGCTCCG-3 ' (SEQ ID NO:11)

Each DPF albumen cDNA PCR product purification after EcoRI and NotI double digestion recombinate according to a conventional method with plasmid pGEX-4T-3 (Pharmacia company) again and be converted into competence intestinal bacteria DE3, thereby obtain three kinds of recombinant vectors pGEX-4T-3/DPF, pGEX-4T-3/DPF-NTP and pGEX-4T-3/DPF-CTP.

The picking DE3 clone that contains recombinant expression vector pGEX-4T-3/DPF, pGEX-4T-3/DPF-NTP or pGEX-4T-3/DPF-CTP is inoculated in 100ml 2 * YTA substratum respectively, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 50 continued shaking culture 6-8 hour, adding behind the 100mM IPTG to 0.1mM 37 ℃ induced 2 hours, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na ₂HPO ₄, 1.8mM KH ₂PO ₄PH7.3) resuspended, add 20% Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, adding 500ul gsh elution buffer (pH 8.0 for 10mM gsh, 50mM Tris-HCl) room temperature left standstill after 30 minutes collects elutriant, repeat wash-out 2-3 time, obtain GST-DPF respectively, GST-DPF-NTP (1-367) and GST-DPF-CTP (368-459) fusion rotein.

The reactive site of embodiment 6 DPF is positioned at C-terminal

In this embodiment, to express the also GST-NTPDPF (1-367) (being GST-NTP367-DPF) and the GST-CTP-DPF (368-459) (being GST-CTP92-DPF) of purifying with GST fusion gene expression system among the embodiment 5, add respectively in the various substratum (liquid), observe the developmental state of mouse fertilized egg.

The result is shown in Fig. 3 and 4.GST-NTP367-DPF does not have the effect (Fig. 3) that overcomes embryonic development blocking-up early; GST-CTP92-DPF has the effect (Fig. 4) that overcomes embryonic development blocking-up early.

Use GST-CTP-DPF antigen further, the antiserum(antisera) that obtains behind the immune bull new zealand white rabbit.The antibody that found that anti-CTP92 DPF has function sealing process (Fig. 5).

This shows that the reactive site of DPF is positioned at C-terminal.

In addition, further experiment shows, above-mentioned short morning of the embryonic development function of rabbit DPF does not have species specificity, all can promote early embryonic development on ox, sheep, pig and mouse.

Embodiment 7 MONOCLONAL ANTIBODIES SPECIFIC FOR

Alcohol precipitation enrichment rabbit uterine tube albumen, the 12%SDS-PAGE electrophoretic separation, cut out 64kD protein band (being DPF albumen), with the immune Balb/C mouse in the fully emulsified back of freund's adjuvant, after the immunity 4 times, get immune mouse spleen cell, under the PEG effect, merge with SD2/0 myeloma cell, screening positive hybridoma cell clone collects hybridoma after the enlarged culturing, inject mouse peritoneal, collect ascites after 9 days, purifying prepares monoclonal antibody.

The result has obtained the monoclonal antibody of anti-DPF.

The Western blot hybridization of embodiment 8 DPF is analyzed

Anti-DPF monoclonal antibody with embodiment 7 preparations is made probe, carries out the Western engram analysis according to a conventional method, and the result as shown in Figure 7.

The result shows that DPF only is present in the rabbit Mucous Epithelium of Fallopian Tube cell, and other tissue of rabbit does not all detect in (brain, lung, the heart, liver,spleen,kidney, small intestine, large intestine, ovary, pancreas and uterus).

Rabbit DPF homolgous molecule is also arranged in embodiment 9 people, the mouse fallopian tube secretions

By the method identical with embodiment 8, make probe with anti-DPF monoclonal antibody, people oviduct tissue is carried out immunoblotting assay, the result is as shown in Figure 8.

Simultaneously, to the Western trace density scan collection of illustrative plates (wherein making probe) of DPF and associated molecule in different plant species (kunming mice and new zealand white rabbit) the uterine tube albumen with anti-DPF monoclonal antibody.The kunming mice that obtains and the uterine tube Western blot densitometric scan collection of illustrative plates of new zealand white rabbit are as shown in Figure 9.

