CN1311439A - Human RTN 4B protein and code sequence, and its preparation and use - Google Patents

Abstract

A human RTN4B protein and coding sequence is an invention to provide a cDNA sequence of human RTN4B. Protein is an isomer of RTN4 with in RTN family members. The present invention is also refering to polypeptide coded by the sequence of nucleotide and manufacturing method and application of polypeptide and polynucleotide.

Description

Human RTN 4 B protein and coded sequence, and method for making and purposes

The present invention relates to the genetic engineering field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of human RTN 4 B, this albumen is the isomeride of the member RTN4 of people RTN family.The invention still further relates to by this nucleotide sequence coded polypeptide, the production method of described polynucleotide and described polypeptide, and the application of these polynucleotide and polypeptide are especially at the method and the kit that detect liver cancer.

RTN family is a new gene family of being found in recent years.Wherein neuroendocrine specific proteins NSP (neuroendocrine-specific protein) back is first member of this family by unified called after RTN1.RTN1 has 3 transcripts, long respectively 3.4Kb, 2.3Kb and 1.8Kb.Its corresponding cDNA NSPA, NSPB, NSPC (code length is 776,356,208 amino acid whose protein respectively) are separated in succession.Same, the genome structure of RTN1 gene is also illustrated.Three RTN1/NSP protein isomers have exclusive separately amino terminal, but identical carboxyl terminal is arranged.Experiment has proved that the existence of these three kinds of transcripts may not be because the variation montage of extron causes, but because exists a plurality of promoters.The common carboxyl terminal of RTN1/NSP has 2 and wears the membrane structure territory, and experiment shows that they are playing the part of important role in contact albumen and endoplasmic reticulum.

According to NSP genome sequence, cDNA sequence and nucleic acid sequence data storehouse relatively, other three people RTN family members (NSP genoid I, II, III) have been found.Wherein two (NSP-like gene I, II) are cloned in succession, and are named as RTN2 and RTN3.RTN2 has the terminal identical isomeride of three C-equally.But RTN3 finds the variation montage originally so far.C-end that it should be noted that RTN2 and RTN3 has also been found two independently transmembrane domains territories, and this is considered to relevant with getting in touch of endoplasmic reticulum.

So far, the function of RTN family is still very not clear.But there is report to find that the RTN1/NSP isomeride can be used as the independent tag thing of difference small-cell carcinoma of the lung (SCLC) and non-small cell lung cancer (non-SCLC).NSPA exists in the SCLC tumour cell, and does not exist in the non-SCLC tumour.By being mark, also can distinguish general n on-SCLC tumour and the non-SCLC that NE differentiation is arranged with the NSP isomeride.

RTN4 is another member who finds recently in the people RTN family.It is similar to RTN1 and RTN2, have equally terminal three the identical isomeride of C-(be RTN4A, 4B, 4C).RTN4 isomeride and RTN1/NSP, RTN2, RTN3 isomeride equally have common C-end, and four C-end all has high homology.This is indicating it as RTN1, has played the part of important role in contact albumen and endoplasmic reticulum.The N-end no tangible homology (＜40%) of RTN4 and other RTN of people, but these zones have some characteristics similar with RTN2 to RTN1, and for example, this area discover is rich in alkaline amino acid residue, proline and serine.In addition, in these zones, also found a large amount of protein phosphorylation sites.And there is report to find that interior RTN1A of body and RTN1B are really by phosphorylation.RTN4A, 4B, the theoretical isoelectric point of 4C is respectively 4.43,4.71 and 9.32, this and RTN1, the isoelectric point that RTN2 estimates is similar substantially.The RH positioning experiment discloses RTN4 between label D2S1786 and D2S2002, and distance D 2S1786 has the 72.9cR value.

Liver cancer is a kind of serious threat human health and the disease in life-span.Yet, present method and the kit that also lacks effective, easy detection liver cancer

An object of the present invention is to provide a kind of new polynucleotide, the isomeride of the member RTN4 of this polynucleotide encoding people RTN family, the isomeride of RTN4 of the present invention is named as human RTN 4 B.

Another object of the present invention provides a kind of new people's RTN family member, and this albumen is named as human RTN 4 B protein.

A further object of the present invention provides a kind of method of utilizing recombinant technique to produce described new people's RTN4B polypeptide.

The present invention also provides this people's the RTN4 nucleotide sequence and the application of polypeptide, especially in the application of liver cancer context of detection.

The present invention also provides a kind of kit that detects liver cancer.

