CN1257920A - Human melanoma growth correlation factor and its coding sequence, preparing process and usage - Google Patents

Abstract

The present invention discloses a new melanoma growth related factor, which is a new protein named as melanoma growth related factor-1 (MGRF-1). The cDNA coding sequence of MGRF-1 protein, polypeptide coded with said sequence, process for preparing the new human MGRF-1 protein and the application of said human MGRF-1 protein and nucleic acid sequence are also disclosed.

Description

New human melanoma growth correlation factor, its encoding sequence and method for making and purposes

The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, polypeptide of the present invention is named as melanoma growth correlation factor 1 (melanoma growth related factor-1 abbreviates " MGRF-1 " as).

Vitamin A acid (RA) can cause kinds of tumor cells cessation of growth cessation and differentiation (Amos B., et a1,1990, Methods Enzymol 190:217-225).The mouse melanin tumor cell strain S91 that handles through RA is divided into benign melanocyte (Lotan R., et al, 1981 Ann.N.Y.Acad.Sci.359:150-170) then again inner cell cessation of growth cessation in 24 hours.The retarding effect of RA by with special intranuclear receptor--retinoic acid receptor (RAR) (retinoic acid receptor, RAR) in conjunction with and mediate.In this approach; RAR also forms the heterodimer mixture with RxR (retinoid X receptor) usually; thereby this mixture is regulated target gene expression level (Glass C.K. by combining with RA response element on the particular target gene; et al; 1991, DNA CellBiol 10:623-638).At present the RAR target gene is also known little about it, and identify that this type of target gene will provide important evidence for the mechanism of causing a disease that discloses melanoma and even other cancers.

For the target gene of identifying that this type of is subjected to the RA regulation and control, human retinoic acid treatments mouse S91 melanoma cell strains such as Spanjaard in 1997, use the method for subtractive hybridization to seek differentially expressed gene (Spanjaard R.A.et al in the S91 cell of retinoic acid treatments front and back then, 1997, Cancer Research 57:5122-5128).Express in the discrepant gene at four that find, the gene of down-regulated expression has two (clone 5d and clone 10d): clone 5d is known cyclin cyclin D1 gene; Clone 10d and people BM28 (CDCL1) dna homolog.The gene of two up-regulateds is clone 7u and clone 13u, and this is two brand-new genes, has encoded respectively to contain 244 and 300 amino acid whose two albumen.Gene 7u almost can not detect expression in the melanoma cell of propagation, and expression amount has increased 7.1 times in the S91 of cessation of growth cessation after retinoic acid treatments cell; For gene 13u, expression amount has increased 2.3 times.The Northern hybridization analysis shows that gene 7u mainly expresses in large intestine; And gene 13u mainly expresses in testis.Although whether gene 7u and 13u are subjected to the direct acting target gene of RA and them inhibited not clear to melanoma, but they be subjected to the RA inductive express phenomenon with and tissue-specific expression pattern illustrate that these two genes may bring into play important physical effect (Spanjaard R.A.et al in melanomatous generative process, 1997, CancerResearch 57:5122-5128), be possible melanoma genes involved.

An object of the present invention is to provide a kind of new polynucleotide, the albumen of this polynucleotide encoding human melanoma growth correlation factor-1, the new people's gene of the present invention is named as people MGRF-1.

Another object of the present invention provides a kind of new human melanoma growth correlation factor, and this albumen is named as people MGRF-1 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people MGRF-1.

The invention still further relates to the application of this people MGRF-1 nucleotide sequence and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people MGRF-1 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 5-805 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 5-805 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has the nucleotide sequence of SEQ ID NO.3 from Nucleotide 5-805 position.

In another aspect of this invention, provide a kind of isolating people MGRF-1 protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

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

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

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people MGRF-1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people MGRF-1 protein-active operationally is connected in expression regulation sequence, form people MGRF-1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 5-805 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people MGRF-1;

(c) under the condition that is fit to expressing human MGRF-1 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people MGRF-1 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 830 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 5-805 position Nucleotide.

