CN1170850C - Human angiogenin-like protein and coding sequence and application thereof - Google Patents
Human angiogenin-like protein and coding sequence and application thereof Download PDFInfo
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- CN1170850C CN1170850C CNB001252828A CN00125282A CN1170850C CN 1170850 C CN1170850 C CN 1170850C CN B001252828 A CNB001252828 A CN B001252828A CN 00125282 A CN00125282 A CN 00125282A CN 1170850 C CN1170850 C CN 1170850C
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Abstract
The present invention discloses novel human angiogenin-like protein, polynucleotide for encoding the polypeptide, and a method for generating the polypeptide by recombination technology. The present invention also discloses a method by which the polypeptide is used for treating various diseases such as cancer, etc.; the present invention also discloses an antagonist for resisting the polypeptide, and a treating function thereof. The present invention also discloses the purpose of the polynucleotide for encoding the novel human angiogenin-like protein.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to the polynucleotide of new coding human angiogenin-like protein, and the polypeptide of this polynucleotide encoding.The invention still further relates to the Use and preparation method of these polynucleotide and polypeptide.
Background technology
Angiogenesis (Angiogenesis) is the key in generation, evolution and the transfer of malignant tumour.Known vascular endothelial growth factor (Vascular endothelial grouth factor, VEGF) and acceptor (VEGFreceptor VEGF R) in tumour, play an important role.VEGF and VEGF R be high expression level in vascular endothelial cell not only, and the part malignant cell expression is arranged also, thereby formation autocrine and neighbour secrete system, supports and quicken the formation and the development of malignant tumour.
Since 1996, find except that the VEGF/VEGFR system, to remain, angiogenesis is had very important effect in angiogenin (Angiopoietin Ang) and acceptor TIE2 system thereof, and with the VEGF/VEGFR system interaction is arranged, the formation of common modulating vascular.Ang is a family, and there is Ang1-5 in the Ang family that has found.Wherein Ang1 has two kinds of sequences, ANGPT1 99.3.19 registration, and registration number NM001146, Ang-1 97.3.26 registration, registration number U83508, Ang-3 have two kinds of sequences.ANGPT3 99.5.7 registration, NM_004673, Ang-1 99.5.24 registration, AF074332.I.e. totally 7 members.In addition, the cDNA (Angiopoietin-related Protein, registration number AF153606) that Korea S has registered an angiogenine associated protein at Genbank in June, 1999 belongs to and does not deliver material.
Cancer is one of principal disease of harm humans health.In order to treat effectively and prophylaxis of tumours, people more and more pay close attention to genetic treatment of tumor at present.Therefore, this area presses for albumen and the agonist/inhibitor thereof that development research has cancer suppressing action.
Summary of the invention
The purpose of this invention is to provide the new PP1158 polypeptide of a class with and fragment, analogue and derivative.PP1158 of the present invention is named as PP1158 albumen.
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 PP1158 polypeptide is provided, it comprises the polypeptide of the aminoacid sequence with SEQ ID NO:2, or its conservative property variation polypeptide or its active fragments or its reactive derivative.
Preferably, this polypeptide is the polypeptide with aminoacid sequence of SEQ ID NO:2.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group: the polynucleotide of the above-mentioned PP1158 polypeptide of (a) encoding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, the polypeptide of this polynucleotide encoding has SEQ ID NO:2 aminoacid sequence.More preferably, the sequence of these polynucleotide is selected from down group: coding region sequence or the full length sequence of SEQ ID NO:3.
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 preparation method of the active polypeptide of human angiogenin-like protein, this method comprises: (a) under the condition that is fit to the expression PP1158, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate and have the active polypeptide of human angiogenin-like protein.
In a fifth aspect of the present invention, provide and above-mentioned PP1158 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-800 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the PP1158 polypeptide of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as cancer and cellular abnormality propagation.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
In the accompanying drawings, Fig. 1 has shown the cancer suppressing action of PP1158 gene PP1158 of the present invention.Wherein set up the contrast of empty carrier control group (PCMV-Script) and non-transformed cell simultaneously.
Fig. 2 has shown the express spectra of human angiogenin-like protein of the present invention (PP1158).
Summary of the invention
The present invention adopts large-scale cDNA clone transfection cancer cell, has on the basis of cancer suppressing action in acquisition, proves new gene through order-checking, further obtains full length cDNA clone. DNA transfection evidence, human angiogenin-like protein of the present invention (PP1158) has the effect that the clone forms that suppresses to cancer cell (HCC).
In one embodiment of the invention, the human angiogenin-like protein gene is the cDNA that separates from placenta, and its sequence and Korea S ARG are basic identical from the later sequence of ATG, but above-mentioned Korea S ARG sequence, it surveys full cDNA, there is no upstream termination codon sequence. The two slightly has difference in section of encoder block ATG end office (EO) sequence, and inventor's ZZZ gene of being cloned into 3 '-the UTR zone has more an extron. The present invention not only has the termination codon sequence of 5 ' upstream, and proves that to malignant tumour namely liver cancer cell growth is inhibited. Therefore, this gene has the treating malignant tumor of being applied to and diagnostic value.
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, " PP1158 of separation or polypeptide " refers to that the PP1158 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 PP1158 of standard. Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel. The purity of PP1158 polypeptide can be used amino acid sequence analysis.
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. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analog of human angiogenin-like protein. As used herein, term " fragment ", " derivative " refer to basically keep the identical biological function of natural human PP1158 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 " human angiogenin-like protein polypeptide " refers to have the polypeptide of the SEQ ID NO.2 sequence of human angiogenin-like protein activity. This term also comprises having and the variant form human angiogenin-like protein identical function, SEQ ID NO.2 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 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 human angiogenin-like protein.
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 human angiogenin-like protein DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-human PP1158 polypeptide to obtain. The present invention also provides other polypeptide, as comprises the fusion (as comprising the fusion of sequence shown in the SEQ ID NO:2) of human angiogenin-like protein polypeptide or its fragment. Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of human angiogenin-like protein polypeptide. Usually, this fragment have the human angiogenin-like protein 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 human angiogenin-like protein or polypeptide. The difference of these analogs and natural human PP1158 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, " human angiogenin-like protein conservative variation polypeptide " refers to compare with the amino acid sequence of SEQ ID NO:2, there are 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 variation polypeptide preferably carry out amino acid substitution according to Table A and produce.
Table A
| 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 (173-1393 position) shown in the SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 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 60%, preferably at least 80%, more preferably at least 85%, the polynucleotide of at least 90% homogeny best.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 95%, be more preferably 97% 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 to separate the polynucleotide of coding human angiogenin-like protein.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
Dna sequence dna of the present invention can obtain with several method.For example, with hybridization technique DNA isolation well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homology nucleotide sequence and 2) antibody screening of expression library to be to detect the dna fragmentation of the clone with common structure feature.
The specific DNA fragment sequence of coding human angiogenin-like protein produces also and can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of required polypeptide.
In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.When the whole aminoacid sequence of the polypeptide product of needs was known, the direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.When if required amino acid whose whole sequence is not known, the direct chemical of dna sequence dna is synthetic to be impossible, and the method for selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) level of the transcript of mensuration human angiogenin-like protein; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.
In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 15 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, the length of probe within 2kb, preferably is within the 1kb usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene DNA sequence information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.
In (4) kind method, detect the protein product of human angiogenin-like protein genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
Use method (Saiki, the et al.Science 1985 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 gene of the present invention that obtains as mentioned above, perhaps the available ordinary method of mensuration of the nucleotide sequence of various dna fragmentations etc. such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467).This class nucleotide sequencing is available commercial sequencing kit etc. also.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
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 human angiogenin-like protein encoding 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 human angiogenin-like protein polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding human angiogenin-like protein 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 human angiogenin-like protein 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: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) 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 human angiogenin-like protein DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring 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 or Bowes melanoma cells 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.Alternative 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.
