CN1990504A - Earthworm protein suppressing cancer cell accretion by road spectrum - Google Patents
Earthworm protein suppressing cancer cell accretion by road spectrum Download PDFInfo
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- CN1990504A CN1990504A CN 200510112490 CN200510112490A CN1990504A CN 1990504 A CN1990504 A CN 1990504A CN 200510112490 CN200510112490 CN 200510112490 CN 200510112490 A CN200510112490 A CN 200510112490A CN 1990504 A CN1990504 A CN 1990504A
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Abstract
The invention discloses a novel protein EFE6 that can widely inhibit the cancer cell growth, polynucleotide sequence coding said protein and method for preparing said protein. The EFE6 protein is separated from earthworm. It can be widely used to inhibit cancer and also possesses esterase activity and fibrinolysis activity.
Description
Technical field
The invention belongs to biotechnology and medical field, but the proteinic polynucleotide sequence that particularly relates to a kind of wide spectrum anticancer growth in the earthworm, and the protein sequence of polynucleotide encoding thus, in addition, also relate to described proteinic preparation method and tumor-suppression activity thereof etc.
Background technology
Earthworm is commonly called as earthworm, has analgesic, relieving convulsion, step-down, effect such as antibacterial and promoting blood circulation and removing blood stasis, has been widely used in the multiple disease of treatment in the traditional Chinese medicine.To the eighties in last century, report successively that again the earthworm extract is inhibited to multiple cancer cells, and in Preliminary Clinical, obtained certain cancer resistant effect (The Fourth Military Medical University's journal, 1986,7 (2): 85; China's clinical tumor, 1991,18 (2): 131; Shanxi Medical College's journal, 1995,26 (2): 81; Acta Biochimica et Biophysica Sinica, 2002,34 (5): 576).
But, the effective ingredient and the mechanism of action thereof of anticancer growth in the earthworm are not but appeared in the newspapers so far.Potential application foreground in view of pressing down the cancer composition in the earthworm presses for it is carried out basis and Application and Development research.
Summary of the invention
But first purpose of the present invention provides the protein of a kind of wide spectrum anticancer growth in the earthworm.
Second purpose of the present invention provides the polynucleotide sequence of code for said proteins.
The 3rd purpose of the present invention provides the described method of protein of preparation.
The 4th purpose of the present invention provides described proteinic cancer purposes and other purposes of pressing down.
In a first aspect of the present invention, a kind of isolating earthworm EFE6 polypeptide is provided, this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or
(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more (1-30 preferably, more preferably 1-20,1-10 best) amino-acid residue formed, and have cancer suppressing function by (a) polypeptides derived.
In a preference of the present invention, this polypeptide is the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.
A second aspect of the present invention provides a kind of isolating polynucleotide, and it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of coding said polypeptide; Or
(b) with polynucleotide (a) complementary polynucleotide.
In a preference of the present invention, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
In another preference, described polynucleotide have the sequence shown in the SEQ ID NO:1.
In another preference, described polynucleotide can be the dna nucleotide sequences that the gene to coding said polypeptide carries out the gene mutation body that obtained behind the artificial rite-directed mutagenesis.
A third aspect of the present invention provides a kind of carrier, and it contains described polynucleotide.
A fourth aspect of the present invention provides a kind of genetically engineered host cell, and it contains described carrier.
A fifth aspect of the present invention provides the preparation method of a peptide species, and this method comprises:
(a) under conditions suitable for the expression, cultivate described host cell; Or
(b) from culture, isolate earthworm EFE6 polypeptide.
A sixth aspect of the present invention, provide a kind of can with described earthworm EFE6 polypeptid specificity bonded antibody.
A seventh aspect of the present invention provides a kind of pharmaceutical composition, and it contains the described polypeptide and the pharmaceutically acceptable carrier of safe and effective amount.
A eighth aspect of the present invention provides the purposes of earthworm EFE6 polypeptide of the present invention, and it is used to:
(a) medicine of preparation treatment cancer;
(b) preparation has the medicine of esterase activity; Or
(c) prepare medicine with fibrinolytic activity.