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

In the present invention, term " people MGRF-1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people MGRF-1 protein-active is as 5-805 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 5-805 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 5-805 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 5-805 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 5-805 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

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

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

In the present invention, term " people MGRF-1 protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of people MGRF-1 protein-active.This term also comprises having and variant form people MGRF-1 identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people MGRF-1 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people MGRF-1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people MGRF-1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people MGRF-1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of people MGRF-1 polypeptide.Usually, this fragment have people MGRF-1 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 1 00 continuous amino acids.

Invention also provides the analogue of people MGRF-1 albumen or polypeptide.The difference of these analogues and natural human MGRF-1 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 can also comprise having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

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

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; Val; Met; Ala; Phe; Nor-leucine	Leu

Leu(L)	Nor-leucine; 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; Nor-leucine	Leu

The present invention also comprises people MGRF-1 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people MGRF-1 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people MGRF-1 nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people MGRF-1.

The present invention also comprises the method that detects people MGRF-1 nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of people MGRF-1 polypeptide.Primer length is generally 20-50 Nucleotide.

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

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

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

On the other hand, the present invention also comprises people MGRF-1 DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people MGRF-1 gene product or fragment.Preferably, refer to that those can combine with people MGRF-1 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people MGRF-1, comprise that also those do not influence the antibody of people MGRF-1 protein function.The present invention also comprise those can with modify or without the people MGRF-1 gene product bonded antibody of modified forms.

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

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people MGRF-1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human MGRF-1 or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In MonoclonalAntibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people MGRF-1 function and the antibody that does not influence people MGRF-1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people MGRF-1 gene product, obtains by immunological technique.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people MGRF-1 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People MGRF-1 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

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

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

In one embodiment of the invention, polynucleotide total length of the present invention is 830 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 5-805 position Nucleotide.These polynucleotide are so to obtain, with SUPERSCRIPT ^TM(this is the plasmid storehouse in people's tire brain cDNA library, available from Gibco company) be template, synthetic forward primer A1:5 ' GGAGATGTTG GACTCGCTGT TGG-3 ' and reverse primer B1:5 '-TCCTGATGAG ACGCTGCTTC CTG-3 ' carry out PCR, obtain the purpose fragment of about 0.9kb.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

The homology retrieval finds that gene of the present invention is with mouse black-in lymphoma genes involved 7u height homology.The consistence of the melanoma genes involved 7u of it and mouse reaches 68% on amino acid levels, and similarity is 71% (Fig. 1).Can infer that gene of the present invention is the homologous gene of murine melanoma genes involved 7u in human body.Mouse 7u gene expression amount after retinoic acid treatments rises, and pointing out it may be the target gene of a RAR-RxR mixture, expresses under the inducing of vitamin A acid, thereby suppresses melanomatous generation.Can infer that thus MGRF-I gene of the present invention also may have the function that suppresses the melanoma generation, be the regulatory gene that the intravital melanoma of people takes place.

In the accompanying drawings, Fig. 1 is the amino acid sequence homologous comparison diagram of people MGRF-1 albumen of the present invention (below) and mouse 7u albumen (top).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " " or ": ".". " above sequence is used to mark the amino acid that is numbered 10 multiple.Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

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

Embodiment 1

The clone and the mensuration of the cDNA sequence of people MGRF-1

1. primer amplification

With SUPERSCRIPT ^TM(this is the plasmid storehouse in people's tire brain cDNA library, available from Gibco company) be template, with a pair of oligonucleotide is primer---A1:5 '-GGAGATGTTG GACTCGCTGT TGG-3 ' (SEQID NO.1) is a forward primer, oligonucleotide B1:5 '-TCCTGATGAG ACGCTGCTTC CTG-3 ' (SEQID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is the purpose fragment of about 0.8kb.

2.PCR the order-checking of product

With pcr amplification product and the pGEM-T that as above obtains ^Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 830bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 5-805 position Nucleotide.

