CN1277259A - Homologous protein of late embryo ample-protein and its code sequence - Google Patents

Abstract

The present invention provides one new kind of human px19 encoding cDNA sequence and the cDNA encoded protein is homologous protein of late embryogenesis abundant (LEA) protein. The present invention also relates to the polypeptide encoded by the nucleotide sequence and the application and production process of the polynucleotide and polypeptide.

Description

The homologous protein of enrichment protein of late stage embryo and encoding sequence thereof

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of people px19, this cDNA encoded protein is the homologue of LEA.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.

Enrichment protein of late stage embryo (Late Embryogenesis Abundant protein, abbreviate " lea protein " as) be that a class extensively is present in the albumen (Biosci.Biotech.Biochem.1997 in the higher plant seed, 61 (8): 1286-1291), the late stage of their seed formation in the development of plants process is assembled gradually, its mRNA is the main mRNA (Plant J.1993,3 (3): 363-369) in the mature seed.The main effect of lea protein is that the protection seed cell is avoided the injury that is caused by mummification and helped the seed of some kind to resist cold pressure (Plant Mol.Biol.1995,29 (1): 11-23).

1981, Dure etc. from cotton seeds, found the earliest lea protein (J.Biochem.1981,20:4162-4168).1988, Baker etc. be separated in the cotton seeds again 6 lea proteins (PlantMol.Biol.1988,11:277-291).In each kind of plant,, cloned multiple coding lea protein gene later on as mouseearcress, soybean, wheat, rye, paddy rice, dragon spruce, corn etc.1996, people such as Niu have cloned first animal lea protein in Gallus gallus (a kind of jungle fowl) (Gene 1996, and 175:187-191), yet its concrete physiological function is not still understood.1998, people such as Stacy were cloned in Bacillus subtilus that (Planta 1998,206:476-478) with plant lea protein height homologous sequence.

Before the present invention, also in the people, do not find or isolate the albumen that contains the LEA motif.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding LEA homologue, lea protein homologue member of the present invention is named as people px19, and it is first people's albumen that contains the LEA motif.

Another object of the present invention provides a kind of new lea protein homologue member, and this albumen is named as people px19 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's px19 polypeptide.

The present invention also provides this people's the px19 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 px19 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 42-701 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 42-701 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 42-701 position.

In another aspect of this invention, provide a kind of isolating people px19 protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

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

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

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people px19 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people px19 protein-active operationally is connected in expression regulation sequence, form people px19 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 42-701 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people px19;

(c) under the condition that is fit to expressing human px19 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people px19 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 755 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 42-701 position Nucleotide.

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

In the present invention, term " people px19 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people px19 protein-active is as 42-701 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 42-701 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 42-701 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 42-701 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 42-701 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people px19 identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

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

In the present invention, term " people px19 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people px19 protein-active.This term also comprises having and variant form people px19 albumen identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several 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 px19 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 px19 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people px19 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people px19 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people px19 polypeptide.Usually, this fragment have people px19 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 px19 albumen or polypeptide.The difference of these analogues and natural human px19 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

In the present invention, " people px19 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr

Thr(T)	Ser	Ser
			Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises people px19 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people px19 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 px19 polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people px19.

The present invention also comprises the method that detects people px19 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 px19 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 px19 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 px19 gene product or fragment.Preferably, refer to that those can combine with people px19 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 px19, comprise that also those do not influence the antibody of people px19 protein function.The present invention also comprise those can with modify or without the people px19 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 px19 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human px19 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 px19 function and the antibody that does not influence people px19 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people px19 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 px19 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 px19 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.

In one embodiment of the invention, the cDNA nucleotide sequence of people px19 is so to obtain, with people's tire brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer--A:5 '-CCTGATGCTGCGCGGGTGCTGAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide B:5 '-GGCAGAGGGGCTAAGCCTAGCTG-3 ' (SEQ ID NO:2) is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after amplified production checked order.