These also have rabbit DPF homolgous molecule in the fallopian tube secretions of confirmer, mouse as a result.

The Northern engram analysis of embodiment 10 people DPF mRNA

With rabbit DPF cDNA is template synthesizing radioactive label probe, and the total RNA of people oviduct tissue that extracts is according to a conventional method carried out the Northern engram analysis, and the result as shown in figure 10.

The result shows that people DPF mRNA is about 3.5kb.

Embodiment 11 purifying natural rabbit DPF

Prepare monoclonal antibody by the mode similar to embodiment 7, difference is to replace as the DPF protein immunogen with DPF-CTP (368-459).

To resist DPF-CTP (368-459) monoclonal antibody and the Sepharose 4B coupling of using bromize hydrogen activating, the preparation affinity column.Then, by the affinity chromatography method natural rabbit DPF of separation and purification albumen from rabbit uterine tube albumen.

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＜110〉Shanghai Family Planning Science and Research Institute.＜120〉its method for making of growth promoting factor and purposes＜130〉012029＜160〉5＜170〉PatentIn version 3.0＜210〉1＜211〉1906＜212〉DNA＜213〉rabbit＜220〉＜221〉CDS＜222〉(53) .. (1477)＜400〉1gatggggaag ctgttgctgt ggcttgggct ggttcttgtg ctggattgca cg atg gcg 58

Met?Ala

1ctg?cta?caa?gct?ggt?gtg?tta?ttt?tac?caa?ctg?gca?cat?agt?cga?ccg??????106Leu?Leu?Gln?Ala?Gly?Val?Leu?Phe?Tyr?Gln?Leu?Ala?His?Ser?Arg?Pro

5???????????????????10??????????????????15ggc?ccc?gcc?gcc?atc?ctg?ccc?cac?gac?ctg?gac?ccg?ttt?ctc?tgc?aca??????154Gly?Pro?Ala?Ala?Ile?Leu?Pro?His?Asp?Leu?Asp?Pro?Phe?Leu?Cys?Thr

20??????????????????25??????????????????30cac?ctg?ata?ttc?gcg?ttt?gcc?tca?atg?aac?gac?aat?gag?atc?gtt?gct??????202His?Leu?Ile?Phe?Ala?Phe?Ala?Ser?Met?Asn?Asp?Asn?Glu?Ile?Val?Ala35??????????????????40??????????????????45??????????????????50aag?gat?gtc?caa?gat?gag?cga?att?ttc?tac?cca?gag?ttc?aac?aaa?ctc??????250Lys?Asp?Val?Gln?Asp?Glu?Arg?Ile?Phe?Tyr?Pro?Glu?Phe?Asn?Lys?Leu

55??????????????????60??????????????????65aaa?gag?agg?aac?agg?gag?ctg?aaa?aca?ctg?ctg?tcc?att?gga?ggg?tgg??????298Lys?Glu?Arg?Asn?Arg?Glu?Leu?Lys?Thr?Leu?Leu?Ser?Ile?Gly?Gly?Trp

70??????????????????75??????????????????80aac?ttc?ggc?aca?acc?agg?ttc?acg?cca?tgc?tgg?tct?tca?ttc?gcc?agc??????346Asn?Phe?Gly?Thr?Thr?Arg?Phe?Thr?Pro?Cys?Trp?Ser?Ser?Phe?Ala?Ser

85??????????????????90??????????????????95cgt?gaa?aaa?ttt?atc?aac?tca?gtt?ata?tcc?ctg?cta?agg?aca?cac?aac??????394Arg?Glu?Lys?Phe?Ile?Asn?Ser?Val?Ile?Ser?Leu?Leu?Arg?Thr?His?Asn

100?????????????????105?????????????????110ttt?gac?ggt?ctc?gac?ctg?ttc?ttc?ttg?tac?cct?ggg?ctc?aga?ggc?agc??????442Phe?Asp?Gly?Leu?Asp?Leu?Phe?Phe?Leu?Tyr?Pro?Gly?Leu?Arg?Gly?Ser115?????????????????120?????????????????125?????????????????130cct?gcc?cat?gac?cgg?tgg?act?ttt?ctc?ttc?tta?gtt?gaa?gag?ctc?ctg??????490Pro?Ala?His?Asp?Arg?Trp?Thr?Phe?Leu?Phe?Leu?Val?Glu?Glu?Leu?Leu