Others of the present invention are because disclosing of this paper technology is conspicuous to those skilled in the art.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention.
Fig. 1 has shown the SDS-PAGE collection of illustrative plates of isolating wide spectrum cancer suppressor protein from earthworm, and wherein swimming lane 1 is a cancer suppressor protein, and swimming lane 2 is a standard molecular weight protein.
Fig. 2 has shown the chromatography collection of illustrative plates of earthworm cancer suppressor protein in HPLC.
Fig. 3 has shown the pcr amplification band of earthworm cancer suppressor protein, and swimming lane 1 is the DNA standard, and swimming lane 2 is a pcr amplification product.
Fig. 4 has shown the gene order and the encoded protein matter sequence thereof of earthworm cancer suppressor protein.
Fig. 5 has shown the building process of earthworm cancer suppressor protein in prokaryotic expression carrier (PGEX4T-1).
Fig. 6 has shown the SDS-PAGE collection of illustrative plates (Fig. 6 A) and the Western collection of illustrative plates (Fig. 6 B) of earthworm cancer suppressor protein expression product.
Fig. 7 has shown the verify restraining effect of several growth of cancer cells of earthworm cancer suppressor protein.
Embodiment
The inventor at first separates the protein that obtains a kind of wide spectrum anticancer growth through extensive and deep research from earthworm, finished the present invention based on this.
More specifically, the inventor at first is separated to a kind of protein of molecular weight 24744.67 daltonian wide spectrum anticancer growths from ring earthworm (Metaphire guillelmi) extract of William chamber, and one section aminoacid sequence having measured this protein N terminal is NH
2-I-L-G-G-Q-D-A-S-P-G-(SEQ ID NO:3), and synthesized corresponding primer on this basis, separating relevant mRNA then from earthworm is template, obtain cDNA through reverse transcription, with PCR method amplification with cloned the gene of earthworm cancer suppressor protein, measured the complete sequence of this gene, then the encoding sequence of earthworm cancer suppressor protein is inserted suitable carrier and change in the proper host cell, express and separate the earthworm cancer suppressor protein of reorganization, and identified by the Westren engram analysis.The inventor is with this albumen called after " EFE6 albumen ".
The inventor also separates having obtained described cancer suppressor protein except separation and purification from the ring earthworm of William chamber has obtained the described cancer suppressor protein from other earthworm kind, for example, and Eisenia foetida (Eiseniafoetida) etc.
In the present invention, term " EFE6 albumen ", " EFE6 polypeptide " or " earthworm cancer suppressor protein EFE6 " are used interchangeably, and all refer to have albumen or the polypeptide of earthworm cancer suppressor protein EFE6 aminoacid sequence (SEQ ID NO:2).They comprise the earthworm cancer suppressor protein EFE6 that contains or do not contain initial methionine.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as 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, " isolating EFE6 albumen or polypeptide " is meant that the EFE6 polypeptide is substantially free of natural relative other protein, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying EFE6 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of EFE6 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 chemosynthesis, or uses recombinant technology 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 the proteic fragment of EFE6, derivative and analogue.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps natural EFE6 albumen of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, derivative or analogue 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 transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the former sequence of protein of this polypeptide of purifying, or with the fusion rotein of the segmental formation of IgG antibody).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " EFE6 polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence of EFE6 protein-active.This term also comprises having and variant form EFE6 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be 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 has also comprised proteic active fragments of EFE6 and/or 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 protein of the DNA of EFE6 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-EFE6 polypeptide to obtain.The present invention also provides other polypeptide, as comprises EFE6 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of EFE6 polypeptide.Usually, this fragment have the EFE6 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 EFE6 albumen or polypeptide.The difference of these analogues and natural EFE6 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, " EFE6 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ IDNO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
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 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding EFE6.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
EFE6 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention protein (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The method of application round pcr DNA amplification/RNA (Saiki etc., Science 1985; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or EFE6 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the EFE6 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention EFE6 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the EFE6 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 BioChem.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 EFE6 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, 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, 293 cells 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.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the protein 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 EFE6 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: directly as pharmacological agent because of similar EFE6 protein function the disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism EFE6 protein function.The peptide molecule that can suppress or stimulate the EFE6 protein function that can be used for seeking therapeutic value with the reorganization EFE6 protein screening peptide library of expressing.