Recombinant polypeptide in the above methods can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.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 human angiogenin-like protein or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: directly as the disease (in particular for suppressing tumor growth) due to pharmacological agent human angiogenin-like protein hypofunction or the forfeiture be used to screen and promote or antibody, polypeptide or other part of antagonism human angiogenin-like protein function.For example, antibody can be used for activating or suppressing the function of human angiogenin-like protein.Generate the peptide molecule that can suppress or stimulate the human angiogenin-like protein function that plain sample protein screening peptide library can be used for seeking therapeutic value with the recombinant human blood vessel of expressing.
The present invention also provides screening of medicaments to identify the method that improves (agonist) or check the medicament of (antagonist) human angiogenin-like protein.Agonist improves human angiogenin-like protein biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can in the presence of medicine, the film preparation of mammalian cell or expressing human PP1158 be cultivated with the human angiogenin-like protein of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of human angiogenin-like protein comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of human angiogenin-like protein can combine and eliminate its function with human angiogenin-like protein, or suppresses the generation of human angiogenin-like protein, or combines with the avtive spot of polypeptide and to make polypeptide can not bring into play biological function.The antagonist of human angiogenin-like protein can be used for therepic use.
In screening during as the compound of antagonist, human angiogenin-like protein can be added during bioanalysis measures, determine by the interaction of measuring between compounds affect human angiogenin-like protein and its acceptor whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.
Polypeptide of the present invention can be directly used in disease treatment, for example, and various malignant tumours and cellular abnormality propagation etc.
Polypeptide of the present invention, and fragment, derivative, analogue or their cell can be used as antigen to produce antibody.These antibody can be polyclone or monoclonal antibody.Polyclonal antibody can obtain by the method with this polypeptide direct injection animal.The technology of preparation monoclonal antibody comprises hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.
Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Human angiogenin-like protein comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's human angiogenin-like protein will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
The polynucleotide of human angiogenin-like protein 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 expression of the human angiogenin-like protein of the nothing expression of human angiogenin-like protein or unusual/non-activity.The human angiogenin-like protein that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic human angiogenin-like protein activity.For example, a kind of human angiogenin-like protein of variation can be the human angiogenin-like protein that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of human angiogenin-like protein expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the human angiogenin-like protein transgenosis to cell.The method that makes up the recombinant viral vector of carrier's PP1158 gene is found in existing document (Sambrook, et al.).The recombinant human blood vessel generates plain sample protein gene and can be packaged in the liposome and be transferred in the cell in addition.
Suppress the oligonucleotide (comprising sense-rna and DNA) of human angiogenin-like protein mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carry out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension.
The present invention also provides the antibody at the human angiogenin-like protein antigenic determinant.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
The antibody of anti-human angiogenin-like protein can be used in the immunohistochemistry technology, detects the human angiogenin-like protein in the biopsy specimen.
With the also available labelled with radioisotope of human angiogenin-like protein bonded monoclonal antibody, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
The disease that antibody among the present invention can be used for treating or prevention is relevant with human angiogenin-like protein.The antibody that gives suitable dosage can stimulate or block the generation or the activity of human angiogenin-like protein.
Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of human angiogenin-like protein high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the human angiogenin-like protein positive cells.
Available human angiogenin-like protein of the production of polyclonal antibody or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
The human angiogenin-like protein monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody of anti-human angiogenin-like protein.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with human angiogenin-like protein bonded peptide molecule obtains.During screening, must carry out mark to the human angiogenin-like protein molecule.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization human angiogenin-like protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The human angiogenin-like protein level that is detected in the test can be with laying down a definition the importance of human angiogenin-like protein in various diseases and be used to the disease of diagnosing human angiogenin-like protein to work.
The polynucleotide of human angiogenin-like protein can be used for the diagnosis and the treatment of human angiogenin-like protein relative disease.Aspect diagnosis, the unconventionality expression of the expression that the polynucleotide of human angiogenin-like protein can be used for detecting human angiogenin-like protein human angiogenin-like protein whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the abnormal expression of human angiogenin-like protein as the human angiogenin-like protein 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 the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect human angiogenin-like protein with the special primer of human angiogenin-like protein.
The sudden change that detects the human angiogenin-like protein gene also can be used for the disease of diagnosing human angiogenin-like protein relevant.The form of human angiogenin-like protein sudden change comprises that the point mutation compared with normal wild type human angiogenin-like protein 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).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.
In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.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.
The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).
The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).
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 Inheritance inMan (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.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
Human angiogenin-like protein 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 come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
In one embodiment of the invention, from the human placenta cDNA library, by to the external DNA transfections screening of liver cancer cell 7721, separate, and by 5 '-RACE and full clone technology obtain the full length cDNA clone of PP1158.In addition, Northern is hybridized proof, and PP1158 gene of the present invention has expression in brain, placenta, the heart, lung, muscle, kidney, pancreas do not have express or a little less than.
In view of PP1158 has the effect of anticancer growth, so it not only has the effect of regulating cell splitted, and has the using value to treating malignant tumor and diagnosis.In addition, because human angiogenin-like protein 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.
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
Embodiment 1
The growth-inhibiting of PP1158 gene pairs human liver cancer cell and the separation of full-length cDNA
Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (Pharmacia company) of purifying.Make up the cDNA library of above-mentioned mRNA with pCMV-script TMXR cDNA library construction test kit (Stratagene company).Wherein ThermoScript II is used MMLV-RT-Superscript II (GIBCO BRL) instead, and reverse transcription reaction carries out at 42 ℃.Transform XL 10-Gold competent cell, obtained the cDNA library of 1 * 106cfu/ μ g cDNA titre.The first round is picking cDNA clone at random, is probe with high abundance cDNA clone with the cDNA clone who has proved cancer inhibitor cell growth function thereafter, screening by hybridization cDNA library, weak positive and negative clone of picking.With Qiagen 96 orifice plate plasmid extraction test kits, carry out the extraction of plasmid DNA by shop instruction.Plasmid DNA and empty carrier transfection simultaneously hepatoma cell line 7721.After the 100ng DNA alcohol precipitation drying, add 6 μ lH2O dissolving, treat transfection.Add 0.74 μ l liposome and 9.3 μ l serum-free mediums in every part of DNA sample, behind the mixing, room temperature was placed 10 minutes.Add 150 μ l serum-free mediums in every pipe, divide equally and add 3 holes and grow in 7721 cells of 96 orifice plates, placed 2 hours for 37 ℃, every hole adds 50 μ l serum-free mediums again, 37 ℃ 24 hours.Every hole is changed 100 μ l and is trained liquid entirely, 37 ℃ 24 hours, change the full training liquid 100 μ l that contain G418,37 ℃ 24～48 hours, the limit is observed, the training liquid that G418 concentration does not wait is changed on the limit.After about 2～3 times, there is the clone to form up to the microscopy cell, counting.Found that PP1158 cDNA clone has the function (it is 42,40,56 that the empty carrier clone forms number, and PP1158 is 0,0,0) that suppresses the clone and form
To finding after the analysis of PP1158 cDNA cloned sequence that gene is still imperfect, adopt Clontech company's SMART RACE cDNA amplification kit (Cat.No.K1811-1), gene specific primer is 5 '-GTGGTCCC TCTGAGTCCCCAACTCC-3 ' and 5 '-CTCAGAAAGGGGGCTTCTCCAGTCG-3 ', by specification is operated, and obtains full-length clone.
The sequential analysis of embodiment 2:PP1158 cDNA
The cDNA total length that embodiment 1 obtains is 1943 Nucleotide, contains the individual Nucleotide of reading frame 1221 (173-1393 position), the albumen of being made up of 406 amino acid (not comprising terminator codon) of encoding.