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 human RTN 4 B protein activity, shows at least 70% homology from the nucleotides sequence of nucleotide 5-1126 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 5-1126 position.Preferably, described sequential coding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO:4.More preferably, this sequence has among the SEQ ID NO:3 nucleotide sequence from nucleotide 5-1126 position.

In another aspect of this invention, provide a kind of human RTN 4 B protein polypeptide of separation, it comprises: polypeptide or its conservative property variation polypeptide or its active fragment or its reactive derivative with SEQ ID NO:4 amino acid sequence.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, the method that provides a kind of generation to have the polypeptide of human RTN 4 B protein activity, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of human RTN 4 B protein activity operationally is connected in expression regulation sequence, form the human RTN 4 B protein expression vector, show at least 70% homology from the nucleotides sequence of nucleotide 5-1126 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 recombinant cell of human RTN 4 B protein;

(c) under the condition that is fit to expressing human RTN4B polypeptide, the recombinant cell in the incubation step (b);

(d) isolate polypeptide with human RTN 4 B protein activity.

In another aspect of this invention, a kind of method whether liver cell canceration takes place that detects is provided, this method comprises: detect the transcript or the RTN4B albumen that whether there are RTN4B in the liver cell, exist the transcript of RTN4B or RTN4B albumen just to show that canceration has taken place this liver cell.In one embodiment, this method is by detecting in the liver cell whether have RTN4B albumen, and it comprises step: the hepatoma carcinoma cell sample is contacted with the specific antibody of anti-RTN4B; Detect whether to have formed immune complex, form immune complex and just represent that canceration has taken place this liver cell.

In another embodiment, this method is the transcript that whether has RTN4B in the liver cell by detecting, and it comprises step: use the RTN4B Auele Specific Primer, with this hepatocellular mRNA of RT-PCR method amplification; Whether have estimate RTN4B amplified production, exist amplified production just to represent that canceration has taken place this liver cell if detecting.

In another embodiment, this method is the transcript that whether has RTN4B in the liver cell by detecting, and it comprises step: use the RTN4B specific probe, with this hepatocellular cDNA sample hybridization; Detect whether the hybridization product is arranged, exist the hybridization product just to represent that canceration has taken place this liver cell.

In another aspect of this invention, also provide a kind of kit that detects liver cancer, it contains: the primer of (1) specific amplification human RTN 4 B is right, the required reagent of reverse transcription mRNA that (2) are optional.The another kind of kit that detects liver cancer contains the specific antibody at human RTN 4 B.The another kind of kit that detects liver cancer, containing can be specifically and the probe of the mRNA hybridization of human RTN 4 B.

In isolated human RTN 4 B polynucleotide total length of the present invention is 781 nucleotide, and its detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 5-1126 position nucleotide.The human RTN 4 B protein total length of coding is 373 amino acid, and sequence is seen SEQ ID NO:4.By research, the present inventor is surprised to find that RTN4B albumen is not expressed in normal liver cell, but RTN4B but expresses in the liver cell of canceration.On the basis of this discovery, the inventor has finished the present invention.

In the present invention, " separation ", " 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 " human RTN 4 B protein (or polypeptide) coded sequence " refer to the encode nucleotide sequence of polypeptide with human RTN 4 B protein activity is as 5-1126 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO:3.This degenerate sequence is meant, is arranged in the encoder block 5-1126 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 5-1126 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO:4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO:3 from the nucleotide sequence of the nucleotide sequence hybridization of nucleotide 5-1126 position.Also term also comprise with SEQ ID NO:3 in from the homology of nucleotide sequence at least 70% of nucleotide 5-1126 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form albumen, SEQ ID NO:4 sequence with the human RTN 4 B identical function.These variant forms comprise (but being not limited to): disappearance, insertion and/or the replacement of several (it is individual to be generally 1-90, and preferably 1-60, more preferably 1-20,1-10 is individual best) nucleotide, and at 5 ＇ and/or several nucleotide of 3 ＇ end interpolation.Coded sequence of the present invention can be DNA or RNA, can be strand or two strands.

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 " human RTN 4 B protein polypeptide " refers to have the SEQ IDNO:4 polypeptide of sequence of human RTN 4 B protein activity.This term also comprises having and the variant form human RTN 4 B 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 and (be generally in 20 in that C end and/or N are terminal, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change the function of protein usually with the close or similar amino acid of performance.Again such as, add the function that or several amino acid also can not change protein usually in that C end and/or N are terminal.This term also comprises the active fragment and the reactive derivative of human RTN 4 B protein.