Derive the aminoacid sequence of people MGRF-1 according to the full length cDNA sequence that obtains, totally 266 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people MGRF-1 of the present invention, in Non-redundant+GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that gene of the present invention is with mouse black-in lymphoma genes involved 7u height homology.The consistence of the melanoma genes involved 7u of it and mouse reaches 68% on amino acid levels, and similarity is 71% (Fig. 1).Can determine that gene of the present invention is the homologous gene of murine melanoma genes involved 7u in human body.

Mouse 7u gene expression amount after retinoic acid treatments rises, and pointing out it may be the target gene of a RAR-RxR mixture, expresses under the inducing of vitamin A acid, thereby suppresses melanomatous generation.Can infer that thus MGRF-I gene of the present invention also may have the function that suppresses the melanoma generation, be the regulation and control genes involved that the intravital melanoma of people takes place.

People MGRF-1 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor MGRF-1 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end with inventor MGRF-1 exchanges with the proteic N end of the 7u of mouse, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor MGRF-1, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor MGRF-1 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people MGRF-1 or the overexpression that suppresses people MGRF-1.People MGRF-1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people MGRF-1 disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.Especially because in S91 cell before and after the retinoic acid treatments, mouse 13u gene is differentially expressed gene (the Spanjaard R.A.et al of up-regulated, 1997, Cancer Research 57:5122-5128), therefore people MGRF-2 of the present invention can be used as a kind of sign that disease prognosis is judged.

Embodiment 3

The expression of people MGRF-1 in intestinal bacteria

The cDNA sequence usefulness of coding people MGRF-1 corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, is that template increases with the pcr amplification product of primer A1B1 among the embodiment 1, with synthetic insertion fragment.

5 ' Oligonucleolide primers sequence is

5′-TIGCGTCGACATGTTGGACTCGCTGTTGG-3′(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of SalI restriction enzyme, and what connect is 19 Nucleotide of the people MGRF-1 encoding sequence that begun by initiator codon;

3 ' Oligonucleolide primers sequence is

5’-GTTCCTGCAGTCACAAGGCGGGGCTCCAT-3’(SEQ?ID?NO.6)，

This primer contains the restriction enzyme site of PstI restriction enzyme, the encoding sequence of translation termination and people MGRF-1.

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

Digest the pQE-9 carrier, insert fragment with SalI and PstI, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).The extracting plasmid, sequence verification result shows that the cDNA of people MGRF-1 inserts the fragment carrier of correctly packing into.

Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people MGRF-1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people MGRF-1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 29KDa.

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

Embodiment 4

The expression of people MGRF-1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with the cDNA sequence of coding people MGRF-1, use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, the pcr amplification product of primer A1B1 is that template increases among the embodiment 1, with the synthetic fragment of inserting.

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

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

This primer contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 19 Nucleotide of the people MGRF-1 encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

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

This primer contains the encoding sequence of the restriction enzyme site of SpeI restriction enzyme, translation termination and people MGRF-1.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

Digest the pcDNA3 carrier, insert fragment with HindIII and SpeI, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, sequence verification result shows that the cDNA of people MGRF-1 inserts the fragment carrier of correctly packing into.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mM TrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 29KDa.

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

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, specific as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people MGRF-1 gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.