A: nucleotide sequence: (SEQ ID NO:1) length: 1943bp
1 GGAGAAGAAG CCGAGCTGAG CGGATCCTCA CACGACTGTG ATCCGATTCT
51 TTCCAGCGGC TTCTGCAACC AAGCGGGTCT TACCCCCGGT CCTCCGCGTC
101 TCCAGTCCTC GCACCTGGAA CCCCAACGTC CCCGAGAGTC CCCGAATCCC
151 CGCTCCCAGG CTACCTAAGA GGATGAGCGG TGCTCCGACG GCCGGGGCAG
201 CCCTGATGCT CTGCGCCGCC ACCGCCGTGC TACTGAGCGC TCAGGGCGGA
251 CCCGTGCAGT CCAAGTCGCC GCGCTTTGCG TCCTGGGACG AGATGAATGT
301 CCTGGCGCAC GGACTCCTGC AGCTCGGCCA GGGGCTGCGC GAACACGCGG
351 AGCGCACCCG CAGTCAGCTG AGCGCGCTGG AGCGGCGCCT GAGCGCGTGC
401 GGGTCCGCCT GTCAGGGAAC CGAGGGGTCC ACCGACCTCC CGTTAGCCCC
451 TGAGAGCCGG GTGGACCCTG AGGTCCTTCA CAGCCTGCAG ACACAACTCA
501 AGGCTCAGAA CAGCAGGATC CAGCAACTCT TCCACAAGGT GGCCCAGCAG
551 CAGCGGCACC TGGAGAAGCA GCACCTGCGA ATTCAGCATC TGCAAAGCCA
601 GTTTGGCCTC CTGGACCACA AGCACCTAGA CCATGAGGTG GCCAAGCCTG
651 CCCGAAGAAA GAGGCTGCCC GAGATGGCCC AGCCAGTTGA CCCGGCTCAC
701 AATGTCAGCC GCCTGCACCG GCTGCCCAGG GATTGCCAGG AGCTGTTCCA
751 GGTTGGGGAG AGGCAGAGTG GACTATTTGA AATCCAGCCT CAGGGGTCTC
801 CGCCATTTTT GGTGAACTGC AAGATGACCT CAGATGGAGG CTGGACAGTA
851 ATTCAGAGGC GCCACGATGG CTCAGTGGAC TTCAACCGGC CCTGGGAAGC
901 CTACAAGGCG GGGTTTGGGG ATCCCCACGG CGAGTTCTGG CTGGGTCTGG
951 AGAAGGTGCA TAGCATCACG GGGGACCGCA ACAGCCGCCT GGCCGTGCAG
1001 CTGCGGGACT GGGATGGCAA CGCCGAGTTG CTGCAGTTCT CCGTGCACCT
1051 GGGTGGCGAG GACACGGCCT ATAGCCTGCA GCTCACTGCA CCCGTGGCCG
1101 GCCAGCTGGG CGCCACCACC GTCCCACCCA GCGGCCTCTC CGTACCCTTC
1151 TCCACTTGGG ACCAGGATCA CGACCTCCGC AGGGACAAGA ACTGCGCCAA
1201 GAGCCTCTCT GGAGGCTGGT GGTTTGGCAC CTGCAGCCAT TCCAACCTCA
1251 ACGGCCAGTA CTTCCGCTCC ATCCCACAGC AGCGGCAGAA GCTTAAGAAG
1301 GGAATCTTCT GGAAGACCTG GCGGGGCCGC TACTACCCGC TGCAGGCCAC
1351 CACCATGTTG ATCCAGCCCA TGGCAGCAGA GGCAGCCTCC TAGCGTCCTG
1401 GCTGGGCCTG GTCCCAGGCC CACGAAAGAC GGTGACTCTT GGCTCTGCCC
1451 GAGGATGTGG CCGTTCCCTG CCTGGGCAGG GGCTCCAAGG AGGGGCCATC
1501 TGGAAACTTG TGGACAGAGA AGAAGACCAC GACTGGAGAA GCCCCCTTTC
1551 TGAGTGCAGG GGGGCTGCAT GCGTTGCCTC CTGAGATCGA GGCTGCAGGA
1601 TATGCTCAGA CTCTAGAGGC GTGGACCAAG GGGCATGGAG CTTCACTCCT
1651 TGCTGGCCAG GGAGTTGGGG ACTCAGAGGG ACCACTTGGG GCCAGCCAGA
1701 CTGGCCTCAA TGGCGGACTC AGTCACATTG ACTGACGGGG ACCAGGGCTT
1751 GTGTGGGTCG AGAGCGCCCT CATGGTGCTG GTGCTGTTGT GTGTAGGTCC
1801 CCTGGGGACA CAAGCAGGCG CCAATGGTAT CTGGGCGGCG TCACAGAGTT
1851 CTTGGAATAA AAGCAACCTC AGAACACTTA AAAAAAAAAA AAAAAAAAAA
1901 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA
B: aminoacid sequence: (SEQ ID NO:2) length: 406 amino acid
1 MSGAPTAGAA LMLCAATAVL LSAQGGPVQS KSPRFASWDE MNVLAHGLLQ
51 LGQGLREHAE RTRSQLSALE RRLSACGSAC QGTEGSTDLP LAPESRVDPE
101 VLHSLQTQLK AQNSRIQQLF HKVAQQQRHL EKQHLRIQHL QSQFGLLDHK
151 HLDHEVAKPA RRKRLPEMAQ PVDPAHNVSR LHRLPRDCQE LFQVGERQSG
201 LFEIQPQGSP PFLVNCKMTS DGGWTVIQRR HDGSVDFNRP WEAYKAGFGD
251 PHGEFWLGLE KVHSITGDRN SRLAVQLRDW DGNAELLQFS VHLGGEDTAY
301 SLQLTAPVAG QLGATTVPPS GLSVPFSTWD QDHDLRRDKN CAKSLSGGWW
351 FGTCSHSNLN GQYFRSIPQQ RQKLKKGIFW KTWRGRYYPL QATTMLIQPM
401 AAEAAS
C: clone number: PP1158 (SEQ ID NO:3)
Start code: 173 ATG stop coding: 1393 TAG
Protein molecular weight: 45211.75
1 G GAG AAG AAG CCG AGC TGA GCG GAT CCT CAC ACG ACT GTG ATC CGA 46
47 TTC TTT CCA GCG GCT TCT GCA ACC AAG CGG GTC TTA CCC CCG GTC CTC 94
95 CGC GTC TCC AGT CCT CGC ACC TGG AAC CCC AAC GTC CCC GAG AGT CCC 142
143 CGA ATC CCC GCT CCC AGG CTA CCT AAG AGG ATG AGC GGT GCT CCG ACG 190
1 Met Ser Gly Ala Pro Thr 6
191 GCC GGG GCA GCC CTG ATG CTC TGC GCC GCC ACC GCC GTG CTA CTG AGC 238
7 Ala Gly Ala Ala Leu Met Leu Cys Ala Ala Thr Ala Val Leu Leu Ser 22
239 GCT CAG GGC GGA CCC GTG CAG TCC AAG TCG CCG CGC TTT GCG TCC TGG 286
23 Ala Gln Gly Gly Pro Val Gln Ser Lys Ser Pro Arg Phe Ala Ser Trp 38
287 GAC GAG ATG AAT GTC CTG GCG CAC GGA CTC CTG CAG CTC GGC CAG GGG 334
39 Asp Glu Met Asn Val Leu Ala His Gly Leu Leu Gln Leu Gly Gln Gly 54
335 CTG CGC GAA CAC GCG GAG CGC ACC CGC AGT CAG CTG AGC GCG CTG GAG 382
55 Leu Arg Glu His Ala Glu Arg Thr Arg Ser Gln Leu Ser Ala Leu Glu 70
383 CGG CGC CTG AGC GCG TGC GGG TCC GCC TGT CAG GGA ACC GAG GGG TCC 430