Enrichment protein of late stage embryo (Late Embryogenesis Abundant protein, lea protein) is that a class extensively is present in the albumen in the higher plant seed, and the late stage that seed forms in the development of plants process is assembled gradually.Their mRNA is the main mRNA (Plant J.1993,3 (3): 363-369) in the mature seed.The mummification effect of nature takes place in seed in ripening process; the main effect of lea protein is exactly to protect seed cell to avoid the injury that is caused by mummification; and significant (Plant Mol.Biol.1995,29 (1): 11-23) in the cold refrigerated physiological action of part kind tolerance.

Lea protein is according to the difference of each member's conservative motif, can be divided into several groups (Biosci.Biotech.Biochem.1997,61 (8): 1286-1291), its general character is to contain a high proportion of hydrophilic amino acid residue and have thermostability (Biosci.Biotech.Biochem.1997,61 (8): 1286-1291), may form simultaneously a kind of ionophore condenses for those are about to ion in the histocyte of mummification dehydration/and crystallization gets ready (Plant J.1993,3 (3): 363-369).

The isolated people px19 of the present invention helps the research of people to the lea protein function, especially to the research of lea protein and homologous protein function thereof in the high animals such as bird and people.In addition, because px19 of the present invention has the natural acid sequence that is derived from the people, therefore compare with the albumen of the same clan that derives from other species, 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.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people px19 of the present invention and Gallus gallus (a kind of jungle fowl) px19 (chpx19).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, condition described in the molecular cloning laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people px19

1. primer amplification

With people's tire brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer--A:5 '-CCTGATGCTGCGCGGGTGCTGAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide B:5 '-GGCAGAGGGGCTAAGCCTAGCTG-3 ' (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A/B 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 purpose Segment A/B of about 750bp.

2.PCR the order-checking of product

Above-mentioned pcr amplification product A/B is connected with pGEM-T carrier (Promega), and transformed into escherichia coli JM103 extracts plasmid with QIAprep Plasmid test kit (QIAGEN).Use SequiThermEXCEL ^TMDna sequencing kit (Epicentre Technologies) inserts fragment to plasmid and checks order, with computer software splicing order, obtain full length cDNA sequence at last, altogether 755bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 42-701 position Nucleotide.

Derive the aminoacid sequence of people px19 according to the full length cDNA sequence that obtains, totally 219 amino-acid residues, its aminoacid sequence sees SEQ IDNO.4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people px19 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 gene that contains the LEA motif and the proteins encoded thereof of they and different sources have homology widely, find relatively that with PCGENE software it and the identity of Gallus gallus (formal name used at school of a kind of jungle fowl) px19 (U31977) on protein level reach 85.6%, other has 4.7% amino acid similarity (Fig. 1).What is more important is at aminoacid sequence of the present invention ₁₇₆KTLVETAKEAKEKAKETA ₁₉₃With ₁₉₆ATEKAKD LASKAATKKQQ ₂₁₃(Gene 1996,175:187-191) to meet the general conserved sequence (A/TAEKAK/RETKD) of LEA.Therefore, people px19 albumen of the present invention can be included into the lea protein homologue, and can infer that it has the general utility functions of lea protein homologue.

Enrichment protein of late stage embryo (lea protein) is that a class extensively is present in the albumen in the higher plant seed.The late stage of their seed formation in the development of plants process is assembled gradually, and therefore a large amount of the existence.Their mRNA is the main mRNA in the mature seed, so their gathering mainly is subjected to the regulation and control (Plant J.1993,3 (3): 363-369) of transcriptional level.The main effect of lea protein is exactly to protect seed cell to avoid the injury that is caused by mummification.Closely plant among the Poncirus trifoliata at hard oranges and tangerines and to find lea protein, its expression can be subjected to inducing of cold environment, significant (Plant Mol.Biol.1995,29 (1): 11-23) in the physiological action of the cold pressure of opposing.