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

Sequence table (1) general information: (i) applicant: Fudan University is denomination of invention (ii): new human melanoma growth correlation factor, its encoding sequence and method for making and purposes be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 830bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3：GGAGATGTTG GACTCGCTGT TGGCCTTGGG CGGCCTGGTG CTGCTTCGGG ATTCCGTGGA 60GTGGGAGGGG CGCAGTCTCT TGAAGGCGCT TGTCAAGAAA TCTGCACTGT GTGGGGAGCA 120AGTGCATATC CTGGGCTGTG AAGTGAGCGA GGAAGAGTTT CGTGAAGGTT TTGACTCTGA 180TATCAACAAT CGGCTGGTTT ACCATGACTT CTTCAGAGAC CCTCTCAACT GGTCAAAAAC 240TGAGGAGGCC TTTCCTGGGG GGCCGCTGGG AGCCTTGAGA GCCATGTGCA AGAGGACAGA 300TCCTGTTCCT GTCACCATTG CTCTCGATTC ACTCAGCTGG CTGCTACTTC GCCTTCCCTG 360CACCACACTC TGCCAGGTCC TGCATGCTGT GGAGCCATCC AGGACTCTTG TCCTGGGTGA 420CAGCTCCTCA GTGGGGAAAG TGAGTGTGCT GGGCTTGCTA CATGAAGAGC TTCATGGACC 480AGGCCCTGTG GGAGCTCTCA GCAGCCTTGC TCAGACTGAG GTGACCCTGG GCGGTACCAT 540GGGCCAGGCC TTCGGCCACA TCCTGTGTCG GAGGCCCCGA CAGCGCCCAA CTGACCAGAC 600TCAGTGGTTC TCCATCCTTC CGGACTTCAG CCTGGATCTC CAAGAGGGGC CCTCTGTAGA 660GTCCCAGCCC TACTCCGATC CTCATATACC CCCGGTGGAT CCCACAACTC ATTTGACCTT 720TAACCATCTT TCTATTGTTT GTGTTAGCCT TACCCTGTCC CTGCCCCACC TTGGTTCCCC 780TTGTCTATGG AGCCCCGCCT TGTGAGCCAG GAAGCAGCGT CTCATCAGGA 830 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 266 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4：Met Leu Asp Ser Leu Leu Ala Leu Gly Gly Leu Val Leu Leu Arg 15Asp Ser Val Glu Trp Glu Gly Arg Ser Leu Leu Lys Ala Leu Val 30Lys Lys Ser Ala Leu Cys Gly Glu Gln Val His Ile Leu Gly Cys 45Glu Val Ser Glu Glu Glu Phe Arg Glu Gly Phe Asp Ser Asp Ile 60Asn Asn Arg Leu Val Tyr His Asp Phe Phe Arg Asp Pro Leu Asn 75Trp Ser Lys Thr Glu Glu Ala Phe Pro Gly Gly Pro Leu Gly Ala 90Leu Arg Ala Met Cys Lys Arg Thr Asp Pro Val Pro Val Thr Ile 105Ala Leu Asp Ser Leu Ser Trp Leu Leu Leu Arg Leu Pro Cys Thr 120Thr Leu Cys Gln Val Leu His Ala Val Glu Pro Ser Arg Thr Leu 135Val Leu Gly Asp Ser Ser Ser Val Gly Lys Val Ser Val Leu Gly 150Leu Leu His Glu Glu Leu His Gly Pro Gly Pro Val Gly Ala Leu 165Ser Ser Leu Ala Gln Thr Glu Val Thr Leu Gly Gly Thr Met Gly 180Gln Ala Phe Gly His Ile Leu Cys Arg Arg Pro Arg Gln Arg Pro 195Thr Asp Gln Thr Gln Trp Phe Ser Ile Leu Pro Asp Phe Ser Leu 210Asp Leu Gln Glu Gly Pro Ser Val Glu Ser Gln Pro Tyr Ser Asp 225Pro His Ile Pro Pro Val Asp Pro Thr Thr His Leu Thr Phe Asn 240His Leu Ser Ile Val Cys Val Ser Leu Thr Leu Ser Leu Pro His 255Leu Gly Ser Pro Cys Leu Trp Ser Pro Ala Leu 266 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:GTTCACTAGT TCACAAGGCG GGGCTCCAT 29

Claims (14)

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

Show at least 70% homology from the nucleotides sequence of Nucleotide 5-805 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 5-805 position.

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

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 5-805 position.

4. isolating people MGRF-1 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

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

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

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

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of people MGRF-1 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people MGRF-1 protein-active operationally is connected in expression regulation sequence, form people MGRF-1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 5-805 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people MGRF-1;

(c) under the condition that is fit to expressing human MGRF-1 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people MGRF-1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 5-805 position among the SEQ ID NO.3.

12. energy and the described people MGRF-1 of claim 4 protein polypeptide specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

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

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