71 Arg Arg Leu Ser Ala Cys Gly Ser Ala Cys Gln Gly Thr Glu Gly Ser 86
431 ACC GAC CTC CCG TTA GCC CCT GAG AGC CGG GTG GAC CCT GAG GTC CTT 478
87 Thr Asp Leu Pro Leu Ala Pro Glu Ser Arg Val Asp Pro Glu Val Leu 102
479 CAC AGC CTG CAG ACA CAA CTC AAG GCT CAG AAC AGC AGG ATC CAG CAA 526
103 His Ser Leu Gln Thr Gln Leu Lys Ala Gln Asn Ser Arg Ile Gln Gln 118
527 CTC TTC CAC AAG GTG GCC CAG CAG CAG CGG CAC CTG GAG AAG CAG CAC 574
119 Leu Phe His Lys Val Ala Gln Gln Gln Arg His Leu Glu Lys Gln His 134
575 CTG CGA ATT CAG CAT CTG CAA AGC CAG TTT GGC CTC CTG GAC CAC AAG 622
135 Leu Arg Ile Gln His Leu Gln Ser Gln Phe Gly Leu Leu Asp His Lys 150
623 CAC CTA GAC CAT GAG GTG GCC AAG CCT GCC CGA AGA AAG AGG CTG CCC 670
151 His Leu Asp His Glu Val Ala Lys Pro Ala Arg Arg Lys Arg Leu Pro 166
671 GAG ATG GCC CAG CCA GTT GAC CCG GCT CAC AAT GTC AGC CGC CTG CAC 718
167 Glu Met Ala Gln Pro Val Asp Pro Ala His Asn Val Ser Arg Leu His 182
719 CGG CTG CCC AGG GAT TGC CAG GAG CTG TTC CAG GTT GGG GAG AGG CAG 766
183 Arg Leu Pro Arg Asp Cys Gln Glu Leu Phe Gln Val Gly Glu Arg Gln 198
767 AGT GGA CTA TTT GAA ATC CAG CCT CAG GGG TCT CCG CCA TTT TTG GTG 814
199 Ser Gly Leu Phe Glu Ile Gln Pro Gln Gly Ser Pro Pro Phe Leu Val 214
815 AAC TGC AAG ATG ACC TCA GAT GGA GGC TGG ACA GTA ATT CAG AGG CGC 862
215 Asn Cys Lys Met Thr Ser Asp Gly Gly Trp Thr Val Ile Gln Arg Arg 230
863 CAC GAT GGC TCA GTG GAC TTC AAC CGG CCC TGG GAA GCC TAC AAG GCG 910
231 His Asp Gly Ser Val Asp Phe Asn Arg Pro Trp Glu Ala Tyr Lys Ala 246
911 GGG TTT GGG GAT CCC CAC GGC GAG TTC TGG CTG GGT CTG GAG AAG GTG 958
247 Gly Phe Gly Asp Pro His Gly Glu Phe Trp Leu Gly Leu Glu Lys Val 262
959 CAT AGC ATC ACG GGG GAC CGC AAC AGC CGC CTG GCC GTG CAG CTG CGG 1006
263 His Ser Ile Thr Gly Asp Arg Asn Ser Arg Leu Ala Val Gln Leu Arg 278
1007 GAC TGG GAT GGC AAC GCC GAG TTG CTG CAG TTC TCC GTG CAC CTG GGT 1054
279 Asp Trp Asp Gly Asn Ala Glu Leu Leu Gln Phe Ser Val His Leu Gly 294
1055 GGC GAG GAC ACG GCC TAT AGC CTG CAG CTC ACT GCA CCC GTG GCC GGC 1102
295 Gly Glu Asp Thr Ala Tyr Ser Leu Gln Leu Thr Ala Pro Val Ala Gly 310
1103 CAG CTG GGC GCC ACC ACC GTC CCA CCC AGC GGC CTC TCC GTA CCC TTC 1150
311 Gln Leu Gly Ala Thr Thr Val Pro Pro Ser Gly Leu Ser Val Pro Phe 326
1151 TCC ACT TGG GAC CAG GAT CAC GAC CTC CGC AGG GAC AAG AAC TGC GCC 1198
327 Ser Thr Trp Asp Gln Asp His Asp Leu Arg Arg Asp Lys Asn Cys Ala 342
1199 AAG AGC CTC TCT GGA GGC TGG TGG TTT GGC ACC TGC AGC CAT TCC AAC 1246
343 Lys Ser Leu Ser Gly Gly Trp Trp Phe Gly Thr Cys Ser His Ser Asn 358
1247 CTC AAC GGC CAG TAC TTC CGC TCC ATC CCA CAG CAG CGG CAG AAG CTT 1294
359 Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro Gln Gln Arg Gln Lys Leu 374
1295 AAG AAG GGA ATC TTC TGG AAG ACC TGG CGG GGC CGC TAC TAC CCG CTG 1342
375 Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg Gly Arg Tyr Tyr Pro Leu 390
1343 CAG GCC ACC ACC ATG TTG ATC CAG CCC ATG GCA GCA GAG GCA GCC TCC 1390
391 Gln Ala Thr Thr Met Leu Ile Gln Pro Met Ala Ala Glu Ala Ala Ser 406
1391 TAG CGT CCT GGC TGG GCC TGG TCC CAG GCC CAC GAA AGA CGG TGA CTC 1438
407 *** 407
1439 TTG GCT CTG CCC GAG GAT GTG GCC GTT CCC TGC CTG GGC AGG GGC TCC 1486
1487 AAG GAG GGG CCA TCT GGA AAC TTG TGG ACA GAG AAG AAG ACC ACG ACT 1534
1535 GGA GAA GCC CCC TTT CTG AGT GCA GGG GGG CTG CAT GCG TTG CCT CCT 1582
1583 GAG ATC GAG GCT GCA GGA TAT GCT CAG ACT CTA GAG GCG TGG ACC AAG 1630
1631 GGG CAT GGA GCT TCA CTC CTT GCT GGC CAG GGA GTT GGG GAC TCA GAG 1678
1679 GGA CCA CTT GGG GCC AGC CAG ACT GGC CTC AAT GGC GGA CTC AGT CAC 1726
1727 ATT GAC TGA CGG GGA CCA GGG CTT GTG TGG GTC GAG AGC GCC CTC ATG 1774
1775 GTG CTG GTG CTG TTG TGT GTA GGT CCC CTG GGG ACA CAA GCA GGC GCC 1822
1823 AAT GGT ATC TGG GCG GCG TCA CAG AGT TCT TGG AAT AAA AGC AAC CTC 1870
1871 AGA ACA CTT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1918
1919 AAA AAA AAA AAA AAA AAA AAA AAA A 1943
D. homology relatively
PP1158 cDNA clone of the present invention contains full length cDNA sequence.