135?????????????????140?????????????????145ttt?gcc?ttc?caa?agg?gag?gcg?cta?ctc?atc?aag?cgt?ccc?cgg?ctg?ctg??????538Phe?Ala?Phe?Gln?Arg?Glu?Ala?Leu?Leu?Ile?Lys?Arg?Pro?Arg?Leu?Leu

150?????????????????155?????????????????160ctc?tcc?gct?gct?gtt?tct?ggg?gtc?cca?cac?atc?atc?caa?aca?tct?tac??????586Leu?Ser?Ala?Ala?Val?Ser?Gly?Val?Pro?His?Ile?Ile?Gln?Thr?Ser?Tyr

165?????????????????170?????????????????175gac?gtg?cgc?ctt?cta?gga?aaa?ctc?ctg?gat?ttc?att?aat?gtc?ttg?tct??????634Asp?Val?Arg?Leu?Leu?Gly?Lys?Leu?Leu?Asp?Phe?Ile?Asn?Val?Leu?Ser

180?????????????????185?????????????????190tat?gac?ttg?cat?gga?agc?tgg?gaa?aag?ttc?aca?ggg?cac?aac?agt?ccc??????682Tyr?Asp?Leu?His?Gly?Ser?Trp?Glu?Lys?Phe?Thr?Gly?His?Asn?Ser?Pro195?????????????????200?????????????????205?????????????????210ctg?ttc?tct?ctg?ccc?gaa?gac?ccc?aaa?tcc?tcg?gca?tat?gcc?atg?aat??????730Leu?Phe?Ser?Leu?Pro?Glu?Asp?Pro?Lys?Ser?Ser?Ala?Tyr?Ala?Met?Asn

215?????????????????220?????????????????225tac?tgg?cga?aag?ctt?ggg?gca?ccc?tca?gag?aag?ctc?atc?atg?ggg?ttc??????778Tyr?Trp?Arg?Lys?Leu?Gly?Ala?Pro?Ser?Glu?Lys?Leu?Ile?Met?Gly?Phe

230?????????????????235?????????????????240ccc?acc?tat?gga?cgc?acc?ttt?cac?ctc?ctc?aaa?gcc?act?aac?cat?ggg??????826Pro?Thr?Tyr?Gly?Arg?Thr?Phe?His?Leu?Leu?Lys?Ala?Thr?Asn?His?Gly

245?????????????????250?????????????????255ctg?cag?gcc?caa?gcg?atc?gga?cca?gca?tct?cca?ggg?aag?tac?acc?aag??????874Leu?Gln?Ala?Gln?Ala?Ile?Gly?Pro?Ala?Ser?Pro?Gly?Lys?Tyr?Thr?Lys

260?????????????????265?????????????????270caa?gct?ggc?ttc?ctg?gct?tat?tat?gag?gtc?tgc?tcc?ttc?gtg?cgg?aag??????922Gln?Ala?Gly?Phe?Leu?Ala?Tyr?Tyr?Glu?Val?Cys?Ser?Phe?Val?Arg?Lys275?????????????????280?????????????????285?????????????????290gcg?aag?agg?cac?tgg?att?gat?tac?cag?tac?gtg?ccc?tat?gct?tac?aag??????970Ala?Lys?Arg?His?Trp?Ile?Asp?Tyr?Gln?Tyr?Val?Pro?Tyr?Ala?Tyr?Lys

295?????????????????300?????????????????305ggg?aag?gag?tgg?gtc?ggc?tat?gac?gac?gcc?atc?agc?ttc?agt?tac?aag?????1018Gly?Lys?Glu?Trp?Val?Gly?Tyr?Asp?Asp?Ala?Ile?Ser?Phe?Ser?Tyr?Lys

310?????????????????315?????????????????320gca?atg?ttt?gtc?aag?aga?aaa?cac?ttc?ggg?ggg?gct?atg?gtg?tgg?aca?????1066Ala?Met?Phe?Val?Lys?Arg?Lys?His?Phe?Gly?Gly?Ala?Met?Val?Trp?Thr