Lea protein is according to the difference of each member's conservative motif, can be divided into several groups (Biosci.Biotech.Biochem.1997,61 (8): 1286-1291), every type lea protein all be from the small-sized multigene family that contains 2-4 member produce and come (Plant J.1993,3 (3): 363-369).The general character of lea protein is to contain a high proportion of hydrophilic amino acid residue and have thermostability.Hydrophilic region contains the height pliable structure, can prevent the gathering that albumen is caused by hydrophobic interaction usually when thermally denature, thereby make albumen have thermostability (Biosci.Biotech.Biochem.1997,61 (8): 1286-1291).In the conservative motif research of a kind of 11 bodies of lea protein, find, 11 bodies contain a large amount of charge residue residues, they may form a kind of ionophore, their existence be ion in those histocytes that are about to mummification dehydration to 5% water content condense/crystallization gets ready (Plant J.1993,3 (3): 363-369).

1996, human subtractive hybridizations such as Niu in birds, found first to include the conservative motif of LEA animal proteinum---(Gene 1996,175:187-191) for px19.Bromodeoxyribouridine can suppress some proteic expression relevant with the embryonic tissue differentiation state and not influence cell fission or other proteic gathering.Niu group checks the transcript that expression amount reduces after stimulating the bird embryonic cell with bromodeoxyribouridine, has found px19.Px19 contains the sequence high conservative in the tumor-necrosis factor glycoproteins of the conservative motif of the 3rd group of LEA and white birch, the Radix Dauci Sativae.Northern blot experiment shows that px19 is higher than other tissue at the expression amount of liver, middle kidney, nephridioduct, hybridization in situ experiment shows that further the px19 transcript concentrates in the hematopoietic cell of liver, and have experiment to show that bromodeoxyribouridine can be suppressed to erythrocytic differentiation by blocking-up synthesizing of oxyphorase, so this prompting px19 may be relevant with hemopoietic function in the early development of bird.Yet the concrete physiological function of px19 is not still understood.We clone's new gene people px19 and bird px19 height homology are first albumen that contains the LEA motif of finding in the people.Illustrated to have important biochemical function in extensive existence of lea protein and all kinds of biologies, our further research lea protein that is found to be provides new clue.

People px19 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 px19 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of the N of inventor px19 end with the px19 of bird exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor px19, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this LEA family).

In addition, inventor px19 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people px19 or the overexpression that suppresses people px19.People px19 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people px19 disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people px19 in intestinal bacteria

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people px19 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.5 and 6) of 3 ' end increase, obtain people px19 cDNA as inserting fragment.

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

5 '-TCAGGTCGACATGGTGAAGTATTTCCTGG-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of SalI restriction enzyme, is 19 Nucleotide of the people px19 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-TTGGAAGCTTCTACACAAACTGTTGCTGC-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people px19 of HindIII restriction enzyme.

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

With SalI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people px19 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).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people px19 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people px19 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-sting isolated purifying protein the zygostyle.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 25KDa.

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

Embodiment 4

The expression of people px19 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people px19 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.7 and 8) of 3 ' end increase, obtain people px19 cDNA as inserting fragment.

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

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

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the people px19 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

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

This primer contains the part encoding sequence of the restriction enzyme site of EcoRI restriction enzyme, translation termination and people px19.

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, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and EcoRI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid is cut and the cDNA fragment of sequence verification people px19 has correctly been inserted carrier with the XmaIII 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 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 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-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 25KDa.