On the other hand, the present invention also comprises EFE6 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 EFE6 gene product or fragment.Preferably, refer to that those can combine with EFE6 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 EFE6, comprise that also those do not influence the antibody of EFE6 protein function.The present invention also comprise those can with modify or without the EFE6 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.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the EFE6 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing EFE6 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler,
Nature.256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the EFE6 protein function and the antibody that does not influence the EFE6 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of EFE6 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 EFE6 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-EFE6 can be used in the immunohistochemistry technology, detects the EFE6 albumen in the biopsy specimen.
The disease that antibody among the present invention can be used for treating or prevention is relevant with EFE6 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of EFE6 or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of EFE6 albumen 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 cell of EFE6 protein positive.
Available EFE6 albumen 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.
Utilize protein of the present invention,, can filter out with EFE6 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Protein of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects, are particularly useful for suppressing the growth of tumour or tumour cell (body is interior or external).
The tumour example includes, but are not limited to: various noumenal tumours, and as lung cancer, small cell lung cancer, liver cancer, carcinoma of the pancreas, cancer of the stomach, esophagus cancer, mastocarcinoma, prostate cancer, carcinoma of testis, colorectal carcinoma, carcinoma of the pancreas, ovarian cancer, bladder cancer, cervical cancer.Preferably, described tumour is selected from down group: lung cancer, liver cancer, mastocarcinoma or prostate cancer.
Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of cancer aspect.When using EFE6 albumen of the present invention, also can use the other treatment agent simultaneously, as taxol etc.
The present invention also provides a kind of pharmaceutical composition, and it contains EFE6 polypeptide of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that EFE6 albumen with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of EFE6 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of EFE6 of the proteic nothing expression of EFE6 or unusual/non-activity.The EFE6 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic EFE6 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the EFE6 transgenosis to cell.The method that structure carries the recombinant viral vector of EFE6 gene is found in existing document (Sambrook etc., molecular cloning experiment guide).The EFE6 gene of recombinating in addition can be packaged in the liposome, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of EFE6 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.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of EFE6 obtains.During screening, must carry out mark to the EFE6 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization EFE6 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The EFE6 protein level that is detected in the test can be with laying down a definition the importance of EFE6 albumen in various diseases and be used to the disease of diagnosing EFE6 albumen to work.
Whether having the proteic method of EFE6 in a kind of test sample is to utilize the proteic specific antibody of EFE6 to detect, and it comprises: sample is contacted with the EFE6 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample EFE6 albumen.
The proteic polynucleotide of EFE6 can be used for the diagnosis and the treatment of EFE6 protein related diseases.Aspect diagnosis, the proteic polynucleotide of EFE6 can be used for detecting the proteic expression of EFE6 EFE6 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of EFE6 as the EFE6 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of EFE6 albumen and also can detect the proteic transcription product of EFE6.
The sudden change that detects the EFE6 gene also can be used for the disease of diagnosing EFE6 albumen relevant.The form of EFE6 protein mutation comprises that the point mutation compared with normal wild type EFE6 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 protein expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of EFE6 prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch MedicalLibrary).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.More specifically, the inventor at first is separated to a kind of protein of molecular weight 24744.67 daltonian wide spectrum anticancer growths from ring earthworm (Metaphire guillelmi) extract of William chamber, and one section aminoacid sequence having measured this protein N terminal is NH
2-I-L-G-G-Q-D-A-S-P-G-(SEQ ID NO:3), and synthesized corresponding primer on this basis, separating relevant mRNA then from earthworm is template, obtain cDNA through reverse transcription, with PCR method amplification with cloned the gene of earthworm cancer suppressor protein, measured the complete sequence of this gene, then the encoding sequence of earthworm cancer suppressor protein is inserted suitable carrier and change in the proper host cell, express and separate the earthworm cancer suppressor protein of reorganization, and identified by the Westren engram analysis.The inventor is with this albumen called after " EFE6 albumen ".