325?????????????????330?????????????????335ctg?gac?atg?gat?gac?gtc?agg?ggc?act?ttc?tgt?ggc?aat?ggc?cct?ttc?????1114Leu?Asp?Met?Asp?Asp?Val?Arg?Gly?Thr?Phe?Cys?Gly?Asn?Gly?Pro?Phe

340?????????????????345?????????????????350ccc?ctt?gtc?tac?aca?ttg?aat?gac?ctt?ctg?gtg?cag?gac?gag?tcc?acc?????1162Pro?Leu?Val?Tyr?Thr?Leu?Asn?Asp?Leu?Leu?Val?Gln?Asp?Glu?Ser?Thr355?????????????????360?????????????????365?????????????????370ccg?aca?cct?ttg?cca?caa?ttc?tgg?ttc?tcc?tca?gct?gtg?aat?ttt?tca?????1210Pro?Thr?Pro?Leu?Pro?Gln?Phe?Trp?Phe?Ser?Ser?Ala?Val?Asn?Phe?Ser

375?????????????????380?????????????????385aga?act?cac?ctg?gcc?gtg?acg?gag?ccg?ctg?acc?act?gat?att?aag?att?????1258Arg?Thr?His?Leu?Ala?Val?Thr?Glu?Pro?Leu?Thr?Thr?Asp?Ile?Lys?Ile

390?????????????????395?????????????????400ttg?ccc?cca?ggg?cgg?agg?cta?tgg?cca?ctg?aga?gca?gtg?gga?tgt?ctg?????1306Leu?Pro?Pro?Gly?Arg?Arg?Leu?Trp?Pro?Leu?Arg?Ala?Val?Gly?Cys?Leu

405?????????????????410?????????????????415aag?cac?cca?gag?gtc?gag?ctg?tgt?ccc?ttg?gca?agc?aca?ctg?tgg?ctc?????1354Lys?His?Pro?Glu?Val?Glu?Leu?Cys?Pro?Leu?Ala?Ser?Thr?Leu?Trp?Leu

420?????????????????425?????????????????430cgg?cag?aga?aga?ctg?agc?ccc?tgg?agt?gaa?gcc?cat?gac?ccc?tgg?caa?????1402Arg?Gln?Arg?Arg?Leu?Ser?Pro?Trp?Ser?Glu?Ala?His?Asp?Pro?Trp?Gln435?????????????????440?????????????????445?????????????????450gat?gac?gat?gac?ctc?tgc?ttg?agg?ttg?aga?ccc?tgc?agt?gaa?gac?cgt?????1450Asp?Asp?Asp?Asp?Leu?Cys?Leu?Arg?Leu?Arg?Pro?Cys?Ser?Glu?Asp?Arg

455?????????????????460?????????????????465gac?tcc?tgg?aag?gaa?ggc?tgt?ggc?cct?tgagagacat?cagtggcccc???????????1497Asp?Ser?Trp?Lys?Glu?Gly?Cys?Gly?Pro

470 475tggtgggcag ctatgtttct gcaaggggac aattcgactt ccgaggcagt caccgcccct 1557aggataggca acattagcgt gcaggtagac gttgggaatg cgctgccgtc ctctgtccct 1617gtcgtctggg gcccaggtct tgtttctgat agctgcttta ttctcactca actcaacgac 1677acttagaaaa aaaaaaaaaa ccctgtctct ccaataaaag aaaactttgc gctggatcag 1737ggagcctaca ttcagcagaa gcaggggcaa gcccttgtgt gtcgtcctga gtgatgttac 1797agaagctgcc tgaccccagc catctgtgaa ctgttcaggg gagaagccac ggagcatcag 1857ttgcttttct gatgaataaa cttagagaca caagaaaaaa aaaaaaaaa 1906＜210〉2＜211〉475＜212〉PRT＜213〉＜400〉2Met Ala Leu Leu Gln Ala Gly Val Leu Phe Tyr Gln Leu Ala His Ser1 5 10 15Arg Pro Gly Pro Ala Ala Ile Leu Pro His Asp Leu Asp Pro Phe Leu