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

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people px19 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 (1) general information: (i) applicant: Fudan University is denomination of invention (ii): the homologous protein of enrichment protein of late stage embryo and encoding sequence thereof be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2:GGCAGAGGGG CTAAGCCTAG CTG 23 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 755bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( iii ) ：SEQ ID NO.3CCTGATGCTG CGCGGGTGCT GAGCCCGCTT CGGCCGGGAC GATGGTGAAG TATTTCCTGG 60GCCAGAGCGT GCTCCGGAGT TCCTGGGACC AAGTGTTCGC CGCCTTCTGG CAGCGGTACC 120CGAATCCCTA TAGCAAACAT GTCTTGACGG AAGACATAGT ACACCGGGAG GTGACCCCTG 180ACCAGAAACT GCTGTCCCGG CGACTCCTGA CCAAGACCAA CAGGATGCCA CGCTGGGCCG 240AGCGACTATT TCCTGCCAAT GTTGCTCACT CGGTGTACGT CCTGGAGGAC TCTATTGTGG 300ACCCACAGAA TCAGACCATG ACTACCTTCA CCTGGAACAT CAACCACGCC CGGCTGATGG 360TGGTGGAGGA ACGATGTGTT TACTGTGTGA ACTCTGACAA CAGTGGCTGG ACTGAAATCC 420GCCGGGAAGC CTGGGTCTCC TCTAGCTTAT TTGGTGTCTC CAGAGCTGTC CAGGAATTTG 480GTCTTGCCCG GTTCAAAAGC AACGTGACCA AGACTATGAA GGGTTTTGAA TATATCTTGG 540CTAAGCTGCA AGGCGAGGCC CCTTCCAAAA CACTTGTTGA GACAGCCAAG GAAGCCAAGG 600AGAAGGCAAA GGAGACGGCA CTGGCAGCTA CAGAGAAGGC CAAGGACCTC GCCAGCAAGG 660CGGCCACCAA GAAGCAGCAG CAGCAGCAAC AGTTTGTGTA GCCAGTCTAC CACCACCACA 720GCACCCCGAG ACAGCTAGGC TTAGCCCCTC TGCCC 755 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 219 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4：Met Val Lys Tyr Phe Leu Gly Gln Ser Val Leu Arg Ser Ser Trp 15Asp Gln Val Phe Ala Ala Phe Trp Gln Arg Tyr Pro Asn Pro Tyr 30Ser Lys His Val Leu Thr Glu Asp Ile Val His Arg Glu Val Thr 45Pro Asp Gln Lys Leu Leu Ser Arg Arg Leu Leu Thr Lys Thr Asn 60Arg Met Pro Arg Trp Ala Glu Arg Leu Phe Pro Ala Asn Val Ala 75His Ser Val Tyr Val Leu Glu Asp Ser Ile Val Asp Pro Gln Asn 90Gln Thr Met Thr Thr Phe Thr Trp Asn Ile Asn His Ala Arg Leu 105Met Val Val Glu Glu Arg Cys Val Tyr Cys Val Asn Ser Asp Asn 120Ser Gly Trp Thr Glu Ile Arg Arg Glu Ala Trp Val Ser Ser Ser 135Leu Phe Gly Val Ser Arg Ala Val Gln Glu Phe Gly Leu Ala Arg 150Phe Lys Ser Asn Val Thr Lys Thr Met Lys Gly Phe Glu Tyr Ile 165Leu Ala Lys Leu Gln Gly Glu Ala Pro Ser Lys Thr Leu Val Glu 180Thr Ala Lys Glu Ala Lys Glu Lys Ala Lys Glu Thr Ala Leu Ala 195Ala Thr Glu Lys Ala Lys Asp Leu Ala Ser Lys Ala Ala Thr Lys 210Lys Gln Gln Gln Gln Gln Gln Phe Val 219 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:TTGGGAATTC CTACACAAAC TGTTGCTGC 29

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people px19 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 42-701 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 42-701 position.

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

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 42-701 position.

4. isolating people px19 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

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

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

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

8. a generation has the method for the polypeptide of people px19 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people px19 protein-active operationally is connected in expression regulation sequence, form people px19 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 42-701 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people px19;

(c) under the condition that is fit to expressing human px19 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people px19 protein-active.

9. energy and the described people px19 of claim 4 protein polypeptide specificity bonded antibody.

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

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