(1) the nucleotide sequence homology relatively
Query=PP1158 (1943 Nucleotide)
GB_PR4:AF153606 AF153606 human angiogenin associated protein
MRNA, complete cds.6/1999 length=1860
Score value=2813bits (1419), predicated value=0.0
Homogeny=1440/1443 (99%), similarity=1440/1443 (99%), breach=3/1443 (0%)
Query：20 gcggatcctcacacgactgtgatccgattctttccagcggcttctgcaaccaagcgggtc 79
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Sbjct：1 gcggatcctcacacgactgtgatccgattctttccagcggcttctgcaaccaagcgggtc 60
Query：80 ttacccccggtcctccgcgtctccagtcctcgcacctggaaccccaacgtccccgagagt 139
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Sbjct：61 ttacccccggtcctccgcgtctccagtcctcgcacctggaaccccaacgtccccgagagt 120
Query：140 ccccgaatccccgctcccaggctacctaagaggatgagcggtgctccgacggccggggca 199
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Sbjct：121 ccccgaatccccgctcccaggctacctaagaggatgagcggtgctccgacggccggggca 180
Query：200 gccctgatgctctgcgccgccaccgccgtgctactgagcgctcagggcggacccgtgcag 259
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Sbjct：181 gccctgatgctctgcgccgccaccgccgtgctactgagcgctcagggcggacccgtgcag 240
Query：260 tccaagtcgccgcgctttgcgtcctgggacgagatgaatgtcctggcgcacggactcctg 319
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Sbjct：241 tccaagtcgccgcgctttgcgtcctgggacgagatgaatgtcctggcgcacggactcctg 300
Query：320 cagctcggccaggggctgcgcgaacacgcggagcgcacccgcagtcagctgagcgcgctg 379
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Sbjct：301 cagctcggccagggg-tgcgcgaacac-cggagcgcacccgcagtcagctgagcgcgctg 358
Query：380 gagcggcgcctgagcgcgtgcgggtccgcctgtcagggaaccgaggggtccaccgacctc 439
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Sbjct：359 gagcg-cgcctgagcgcgtgcgggtccgcctgtcagggaaccgaggggtccaccgacctc 417
Query：440 ccgttagcccctgagagccgggtggaccctgaggtccttcacagcctgcagacacaactc 499
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Sbjct：418 ccgttagcccctgagagccgggtggaccctgaggtccttcacagcctgcagacacaactc 477
Query：500 aaggctcagaacagcaggatccagcaactcttccacaaggtggcccagcagcagcggcac 559
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Sbjct：478 aaggctcagaacagcaggatccagcaactcttccacaaggtggcccagcagcagcggcac 537
Query：560 ctggagaagcagcacctgcgaattcagcatctgcaaagccagtttggcctcctggaccac 619
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Sbjct：538 ctggagaagcagcacctgcgaattcagcatctgcaaagccagtttggcctcctggaccac 597
Query：620 aagcacctagaccatgaggtggccaagcctgcccgaagaaagaggctgcccgagatggcc 679
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Sbjct：598 aagcacctagaccatgaggtggccaagcctgcccgaagaaagaggctgcccgagatggcc 657
Query：680 cagccagttgacccggctcacaatgtcagccgcctgcaccggctgcccagggattgccag 739
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Sbjct：658 cagccagttgacccggctcacaatgtcagccgcctgcaccggctgcccagggattgccag 717
Query：740 gagctgttccaggttggggagaggcagagtggactatttgaaatccagcctcaggggtct 799
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Sbjct：718 gagctgttccaggttggggagaggcagagtggactatttgaaatccagcctcaggggtct 777
Query：800 ccgccatttttggtgaactgcaagatgacctcagatggaggctggacagtaattcagagg 859
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Sbjct：778 ccgccatttttggtgaactgcaagatgacctcagatggaggctggacagtaattcagagg 837
Query：860 cgccacgatggctcagtggacttcaaccggccctgggaagcctacaaggcggggtttggg 919
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：838 cgccacgatggctcagtggacttcaaccggccctgggaagcctacaaggcggggtttggg 897
Query：920 gatccccacggcgagttctggctgggtctggagaaggtgcatagcatcacgggggaccgc 979
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：898 gatccccacggcgagttctggctgggtctggagaaggtgcatagcatcacgggggaccgc 957
Query：980 aacagccgcctggccgtgcagctgcgggactgggatggcaacgccgagttgctgcagttc 1039
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：958 aacagccgcctggccgtgcagctgcgggactgggatggcaacgccgagttgctgcagttc 1017
Query：1040 tccgtgcacctgggtggcgaggacacggcctatagcctgcagctcactgcacccgtggcc 1099
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1018 tccgtgcacctgggtggcgaggacacggcctatagcctgcagctcactgcacccgtggcc 1077
Query：1100 ggccagctgggcgccaccaccgtcccacccagcggcctctccgtacccttctccacttgg 1159
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1078 ggccagctgggcgccaccaccgtcccacccagcggcctctccgtacccttctccacttgg 1137
Query：1160 gaccaggatcacgacctccgcagggacaagaactgcgccaagagcctctctggaggctgg 1219
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1138 gaccaggatcacgacctccgcagggacaagaactgcgccaagagcctctctggaggctgg 1197
Query：1220 tggtttggcacctgcagccattccaacctcaacggccagtacttccgctccatcccacag 1279
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1198 tggtttggcacctgcagccattccaacctcaacggccagtacttccgctccatcccacag 1257
Query：1280 cagcggcagaagcttaagaagggaatcttctggaagacctggcggggccgctactacccg 1339
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1258 cagcggcagaagcttaagaagggaatcttctggaagacctggcggggccgctactacccg 1317
Query：1340 ctgcaggccaccaccatgttgatccagcccatggcagcagaggcagcctcctagcgtcct 1399
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1318 ctgcaggccaccaccatgttgatccagcccatggcagcagaggcagcctcctagcgtcct 1377
Query：1400 ggctgggcctggtcccaggcccacgaaagacggtgactcttggctctgcccgaggatgtg 1459
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1378 ggctgggcctggtcccaggcccacgaaagacggtgactcttggctctgcccgaggatgtg 1437
Query：1460 gcc 1462
|||
Sbjct：1438 gcc 1440
Score value=680bits (343), predicated value=0.0
Homogeny=353/355 (99%), similarity=353/355 (99%), breach=1/355 (0%)
Query：1523 aagaccacgactggagaagccccctttctgagtgcaggggggctgcatgcgttgcctcct 1582
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1441 aagaccacgactggagaagccccctttctgagtgcaggggggctgcatgcgttgcctcct 1500
Query：1583 gagatcgaggctgcaggatatgctcagactctagaggcgtggaccaaggggcatggagct 1642
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1501 gagatcgaggctgcaggatatgctcagactctagaggcgtggaccaaggggcatggagct 1560
Query：1643 tcactccttgctggccagggagttggggactcagagggaccacttggggccagccagact 1702
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1561 tcactccttgctggccagggagttggggactcagagggaccacttggggccagccagact 1620
Query：1703 ggcctcaatggcggactcagtcacattgactgacggggaccagggcttgtgtgggtcgag 1762
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1621 ggcctcaatggcggactcagtcacattgactgacggggaccagggcttgtgtgggtcgag 1680
Query：1763 agcgccctcatggtgctggtgctgttgtgtgtaggtcccctggggacacaagcaggcgcc 1822
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1681 agcgccctcatggtgctggtgctgttgtgtgtaggtcccctggggacacaagcaggcgcc 1740
Query：1823 aatggtatctgggcggcg-tcacagagttcttggaataaaagcaacctcagaaca 1876
|||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1741 aatggtatctgggcggagctcacagagttcttggaataaaagcaacctcagaaca 1795
Score value=71.9bits (36), predicated value=7e-10
Homogeny=36/36 (100%), similarity=36/36 (100%)
Query：1841 tcacagagttcttggaataaaagcaacctcagaaca 1876
||||||||||||||||||||||||||||||||||||
Sbjct：1820 tcacagagttcttggaataaaagcaacctcagaaca 1855
(2) comparison of aminoacid sequence and genes involved
Query=PP1158AA (406 amino acid)