The inventor also separates having obtained described cancer suppressor protein except separation and purification from the ring earthworm of William chamber has obtained the described cancer suppressor protein from other earthworm kind, for example, and Eisenia foetida (Eiseniafoetida) etc.Protein of the present invention can be treatment cancer relative disease new treatment approach is provided, thereby has great application prospect.
Other biological activity of earthworm cancer suppressor protein EFE6 of the present invention is meant that this protein also has esterase activity and hydrolysis of fibrin and the fibrinogenic fibrinolytic activity of hydrolysis BAEE (enzoyl arginine ethyl ester).
Major advantage of the present invention is:
Be purified to the cancer suppressor protein of single purity from numerous and diverse earthworm extract, the normal cell that this cancer suppressor protein confrontation is cultivated is nontoxic or low toxicity.The gene of earthworm cancer suppressor protein is cloned in the present invention simultaneously, assay determination gene order, and successfully express, this has not only created good condition for the gene engineering product of development research earthworm cancer suppressor protein, also provide necessary chemical structure data, thereby had great application prospect for exploitation earthworm cancer suppressor protein injection is applied to the clinical treatment tumour disease.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The cancer suppressor protein that embodiment 1 separation and purification from earthworm is natural
The inventor has mainly adopted William chamber ring earthworm to carry out extracting and separation and purification in experiment, has obtained to have the component of tumor-suppression activity.Adopt Eisenia foetida in addition, also obtained identical result.
Winning earthworm with the William chamber is example, and it is some to get fresh and alive earthworm, cleans after machinery is smashed to pieces, adds ice-cold acetone or alcohol dehydration, and gained precipitates and dries, and is thick earthworm powder, stores in-20 ℃ of refrigerators standby.
It is some to get thick earthworm powder, the water logging bubble that adds two volumes spends the night, next day is with the 5000rmp frozen centrifugation, collect supernatant liquor and the dialysis tubing of packing into to the water 24h that dialyses, centrifugal at 5000rmp again, supernatant liquor becomes lyophilized powder through lyophilize, a little dissolves with damping fluid to get lyophilized powder during purifying, separates with gel permeation chromatography through ion-exchange then, obtains single cancer suppressor protein component, particularly, be defined as single component according to following two kinds of authentication methods.
The inventor has carried out the HPLC analysis to the cancer suppressor protein component that obtains, and the result is a simple spike, sees Fig. 2.
The inventor has carried out SDS-PAGE to the cancer suppressor protein component that obtains and has analyzed to single band, sees Fig. 1.
Through 10 circulations, the n terminal amino acid sequence that records is ILGGQDASPG to the earthworm cancer suppressor protein quality sample of purifying on the PE491 protein sequencer.
The N-terminal sequence of the earthworm cancer suppressor protein that records according to embodiment 2, design upstream oligonucleotide degenerate primer is:
5 ' AT (T/C/A) CT (T/C/A/G) GG (T/C/A/G) GG (T/C/A/G) CA (A/G) GA (T/C) GC3 ' (SEQ ID NO:4) (being merger Nucleotide in the bracket), because lack the C-terminal amino acid sequence information of earthworm cancer suppressor protein, downstream primer is selected OligdT for use
20, primer is synthetic entrusts Shanghai living worker bio-engineering corporation to finish.