20??????????????????25??????????????????30Cys?Thr?His?Leu?Ile?Phe?Ala?Phe?Ala?Ser?Met?Asn?Asp?Asn?Glu?Ile

35??????????????????40??????????????????45Val?Ala?Lys?Asp?Val?Gln?Asp?Glu?Arg?Ile?Phe?Tyr?Pro?Glu?Phe?Asn

50??????????????????55??????????????????60Lys?Leu?Lys?Glu?Arg?Asn?Arg?Glu?Leu?Lys?Thr?Leu?Leu?Ser?Ile?Gly65??????????????????70??????????????????75??????????????????80Gly?Trp?Asn?Phe?Gly?Thr?Thr?Arg?Phe?Thr?Pro?Cys?Trp?Ser?Ser?Phe

85??????????????????90??????????????????95Ala?Ser?Arg?Glu?Lys?Phe?Ile?Asn?Ser?Val?Ile?Ser?Leu?Leu?Arg?Thr

100?????????????????105?????????????????110His?Asn?Phe?Asp?Gly?Leu?Asp?Leu?Phe?Phe?Leu?Tyr?Pro?Gly?Leu?Arg

115?????????????????120?????????????????125Gly?Ser?Pro?Ala?His?Asp?Arg?Trp?Thr?Phe?Leu?Phe?Leu?Val?Glu?Glu

130?????????????????135?????????????????140Leu?Leu?Phe?Ala?Phe?Gln?Arg?Glu?Ala?Leu?Leu?Ile?Lys?Arg?Pro?Arg145?????????????????150?????????????????155?????????????????160Leu?Leu?Leu?Ser?Ala?Ala?Val?Ser?Gly?Val?Pro?His?Ile?Ile?Gln?Thr

165?????????????????170?????????????????175Ser?Tyr?Asp?Val?Arg?Leu?Leu?Gly?Lys?Leu?Leu?Asp?Phe?Ile?Asn?Val

180?????????????????185?????????????????190Leu?Ser?Tyr?Asp?Leu?His?Gly?Ser?Trp?Glu?Lys?Phe?Thr?Gly?His?Asn

195?????????????????200?????????????????205Ser?Pro?Leu?Phe?Ser?Leu?Pro?Glu?Asp?Pro?Lys?Ser?Ser?Ala?Tyr?Ala

210?????????????????215?????????????????220Met?Asn?Tyr?Trp?Arg?Lys?Leu?Gly?Ala?Pro?Ser?Glu?Lys?Leu?Ile?Met225?????????????????230?????????????????235?????????????????240Gly?Phe?Pro?Thr?Tyr?Gly?Arg?Thr?Phe?His?Leu?Leu?Lys?Ala?Thr?Asn

245?????????????????250?????????????????255His?Gly?Leu?Gln?Ala?Gln?Ala?Ile?Gly?Pro?Ala?Ser?Pro?Gly?Lys?Tyr

260?????????????????265?????????????????270Thr?Lys?Gln?Ala?Gly?Phe?Leu?Ala?Tyr?Tyr?Glu?Val?Cys?Ser?Phe?Val

275?????????????????280?????????????????285Arg?Lys?Ala?Lys?Arg?His?Trp?Ile?Asp?Tyr?Gln?Tyr?Val?Pro?Tyr?Ala

290?????????????????295?????????????????300Tyr?Lys?Gly?Lys?Glu?Trp?Val?Gly?Tyr?Asp?Asp?Ala?Ile?Ser?Phe?Ser305?????????????????310?????????????????315?????????????????320Tyr?Lys?Ala?Met?Phe?Val?Lys?Arg?Lys?His?Phe?Gly?Gly?Ala?Met?Val

325?????????????????330?????????????????335Trp?Thr?Leu?Asp?Met?Asp?Asp?Val?Arg?Gly?Thr?Phe?Cys?Gly?Asn?Gly

340?????????????????345?????????????????350Pro?Phe?Pro?Leu?Val?Tyr?Thr?Leu?Asn?Asp?Leu?Leu?Val?Gln?Asp?Glu

355?????????????????360?????????????????365Ser?Thr?Pro?Thr?Pro?Leu?Pro?Gln?Phe?Trp?Phe?Ser?Ser?Ala?Val?Asn