Produce the sequence that significantly alignment is arranged: (bits) value
NG27 585 e-171
Gi|4557315|ref|NP_001138.1|| angiogenin 2 167 3e-45
Gi|4378598|gb|AAD19608.1| angiogenin Y1 162 7e-44
Gi|4502087|ref|NP_001137.1|| angiogenin 1 149 6e-40
NG27 length=410
Score value=585bits (1493), predicated value=e-171
Homogeny=286/390 (73%), similarity=318/390 (81%), breach=6/390 (1%)
Query：22 SAQGGPVQSKSPRFASWDEMNVLAHXXXXXXXXXREHAERTRSQLSALERRLSACGSACQ 81
SAQG P Q + PRFASWDEMN+LAH REH ERTR QL ALERR++ACG+ACQ
Sbjct：22 SAQGRPAQPEPPRFASWDEMNLLAHGLLQLGHGLREHVERTRGQLGALERRMAACGNACQ 81
Query：82 GTEGSTDLPLAP-ESRVD----PEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLR 136
G +G D P E RV PE L SLQTQLKAQNS+IQQLF KVAQQQR+L KQ+LR
Sbjct：82 GPKGK-DAPFKDSEDRVPEGQTPETLQSLQTQLKAQNSKIQQLFQKVAQQQRYLSKQNLR 140
Query：137 IQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGE 196
IQ+LQSQ LL HLD+ V K +R KRLP+M Q + N + LHR PRDCQELFQ GE
Sbjct：141 IQNLQSQIDLLAPTHLDNGVDKTSRGKRLPKMTQLIGLTPNATHLHRPPRDCQELFQEGE 200
Query：197 RQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFW 256
R SGLF+IQP GSPPFLVNC+MTSDGGWTVIQRR +GSVDFN+ WEAYK GFGDP GEFW
Sbjct：201 RHSGLFQIQPLGSPPFLVNCEMTSDGGWTVIQRRLNGSVDFNQSWEAYKDGFGDPQGEFW 260
Query：257 LGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATT 316
LGLEK+HSITG+R S+LAVQL+DWDGNA+LLQF +HLGGEDTAYSLQLT P A +LGAT
Sbjct：261 LGLEKMHSITGNRGSQLAVQLQDWDGNAKLLQFPIHLGGEDTAYSLQLTEPTANELGATN 320
Query：317 VPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKK 376
V P+GLS+PFSTWDQDHDLR D NCAKSLSGGWWFGTCSHSNLNGQYF SIP+QRQ+ KK
Sbjct：321 VSPNGLSLPFSTWDQDHDLRGDLNCAKSLSGGWWFGTCSHSNLNGQYFHSIPRQRQERKK 380
Query：377 GIFWKTWRGRYYPLQATTMLIQPMAAEAAS 406
GIFWKTW+GRYYPLQATT+LIQPM A AAS
Sbjct：381 GIFWKTWKGRYYPLQATTLLIQPMEATAAS 410
Gi|4557315|ref|NP_001138.1|| angiogenin 2 length=496
Score value=167bits (418), predicated value=3e-45
Homogeny=93/221 (42%), similarity=127/221 (57%), breach=16/221 (7%)
Query：186 RDCQELFQVGERQSGLFEIQ-PQGSPPFLVNCKMTSDGG-WTVIQRRHDGSVDFNRPWEA 243
RDC E+F+ G +G++ + P + C M + GG WT+IQRR DGSVDF R W+
Sbjct：282 RDCAEVFKSGHTTNGIYTLTFPNSTEEIKAYCDMEAGGGGWTIIQRREDGSVDFQRTWKE 341
Query：244 YKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGN-AELLQFSVHLGGEDTAYSL 302
YK GFG+P GE+WLG E V +T + L + L+DW+GN A L +L E+ Y +
Sbjct：342 YKVGFGNPSGEYWLGNEFVSQLTNQQRYVLKIHLKDWEGNEAYSLYEHFYLSSEELNYRI 401
Query：303 QL--TAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLN 360
L AG++ + + P + FST D D+D K C++ L+GGWWF C SNLN
Sbjct：402 HLKGLTGTAGKISSISQPGN----DFSTKDGDNDKCICK-CSQMLTGGWWFDACGPSNLN 456
Query：361 GQYFRSIPQQRQKLKK--GIFWKTWRGRYYPLQATTMLIQP 399
G Y+ QRQ K GI W W+G Y L+ATTM+I+P
Sbjct：457 GMYY----PQRQNTNKFNGIKWYYWKGSGYSLKATTMMIRP 493
Score value=22.3bits (46), predicated value=0.11
Homogeny=10/34 (29%), similarity=18/34 (52%)
Query：122 KVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHE 155
++ QQR++ K HL+ + L +H +L E
Sbjct：362 QLTNQQRYVLKIHLKDWEGNEAYSLYEHFYLSSE 395
Gi|4378598|gb|AAD19608.1| angiogenin Y1 length=491
Score value=162bits (406), predicated value=7e-44
Homogeny=88/218 (40%), similarity=128/218 (58%), breach=8/218 (3%)
Query：186 RDCQELFQVGERQSGLFEIQPQGSP-PFLVNCKMTSD-GGWTVIQRRHDGSVDFNRPWEA 243
+DCQ+ + G SG++ I+P+ S P + C+ + D GGWTVIQ+R DGSV+F R WE
Sbjct：278 KDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTDGSVNFFRNWEN 337
Query：244 YKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQF-SVHLGGEDTAYSL 302
YK GFG+ GE+WLGLE ++ ++ N +L ++L DW ++ S L E Y L
Sbjct：338 YKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAEYSSFRLEPESEFYRL 397
Query：303 QLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQ 362
+L G G + + +G F+T D+D D+ NCA GGWW+ C+HSNLNG
Sbjct：398 RL-GTYQGNAGDSMMWHNGKQ--FTTLDRDKDMYAG-NCAHFHKGGWWYNACAHSNLNGV 483
Query：363 YFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPM 400
++R R K + GIFW +RG Y L+A M+I+P+
Sbjct：454 WYRG-GHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPI 490
Score value=21.2bits (43), predicated value=0.24
Homogeny=26/111 (23%), similarity=47/111 (41%), breach=10/111 (9%)
Query：105 LQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKR 164
L+ + + NSR+ QL+ ++ + L + L+++ ++ E+ K A R R
Sbjct：119 LRKESRNMNSRVTQLYMQLLHEIIRKRDNSLELSQLENKI-----LNVTTEMLKMATRYR 173
Query：165 LPEM--AQPVDPAHNVS-RLHRLPRDCQELF--QVGERQSGLFEIQPQGSP 210
E+ A D +N S + L C +F Q L ++ PQ P
Sbjct：174 ELEVKYASLTDLVNNQSVMITLLEEQCLRIFSRQDTHVSPPLVQVVPQHIP 224
Gi|4502087|ref|NP_001137.1|| angiogenin 1 length=192
Score value=149bits (372), predicated value=6e-40
Homogeny=85/194 (43%), similarity=110/194 (55%), breach=11/194 (5%)
Query：211 PFLVNCKM-TSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDR 269
P V C M + GGWTVIQ R DGS+DF R W+ YK GFG+P GE+WLG E + +IT R
Sbjct：4 PKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQR 63
Query：270 NSRLAVQLRDWDGNAELLQFS-VHLGGEDTAYSLQLTAPVAGQLGATTVPPSGL--SVPF 326
L ++L DW+GN Q+ H+G E Y L L G G S + F
Sbjct：64 QYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYL----KGHTGTAGKQSSLILHGADF 119
Query：327 STWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGR 386
ST D D+D K CA L+GGWWF C SNLNG ++ + Q KL GI W ++G
Sbjct：120 STKDADNDNCMCK-CALMLTGGWWFDACGPSNLNGMFY-TAGQNHGKL-NGIKWHYFKGP 176
Query：387 YYPLQATTMLIQPM 400
Y L++TTM+I+P+
Sbjct：177 SYSLRSTTMMIRPL 190
Embodiment 3: become the knurl experiment in PP1158 gene transformation cell (L929) body
Male nude mouse with 8 ages in week is divided into experimental group (Elpp1158) at random, empty carrier control group (PCMV-Script) and non-transformed cell contrast (Control) three groups, and every group is respectively 4,6 and 6 nude mices.
Every the right back neck veutro of nude mice subcutaneous vaccination transformant 2 * 10
6Individual, it during the experimental observation 4 weeks, observation period finishes the back and puts to death mouse, take out Subcutaneous tumor, its weight of weighing compares, experimental group (Elpp1158) has been compared utmost point significant difference (p＜0.01) with empty carrier control group (PCMV-Script) as a result, and the effect (see Table 1 and Fig. 1) of obvious inhibition transformed cell growth is promptly arranged.
Table 1: transformant (L929) becomes knurl experimental tumor size comparison sheet
| Numbering | Elpp1158 | The PCMV-Script carrier | The non-processor groups of cells |
| 1 | 0.3 | 3.9 | 0.9 |
| 2 | 0.7 | 3.7 | 0.6 |
| 3 | 0.7 | 3.3 | 0.2 |
| 4 | 0.3 | 3 | 0 |
| 5 | 2.8 | 0 | |
| 6 | 2.1 | 0 | |
| Mean value ± SD | 0.5* | 3.133+0.653 | 0.283 |
*：p＜0.01
Embodiment 4: the mRNA of PP1158 cDNA in various tissues expresses
With the PP1158 gene is probe, hybridizes in multiple tissue mRNA diaphragm (Clontech).The result finds that the PP1158 gene has expression in brain, placenta as shown in the figure, does not have at the heart, lung, muscle, kidney, pancreas and expresses or more weak (see figure 2).