The extraction of embodiment 4 total RNA
Get the earthworm body cavity 100mg of inner tissue, in liquid nitrogen, pulverize, add 1ml Trizol reagent and continue to grind to form homogenate, be transferred to then in the centrifuge tube, 4 ℃, the centrifugal 10min of 12000g, supernatant liquor sucks new centrifuge tube, and 25 ℃ of water-bath 5min add the 0.2ml chloroform, 15s fully vibrates, 25 ℃ of water-bath 5min, 4 ℃ of centrifugal 15min of 12000g get upper water addition 0.5ml Virahol, 25 ℃ of water-bath 10min, 4 ℃ of centrifugal 10min of 12000g, collecting precipitation is with 1ml 75% washing with alcohol, 4 ℃ of centrifugal 5min of 7500g, be open to air drying 5-10min, ethanol is fully volatilized, add 100 μ l water dissolution, 58 ℃ of water-bath 10min ,-70 ℃ of preservations.
Embodiment 5 RT-PCR amplification earthworm cancer suppressor protein plasmagene
Total RNA is a template with the earthworm that makes among the embodiment 4, utilizes the test kit (kit) of Invitrogen company to carry out reverse transcription, is undertaken by process specifications.
Use the synthetic primer among the embodiment 3, from above-mentioned gained cDNA is template, carry out pcr amplification, total reaction system 25 μ l, wherein template 1 μ l, upstream primer 2 μ l (250pM/ μ l), OligdT (10pM/ μ l) 0.5 μ l, high-fidelity enzyme 0.5 μ l (5u/ μ l), dNTP (2.5mM) 2 μ l, reaction conditions is 94 ℃ of sex change 3min of elder generation, enter circulation then, 94 ℃ of 25s, 51 ℃ of annealing 45s, 72 ℃ are extended 1.5min, carry out 31 circulations, last 72 ℃ of insulation 10min get the PCR product and carry out 1% agarose electrophoresis inspection, amplify the DNA sheet glue of a treaty 1000bp, see Fig. 3.Product is with the chloroform extracting, and ethanol sedimentation reclaims, and is dissolved in the aqua sterilisa of 10 μ l.
The gene clone of embodiment 6 earthworm cancer suppressor proteins.
Get PCR recovery product 2 μ l and be connected with T-carrier 1 μ l, transform, through blue hickie screening, enzyme is cut with PCR and is identified, obtains positive colony.
The evaluation of embodiment 7 earthworm cancer suppressor protein plasmagenes
The positive colony that obtains among the embodiment 6 entrusts Bo Ya company to measure, and gene complete sequence and encoded protein matter sequence are seen SEQ ID NO:1 and SEQ ID NO:2, or see Fig. 4.CDNA coding 717 Nucleotide (unlisted initiator codon and terminator codon in the sequence), i.e. 239 amino acid of this maturation protein.
Through this proteinic molecular weight of computer software analysis is 24744.67, and iso-electric point is 4.28.
The preparation of the anti-earthworm cancer suppressor protein of embodiment 8 rabbits antibody
Get body weight and be one of male new zealand rabbit about 2 kilograms, add Freund's complete adjuvant with 1mg earthworm cancer suppressor protein quality sample and grind to form emulsion state, at the rabbit neck part multi-point injection, after raising two weeks, add Freund's incomplete adjuvant with 1mg earthworm cancer suppressor protein and be ground into emulsion state, again at the rabbit neck part multi-point injection, after one month again 1mg earthworm cancer suppressor protein add Freund's incomplete adjuvant booster immunization once more, raise the two weeks rear neck artery and get blood, 4 ℃ of standing over night, 2000 left the heart 3 minutes, and supernatant liquor is the anti-earthworm cancer suppressor protein of a rabbit antibody.
The genetic expression and the evaluation of embodiment 9 earthworm cancer suppressor proteins
For the earthworm cancer suppressor protein of examining this coded by said gene is exactly the earthworm cancer suppressor protein of natural extract, the translation district of this gene is cloned among the EcoRI and XholI restriction enzyme site of prokaryotic expression carrier PGEX4T-1 (available from Pharmacia Biotech), sees Fig. 5.Then recombinant vectors is transformed in the e. coli bl21 (DE3) and inducible gene expression.