370?????????????????375?????????????????????380Phe?Ser?Arg?Thr?His?Leu?Ala?Val?Thr?Glu?Pro?Leu?Thr?Thr?Asp?Ile385?????????????????390?????????????????395?????????????????400Lys?Ile?Leu?Pro?Pro?Gly?Arg?Arg?Leu?Trp?Pro?Leu?Arg?Ala?Val?Gly

405?????????????????410?????????????????415Cys?Leu?Lys?His?Pro?Glu?Val?Glu?Leu?Cys?Pro?Leu?Ala?Ser?Thr?Leu

420?????????????????425?????????????????430Trp?Leu?Arg?Gln?Arg?Arg?Leu?Ser?Pro?Trp?Ser?Glu?Ala?His?Asp?Pro

435?????????????????440?????????????????445Trp?Gln?Asp?Asp?Asp?Asp?Leu?Cys?Leu?Arg?Leu?Arg?Pro?Cys?Ser?Glu

450 455 460Asp Arg Asp Ser Trp Lys Glu Gly Cys Gly Pro465,470 475＜210〉3＜211〉459＜212〉PRT＜213〉rabbit＜400〉3Arg Pro Gly Pro Ala Ala Ile Leu Pro His Asp Leu Asp Pro Phe Leu1,5 10 15Cys Thr His Leu Ile Phe Ala Phe Ala Ser Met Asn Asp Asn Glu Ile

20??????????????????25??????????????????30Val?Ala?Lys?Asp?Val?Gln?Asp?Glu?Arg?Ile?Phe?Tyr?Pro?Glu?Phe?Asn

35??????????????????40??????????????????45Lys?Leu?Lys?Glu?Arg?Asn?Arg?Glu?Leu?Lys?Thr?Leu?Leu?Ser?Ile?Gly

50??????????????????55??????????????????60Gly?Trp?Asn?Phe?Gly?Thr?Thr?Arg?Phe?Thr?Pro?Cys?Trp?Ser?Ser?Phe65??????????????????70??????????????????75??????????????????80Ala?Ser?Arg?Glu?Lys?Phe?Ile?Asn?Ser?Val?Ile?Ser?Leu?Leu?Arg?Thr

85??????????????????90??????????????????95His?Asn?Phe?Asp?Ala?Leu?Asp?Leu?Phe?Phe?Leu?Tyr?Pro?Gly?Leu?Arg

100?????????????????105?????????????????110Gly?Ser?Pro?Ala?His?Asp?Arg?Trp?Thr?Phe?Leu?Phe?Leu?Val?Glu?Glu

115?????????????????120?????????????????125Leu?Leu?Phe?Ala?Phe?Gln?Arg?Glu?Ala?Leu?Leu?Ile?Lys?Arg?Pro?Arg

130?????????????????135?????????????????140Leu?Leu?Leu?Ser?Ala?Ala?Val?Ser?Gly?Val?Pro?His?Ile?Ile?Gln?Thr145?????????????????150?????????????????155?????????????????160Ser?Tyr?Asp?Val?Arg?Leu?Leu?Gly?Lys?Leu?Leu?Asp?Phe?Ile?Asn?Val

165??????????????????170?????????????????175Leu?Ser?Tyr?Asp?Leu?His?Gly?Ser?Trp?Glu?Lys?Phe?Thr?Gly?His?Asn

180?????????????????185?????????????????190Ser?Pro?Leu?Phe?Ser?Leu?Pro?Glu?Asp?Pro?Lys?Ser?Ser?Ala?Tyr?Ala

195?????????????????200?????????????????205Met?Asn?Tyr?Trp?Arg?Lys?Leu?Gly?Ala?Pro?Ser?Glu?Lys?Leu?Ile?Met

210?????????????????215?????????????????220Gly?Phe?Pro?Ser?Tyr?Gly?Arg?Thr?Phe?His?Leu?Leu?Lys?Ala?Asn?Thr225?????????????????230?????????????????235?????????????????240His?Gly?Leu?Gln?Ala?Gln?Ala?Ile?Gly?Pro?Ala?Ser?Pro?Gly?Lys?Tyr