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: Shanghai Inst. of Tumor
(ii) denomination of invention: human angiogenin-like protein and encoding sequence and uses thereof
(iii) sequence number: 3
(2) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 1943bp
(B) type: Nucleotide
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(ix) sequence description: SEQ ID NO.1:
GGAGAAGAAG CCGAGCTGAG CGGATCCTCA CACGACTGTG ATCCGATTCT TTCCAGCGGC 60
TTCTGCAACC AAGCGGGTCT TACCCCCGGT CCTCCGCGTC TCCAGTCCTC GCACCTGGAA 120
CCCCAACGTC CCCGAGAGTC CCCGAATCCC CGCTCCCAGG CTACCTAAGA GGATGAGCGG 180
TGCTCCGACG GCCGGGGCAG CCCTGATGCT CTGCGCCGCC ACCGCCGTGC TACTGAGCGC 240
TCAGGGCGGA CCCGTGCAGT CCAAGTCGCC GCGCTTTGCG TCCTGGGACG AGATGAATGT 300
CCTGGCGCAC GGACTCCTGC AGCTCGGCCA GGGGCTGCGC GAACACGCGG AGCGCACCCG 360
CAGTCAGCTG AGCGCGCTGG AGCGGCGCCT GAGCGCGTGC GGGTCCGCCT GTCAGGGAAC 420
CGAGGGGTCC ACCGACCTCC CGTTAGCCCC TGAGAGCCGG GTGGACCCTG AGGTCCTTCA 480
CAGCCTGCAG ACACAACTCA AGGCTCAGAA CAGCAGGATC CAGCAACTCT TCCACAAGGT 540
GGCCCAGCAG CAGCGGCACC TGGAGAAGCA GCACCTGCGA ATTCAGCATC TGCAAAGCCA 600
GTTTGGCCTC CTGGACCACA AGCACCTAGA CCATGAGGTG GCCAAGCCTG CCCGAAGAAA 660
GAGGCTGCCC GAGATGGCCC AGCCAGTTGA CCCGGCTCAC AATGTCAGCC GCCTGCACCG 720
GCTGCCCAGG GATTGCCAGG AGCTGTTCCA GGTTGGGGAG AGGCAGAGTG GACTATTTGA 780
AATCCAGCCT CAGGGGTCTC CGCCATTTTT GGTGAACTGC AAGATGACCT CAGATGGAGG 840
CTGGACAGTA ATTCAGAGGC GCCACGATGG CTCAGTGGAC TTCAACCGGC CCTGGGAAGC 900
CTACAAGGCG GGGTTTGGGG ATCCCCACGG CGAGTTCTGG CTGGGTCTGG AGAAGGTGCA 960
TAGCATCACG GGGGACCGCA ACAGCCGCCT GGCCGTGCAG CTGCGGGACT GGGATGGCAA 1020
CGCCGAGTTG CTGCAGTTCT CCGTGCACCT GGGTGGCGAG GACACGGCCT ATAGCCTGCA 1080
GCTCACTGCA CCCGTGGCCG GCCAGCTGGG CGCCACCACC GTCCCACCCA GCGGCCTCTC 1140
CGTACCCTTC TCCACTTGGG ACCAGGATCA CGACCTCCGC AGGGACAAGA ACTGCGCCAA 1200
GAGCCTCTCT GGAGGCTGGT GGTTTGGCAC CTGCAGCCAT TCCAACCTCA ACGGCCAGTA 1260
CTTCCGCTCC ATCCCACAGC AGCGGCAGAA GCTTAAGAAG GGAATCTTCT GGAAGACCTG 1320
GCGGGGCCGC TACTACCCGC TGCAGGCCAC CACCATGTTG ATCCAGCCCA TGGCAGCAGA 1380
GGCAGCCTCC TAGCGTCCTG GCTGGGCCTG GTCCCAGGCC CACGAAAGAC GGTGACTCTT 1440
GGCTCTGCCC GAGGATGTGG CCGTTCCCTG CCTGGGCAGG GGCTCCAAGG AGGGGCCATC 1500
TGGAAACTTG TGGACAGAGA AGAAGACCAC GACTGGAGAA GCCCCCTTTC TGAGTGCAGG 1560
GGGGCTGCAT GCGTTGCCTC CTGAGATCGA GGCTGCAGGA TATGCTCAGA CTCTAGAGGC 1620
GTGGACCAAG GGGCATGGAG CTTCACTCCT TGCTGGCCAG GGAGTTGGGG ACTCAGAGGG 1680
ACCACTTGGG GCCAGCCAGA CTGGCCTCAA TGGCGGACTC AGTCACATTG ACTGACGGGG 1740
ACCAGGGCTT GTGTGGGTCG AGAGCGCCCT CATGGTGCTG GTGCTGTTGT GTGTAGGTCC 1800
CCTGGGGACA CAAGCAGGCG CCAATGGTAT CTGGGCGGCG TCACAGAGTT CTTGGAATAA 1860
AAGCAACCTC AGAACACTTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1920
AAAAAAAAAA AAAAAAAAAA AAA 1943
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 406 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: polypeptide
(ix) sequence description: SEQ ID NO.2:
MSGAPTAGAA LMLCAATAVL LSAQGGPVQS KSPRFASWDE MNVLAHGLLQ 50
LGQGLREHAE RTRSQLSALE RRLSACGSAC QGTEGSTDLP LAPESRVDPE 100
VLHSLQTQLK AQNSRIQQLF HKVAQQQRHL EKQHLRIQHL QSQFGLLDHK 150
HLDHEVAKPA RRKRLPEMAQ PVDPAHNVSR LHRLPRDCQE LFQVGERQSG 200
LFEIQPQGSP PFLVNCKMTS DGGWTVIQRR HDGSVDFNRP WEAYKAGFGD 250
PHGEFWLGLE KVHSITGDRN SRLAVQLRDW DGNAELLQFS VHLGGEDTAY 300
SLQLTAPVAG QLGATTVPPS GLSVPFSTWD QDHDLRRDKN CAKSLSGGWW 350
FGTCSHSNLN GQYFRSIPQQ RQKLKKGIFW KTWRGRYYPL QATTMLIQPM 400
AAEAAS 406
(2) information of SEQ ID NO.3
(i) sequence signature:
(A) length: 1943bp
(B) type: Nucleotide
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(ix) sequence description: SEQ ID NO.3:
1 G GAG AAG AAG CCG AGC TGA GCG GAT CCT CAC ACG ACT GTG ATC CGA 46
47 TTC TTT CCA GCG GCT TCT GCA ACC AAG CGG GTC TTA CCC CCG GTC CTC 94
95 CGC GTC TCC AGT CCT CGC ACC TGG AAC CCC AAC GTC CCC GAG AGT CCC 142
143 CGA ATC CCC GCT CCC AGG CTA CCT AAG AGG ATG AGC GGT GCT CCG ACG 190
1 Met Ser Gly Ala Pro Thr 6
191 GCC GGG GCA GCC CTG ATG CTC TGC GCC GCC ACC GCC GTG CTA CTG AGC 238
7 Ala Gly Ala Ala Leu Met Leu Cys Ala Ala Thr Ala Val Leu Leu Ser 22
239 GCT CAG GGC GGA CCC GTG CAG TCC AAG TCG CCG CGC TTT GCG TCC TGG 286
23 Ala Gln Gly Gly Pro Val Gln Ser Lys Ser Pro Arg Phe Ala Ser Trp 38
287 GAC GAG ATG AAT GTC CTG GCG CAC GGA CTC CTG CAG CTC GGC CAG GGG 334
39 Asp Glu Met Asn Val Leu Ala His Gly Leu Leu Gln Leu Gly Gln Gly 54
335 CTG CGC GAA CAC GCG GAG CGC ACC CGC AGT CAG CTG AGC GCG CTG GAG 382
55 Leu Arg Glu His Ala Glu Arg Thr Arg Ser Gln Leu Ser Ala Leu Glu 70
383 CGG CGC CTG AGC GCG TGC GGG TCC GCC TGT CAG GGA ACC GAG GGG TCC 430
71 Arg Arg Leu Ser Ala Cys Gly Ser Ala Cys Gln Gly Thr Glu Gly Ser 86
431 ACC GAC CTC CCG TTA GCC CCT GAG AGC CGG GTG GAC CCT GAG GTC CTT 478
87 Thr Asp Leu Pro Leu Ala Pro Glu Ser Arg Val Asp Pro Glu Val Leu 102
479 CAC AGC CTG CAG ACA CAA CTC AAG GCT CAG AAC AGC AGG ATC CAG CAA 526
103 His Ser Leu Gln Thr Gln Leu Lys Ala Gln Asn Ser Arg Ile Gln Gln 118
527 CTC TTC CAC AAG GTG GCC CAG CAG CAG CGG CAC CTG GAG AAG CAG CAC 574
119 Leu Phe His Lys Val Ala Gln Gln Gln Arg His Leu Glu Lys Gln His 134
575 CTG CGA ATT CAG CAT CTG CAA AGC CAG TTT GGC CTC CTG GAC CAC AAG 622
135 Leu Arg Ile Gln His Leu Gln Ser Gln Phe Gly Leu Leu Asp His Lys 150
623 CAC CTA GAC CAT GAG GTG GCC AAG CCT GCC CGA AGA AAG AGG CTG CCC 670
151 His Leu Asp His Glu Val Ala Lys Pro Ala Arg Arg Lys Arg Leu Pro 166
671 GAG ATG GCC CAG CCA GTT GAC CCG GCT CAC AAT GTC AGC CGC CTG CAC 718
167 Glu Met Ala Gln Pro Val Asp Pro Ala His Asn Val Ser Arg Leu His 182
719 CGG CTG CCC AGG GAT TGC CAG GAG CTG TTC CAG GTT GGG GAG AGG CAG 766