Carry out SDS-PAGE collection of illustrative plates and Western hybridization evaluation with the rabbit that makes among the embodiment 8 anti-earthworm cancer suppressor protein antibody and expression product.
The results are shown in Figure 6, by the result as seen, expressed protein can combine to specificity with earthworm cancer suppressor protein antibody.
The verify restraining effect of cancer cells of multiple cultivation of embodiment 10 earthworm cancer suppressor proteins
In the present embodiment, selected for use the cancer cells of following several routines to carry out inhibition test: SMCC7721 (human liver cancer cell) respectively, SW620 (human colon cancer cell), H08910 (Proliferation of Human Ovarian Cell), HepG2 (human liver cell liver cancer), A549 (human lung carcinoma cell), BCAP-37 (human breast cancer cell), HeLa (human cervical carcinoma cell), BGC823 (the low differentiation of people adenocarcinoma of stomach cell), T24 (human bladder cancer cell) and L1210 (mouse leukemia cell).Above-mentioned cancer cells is available from Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences's cell bank, or can be available from U.S. ATCC.
Various cells are divided into control group and test group, and the proteic final concentration of EFE6 is 50 μ g/ml in the test group.After cultivating in 24 hours, compare with cellular control unit.
Found that, except L1210 (mouse leukemia cell), other each add that obvious inhibition all takes place growth of cancer cells in the proteic test group of EFE6, visible EFE6 albumen has tangible cancer resistant effect, but it is to the no effect of L1210 (mouse leukemia cell).Concrete outcome is seen Fig. 7.
Other bioactive mensuration of embodiment 11 earthworm cancer suppressor proteins
1. detection esterase activity
With BAEE is the esterase activity that substrate has been measured the earthworm cancer suppressor protein, and the result shows earthworm cancer suppressor protein hydrolysis BAEE.Measure used BAEE and be dissolved in pH 7.8, in the 0.05M Tris-HCl damping fluid, concentration is 0.8 * 10
-3M gets the 2.9ml substrate solution and adds 0.1ml EFE6 albumen (final concentration is 10 mcg/ml).
Measure optical density(OD) and change under 253nM, tiring of EFE6 proteolysis BAEE is 100 unit of activity/milligram protein as calculated.
2. detection fibrinolytic
Press the described fibrin plate method of Deogny (Clinica Chimica Acta, 1975,60:85) measured the fibrinolytic of earthworm cancer suppressor protein, calculate the size of fibrinolytic by the area of measuring solusphere, the result shows that the earthworm cancer suppressor protein has fibrinolytic, vigor reaches 25,000mm
2/ mg EFE6 albumen.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉a kind of earthworm protein and encoding sequence thereof of wide spectrum anticancer growth
<130>059726
<160>4
<170>PatentIn version 3.3
<210>1
<211>717
<212>DNA
＜213〉William chamber ring earthworm (Metaphire guillelmi)
<400>1
atcctggggg gacaggatgc ctctcctgga gagttcccgt ggcaactgtc ccagttgaga 60
ggtggaagcc acagctgcgg agcctctctc ctccacgcga cagctgctct cagtgccgcc 120
cactgcgtcg acggagcact cgtatcatcc gttgatgtca tcgccggtct tcatcggcga 180
tcagatctta ctggaacaca gacttcagct gctgcgtcat tcagcattaa cgtgaactac 240
gataacggag agggtacatt cgcacatgat atctccatca tccacctcgc cacagccatc 300
gacgcctcgc cagccaacat cgcgtttttg acccttcctc cagacaacag ctatcagttt 360
actggtgaca cctgcactct cagtggatgg ggacgtacat ctgccagtaa cattcttccg 420