245?????????????????250?????????????????255Thr?Lys?Gln?Ala?Gly?Phe?Leu?Ala?Tyr?Tyr?Glu?Val?Cys?Ser?Phe?Val

260?????????????????265?????????????????270Arg?Lys?Ala?Lys?Arg?His?Trp?Ile?Asp?Tyr?Gln?Tyr?Val?Pro?Tyr?Ala

275?????????????????280?????????????????285Tyr?Lys?Gly?Lys?Glu?Trp?Val?Gly?Tyr?Asp?Asp?Ala?Ile?Ser?Phe?Ser

290?????????????????295?????????????????300Tyr?Lys?Ala?Met?Phe?Val?Lys?Arg?Lys?His?Phe?Gly?Gly?Ala?Met?Val305?????????????????310?????????????????315?????????????????320Trp?Thr?Leu?Asp?Met?Asp?Asp?Val?Arg?Gly?Thr?Phe?Cys?Gly?Asn?Gly

325?????????????????330?????????????????335Pro?Phe?Pro?Leu?Val?Tyr?Thr?Leu?Asn?Asp?Leu?Leu?Val?Gln?Asp?Glu

340?????????????????345?????????????????350Ser?Thr?Pro?Thr?Pro?Leu?Pro?Gln?Phe?Trp?Phe?Ser?Ser?Ala?Val?Asn

355?????????????????360?????????????????365Phe?Ser?Arg?Thr?His?Leu?Ala?Val?Thr?Glu?Pro?Leu?Thr?Thr?Asp?Ile

370?????????????????375?????????????????380Lys?Ile?Leu?Pro?Pro?Gly?Arg?Arg?Leu?Trp?Pro?Leu?Arg?Ala?Val?Gly385?????????????????390?????????????????395?????????????????400Cys?Leu?Lys?His?Pro?Glu?Val?Glu?Leu?Cys?Pro?Leu?Ala?Ser?Thr?Leu

405?????????????????410?????????????????415Trp?Leu?Arg?Gln?Arg?Arg?Leu?Ser?Pro?Trp?Ser?Glu?Ala?His?Asp?Pro

420?????????????????425?????????????????430Trp?Gln?Asp?Asp?Asp?Asp?Leu?Cys?Leu?Arg?Leu?Arg?Pro?Cys?Ser?Glu

435?????????????????440?????????????????445Asp?Arg?Asp?Ser?Trp?Lys?Glu?Gly?Cys?Gly?Pro

450 455＜210〉4＜211〉20＜212〉DNA＜213〉＜400〉4gctggttctt gtgctggatt 20＜210〉5＜211〉20＜212〉DNA＜213〉＜400〉5gaaaagcaac tgatgctccg 20＜210〉6＜211〉29＜212〉DNA＜213〉＜400〉6tagaattctt accaactggc acatagtcg 29＜210〉7＜211〉30＜212〉DNA＜213〉＜400〉7ttgcggccgc gaaaagcaac tgatgctccg 30＜210〉8＜211〉29＜212〉DNA＜213〉＜400〉8tagaattctt accaactggc acatagtcg 29＜210〉9＜211〉31＜212〉DNA＜213〉＜400〉9ttgcggccgc aggtgagttc ttgaaaaatt c 31＜210〉10＜211〉30＜212〉DNA＜213〉＜400〉10cggaattctt caagaactca cctggccgtg 30＜210〉11＜211〉30＜212〉DNA＜213〉＜400〉11ttgcggccgc gaaaagcaac tgatgctccg 30

Claims (11)

1. an isolating DPF polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 or 3 aminoacid sequences.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or 3.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 53-1477 position among the SEQ ID NO:1;

(b) has the sequence of 101-1477 position among the SEQ ID NO:1;

(c) has the sequence of 1-1906 position among the SEQ ID NO:1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. the preparation method with polypeptide of DPF protein-active is characterized in that, this method comprises:

(a) being fit to express under the proteic condition of DPF, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with DPF protein-active.

9. energy and the described DPF protein-specific of claim 1 bonded antibody.

10. whether there is the proteic method of DPF in a test sample, it is characterized in that, comprising:

The described antibody of sample and claim 9 is contacted,

Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DPF albumen.

11. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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