183 Arg Leu Pro Arg Asp Cys Gln Glu Leu Phe Gln Val Gly Glu Arg Gln 198
767 AGT GGA CTA TTT GAA ATC CAG CCT CAG GGG TCT CCG CCA TTT TTG GTG 814
199 Ser Gly Leu Phe Glu Ile Gln Pro Gln Gly Ser Pro Pro Phe Leu Val 214
815 AAC TGC AAG ATG ACC TCA GAT GGA GGC TGG ACA GTA ATT CAG AGG CGC 862
215 Asn Cys Lys Met Thr Ser Asp Gly Gly Trp Thr Val Ile Gln Arg Arg 230
863 CAC GAT GGC TCA GTG GAC TTC AAC CGG CCC TGG GAA GCC TAC AAG GCG 910
231 His Asp Gly Ser Val Asp Phe Asn Arg Pro Trp Glu Ala Tyr Lys Ala 246
911 GGG TTT GGG GAT CCC CAC GGC GAG TTC TGG CTG GGT CTG GAG AAG GTG 958
247 Gly Phe Gly Asp Pro His Gly Glu Phe Trp Leu Gly Leu Glu Lys Val 262
959 CAT AGC ATC ACG GGG GAC CGC AAC AGC CGC CTG GCC GTG CAG CTG CGG 1006
263 His Ser Ile Thr Gly Asp Arg Asn Ser Arg Leu Ala Val Gln Leu Arg 278
1007 GAC TGG GAT GGC AAC GCC GAG TTG CTG CAG TTC TCC GTG CAC CTG GGT 1054
279 Asp Trp Asp Gly Asn Ala Glu Leu Leu Gln Phe Ser Val His Leu Gly 294
1055 GGC GAG GAC ACG GCC TAT AGC CTG CAG CTC ACT GCA CCC GTG GCC GGC 1102
295 Gly Glu Asp Thr Ala Tyr Ser Leu Gln Leu Thr Ala Pro Val Ala Gly 310
1103 CAG CTG GGC GCC ACC ACC GTC CCA CCC AGC GGC CTC TCC GTA CCC TTC 1150
311 Gln Leu Gly Ala Thr Thr Val Pro Pro Ser Gly Leu Ser Val Pro Phe 326
1151 TCC ACT TGG GAC CAG GAT CAC GAC CTC CGC AGG GAC AAG AAC TGC GCC 1198
327 Ser Thr Trp Asp Gln Asp His Asp Leu Arg Arg Asp Lys Asn Cys Ala 342
1199 AAG AGC CTC TCT GGA GGC TGG TGG TTT GGC ACC TGC AGC CAT TCC AAC 1246
343 Lys Ser Leu Ser Gly Gly Trp Trp Phe Gly Thr Cys Ser His Ser Asn 358
1247 CTC AAC GGC CAG TAC TTC CGC TCC ATC CCA CAG CAG CGG CAG AAG CTT 1294
359 Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro Gln Gln Arg Gln Lys Leu 374
1295 AAG AAG GGA ATC TTC TGG AAG ACC TGG CGG GGC CGC TAC TAC CCG CTG 1342
375 Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg Gly Arg Tyr Tyr Pro Leu 390
1343 CAG GCC ACC ACC ATG TTG ATC CAG CCC ATG GCA GCA GAG GCA GCC TCC 1390
391 Gln Ala Thr Thr Met Leu Ile Gln Pro Met Ala Ala Glu Ala Ala Ser 406
1391 TAG CGT CCT GGC TGG GCC TGG TCC CAG GCC CAC GAA AGA CGG TGA CTC 1438
407 *** 407
1439 TTG GCT CTG CCC GAG GAT GTG GCC GTT CCC TGC CTG GGC AGG GGC TCC 1486
1487 AAG GAG GGG CCA TCT GGA AAC TTG TGG ACA GAG AAG AAG ACC ACG ACT 1534
1535 GGA GAA GCC CCC TTT CTG AGT GCA GGG GGG CTG CAT GCG TTG CCT CCT 1582
1583 GAG ATC GAG GCT GCA GGA TAT GCT CAG ACT CTA GAG GCG TGG ACC AAG 1630
1631 GGG CAT GGA GCT TCA CTC CTT GCT GGC CAG GGA GTT GGG GAC TCA GAG 1678
1679 GGA CCA CTT GGG GCC AGC CAG ACT GGC CTC AAT GGC GGA CTC AGT CAC 1726
1727 ATT GAC TGA CGG GGA CCA GGG CTT GTG TGG GTC GAG AGC GCC CTC ATG 1774
1775 GTG CTG GTG CTG TTG TGT GTA GGT CCC CTG GGG ACA CAA GCA GGC GCC 1822
1823 AAT GGT ATC TGG GCG GCG TCA CAG AGT TCT TGG AAT AAA AGC AAC CTC 1870
1871 AGA ACA CTT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1918
1919 AAA AAA AAA AAA AAA AAA AAA AAA A 1943
Claims (9)
1. isolating PP1158 polypeptide is characterized in that this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through disappearance, insertion or the replacement of 1-10 amino-acid residue, and have suppress hepatoma cell line 7721 clone's formation functions by (a) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with aminoacid sequence of SEQ ID NO:2.
3. isolating polynucleotide is characterized in that, are selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of SEQID NO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:
Coding region sequence or the full length sequence of SEQ ID NO:3.
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 is a kind of host cell that is selected from down group:
(a) host cell that transforms or transduce with the described carrier of claim 6;
(b) host cell that transforms or transduce with the described polynucleotide of claim 3.
8. the preparation method with the active polypeptide of PP1158 is characterized in that, this method comprises:
(a) being fit to express under the condition of PP1158, cultivate the described host cell of claim 7;
(b) from culture, isolate and have the active polypeptide of PP1158.
9. 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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| CNB001252828A CN1170850C (en) | 2000-09-19 | 2000-09-19 | Human angiogenin-like protein and coding sequence and application thereof |
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| CNB001252828A CN1170850C (en) | 2000-09-19 | 2000-09-19 | Human angiogenin-like protein and coding sequence and application thereof |
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| CN1343725A CN1343725A (en) | 2002-04-10 |
| CN1170850C true CN1170850C (en) | 2004-10-13 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20050123925A1 (en) | 2002-11-15 | 2005-06-09 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
| MX2007000783A (en) | 2004-07-20 | 2007-04-09 | Genentech Inc | Compositions and methods of using angiopoietin-like 4 protein. |
| US8604185B2 (en) | 2004-07-20 | 2013-12-10 | Genentech, Inc. | Inhibitors of angiopoietin-like 4 protein, combinations, and their use |
| ZA200701183B (en) | 2004-07-20 | 2008-05-28 | Genentech Inc | Inhibitors of angiopoietin-like 4 protein, combinations, an their use |
| CN1952129B (en) * | 2005-10-20 | 2011-09-21 | 上海市肿瘤研究所 | Angptl4 deletion mutant and application thereof |
| CN113491643B (en) * | 2020-03-20 | 2024-04-12 | 湖州美易得生物科技有限公司 | Antipruritic use of angiogenin |
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