gacacgctgc agaaagtcga cattgaagta atcagcacag acgagtgcaa cgttcgtatg 480
gctaccgtca ttggtgccga ctgcactgat aatcaaatcg cagttttcga tgttgccgag 540
aacgagggat cgtgcaacgg tgacagcgga ggcccgatga actgccagct caatggtcag 600
actgtggttg ccggaataac gtcgtgggga atccagtcgg gcggcgcctg tgcaccatcc 660
tacccatctg tctacaccag gacttcagcc taccttcaat ggatcagcga caaccaa 717
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<212>PRT
＜213〉William chamber ring earthworm (Metaphire guillelmi)
<400>2
Ile Leu Gly Gly Gln Asp Ala Ser Pro Gly Glu Phe Pro Trp Gln Leu
1 5 10 15
Ser Gln Leu Arg Gly Gly Ser His Ser Cys Ala Ala Ser Leu Leu His
20 25 30
Ala Thr Ala Ala Leu Ser Ala Ala His Cys Val Asp Gly Ala Leu Val
35 40 45
Ser Ser Val Asp Val Ile Ala Gly Leu His Arg Arg Ser Asp Leu Thr
50 55 60
Gly Thr Gln Thr Ser Ala Ala Ala Ser Phe Ser Ile Asn Val Asn Tyr
65 70 75 80
Asp Ash Gly Glu Gly Thr Phe Ala His Asp Ile Ser Ile Ile His Leu
85 90 95
Ala Thr Ala Ile Asp Ala Ser Pro Ala Asn Ile Ala Phe Leu Thr Leu
100 105 110
Pro Pro Asp Asn Ser Tyr Gln Phe Thr Gly Asp Thr Cys Thr Leu Ser
115 120 125
Gly Trp Gly Arg Thr Ser Ala Ser Asn Ile Leu Pro Asp Thr Leu Gln
130 135 140
Lys Val Asp Ile Glu Val Ile Ser Thr Asp Glu Cys Asn Val Arg Met
145 150 155 160
Ala Thr Val Ile Gly Ala Asp Cys Thr Asp Asn Gln Ile Ala Val Phe
165 170 175
Asp Val Ala Glu Asn Glu Gly Ser Cys Asn Gly Asp Ser Gly Gly Pro
180 185 190
Met Asn Cys Gln Leu Asn Gly Gln Thr Val Val Ala Gly Ile Thr Ser
195 200 205
Trp Gly Ile Gln Ser Gly Gly Ala Cys Ala Pro Ser Tyr Pro Ser Val
210 215 220
Tyr Thr Arg Thr Ser Ala Tyr Leu Gln Trp Ile Ser Asp Asn Gln
225 230 235
<210>3
<211>10
<212>PRT
＜213〉William chamber ring earthworm (Metaphire guillelmi)
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＜213〉primer
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＜223〉n=a, t, c or g
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Claims (10)
1. isolating earthworm polypeptide is characterized in that this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have cancer suppressing function by (a) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding; Or
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.
5. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
6. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 5.
7. the preparation method of the described polypeptide of claim 1 is characterized in that, this method comprises:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 6;
(b) from culture, isolate the described earthworm polypeptide of claim 1.
8. energy and the described earthworm polypeptid specificity of claim 1 bonded antibody.
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.
10. the purposes of the described earthworm polypeptide of claim 1 is characterized in that, described earthworm polypeptide is used for:
(a) medicine of preparation treatment cancer;
(b) preparation has the medicine of esterase activity; Or
(c) prepare medicine with fibrinolytic activity.
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| CN1563387A (en) * | 2004-04-14 | 2005-01-12 | 绿色生命实验室有限公司 | Earthworm fibrinolytic enzyme gene and genetic engineering strain, and construction and application thereof |
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| CN105884876A (en) * | 2015-01-26 | 2016-08-24 | 中国科学院上海生命科学研究院 | Earthworm polypeptide and coded sequence and application thereof |
| CN105884876B (en) * | 2015-01-26 | 2019-12-10 | 中国科学院上海生命科学研究院 | Earthworm polypeptide, its coding sequence and application |
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