The variant form of this polypeptide comprises: homologous sequence, conservative property variant, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of human RTN 4 B DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-human RTN 4 B polypeptide to obtain.The present invention also provides other polypeptide, as comprises the fusion of human RTN 4 B polypeptide or its fragment.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of human RTN 4 B polypeptide.Usually, this fragment have the human RTN 4 B peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analog of human RTN 4 B protein or polypeptide.The difference of these analogs and natural human RTN4B polypeptide can be the difference on the amino acid 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 genetic variant natural or that induce.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagen and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars.Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(as D-amino acid), and the analog with that non-natural exists 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 acetylation 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 phosphotyrosine, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

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

Table 1

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

Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises the antisense sequences of human RTN 4 B polypeptid coding sequence and fragment thereof.This antisense sequences can be used for suppressing the expression of human RTN 4 B in the cell.For example be used for suppressing the expression of hepatoma carcinoma cell RTN4B.

The present invention also comprises a kind of nucleic acid molecules that can be used as probe, and this molecule has 8-100 of human RTN 4 B polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecules of the human RTN 4 B of encoding.

The present invention also comprises the method that detects the human RTN 4 B nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detector probe combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the coded sequence of human RTN 4 B polypeptide, and can be positioned at the both sides or the centre of this coded sequence.Primer length is generally 20-50 nucleotide.

Because in liver cell, the expression of RTN4B is the sign that canceration takes place liver cell, therefore can whether there be the transcript of RTN4B or RTN4B albumen to determine whether liver cell canceration has taken place by detecting in the liver cell.

In the correlativity of having known certain gene and certain disease, this area has many existing detection techniques available, difference only is that used primer and/or probe have the nucleotide sequence derived from RTN4B, and perhaps used antibody is the antibody to the RTN4B protein-specific.

As exemplifying, a kind ofly detect the method whether liver cell canceration takes place, comprising: detect the transcript or the RTN4B albumen that whether there are RTN4B in the liver cell, exist the transcript of RTN4B or RTN4B albumen just to show that canceration has taken place this liver cell.Particularly, (1) generally includes step: the hepatoma carcinoma cell sample is contacted with the specific antibody of anti-RTN4B when by detecting in the liver cell whether have RTN4B albumen; Detect whether to have formed immune complex, form immune complex and just represent that canceration has taken place this liver cell.(2) when by detecting when whether having the transcript of RTN4B in the liver cell, it generally includes step: use the RTN4B Auele Specific Primer, with RT-PCR method this hepatocellular mRNA that increases; Whether have estimate RTN4B amplified production, exist amplified production just to represent that canceration (pcr amplification method) has taken place this liver cell if detecting.Perhaps, it comprises step: use the RTN4B specific probe, whether with this hepatocellular cDNA sample hybridization, detecting has the hybridization product, exists the hybridization product just to represent that canceration (hybrid method) has taken place this liver cell.

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

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

In the present invention, term " host cell " comprises prokaryotic and eukaryotic.The example of prokaryotic host cell commonly used comprises Escherichia coli, hay bacillus etc.Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises human RTN 4 B 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 human RTN 4 B gene outcome or fragment.Preferably, refer to that those can combine with human RTN 4 B gene outcome 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 molecule of human RTN 4 B protein, comprise that also those do not influence the antibody of human RTN 4 B protein function.The present invention also comprise those can with the antibody of modifying or combining without the human RTN 4 B gene outcome of modified forms.

The present invention not only comprises complete monoclonal 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 human RTN 4 B gene outcome of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human RTN4B or its cell with antigenic fragment 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.Immuno1.6:511,1976; 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 human RTN 4 B function and the antibody that does not influence the human RTN 4 B function.Each antibody-like of the present invention can utilize the fragment or the functional areas of human RTN 4 B gene outcome, obtains by the routine immunization technology.These fragments or functional areas can utilize recombinant methods or utilize Peptide synthesizer synthetic.The antibody that combines with the unmodified form of human RTN 4 B gene outcome can come immune animal and produces with the gene outcome of producing in the prokaryotic (for example E.Coli); The antibody that combines with the posttranslational modification form (as the albumen or the polypeptide of glycosylation or phosphorylation) can come immune animal and obtains with the gene outcome that produces in the eukaryotic (for example yeast or insect cell).

Human RTN 4 B 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 conventional 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 conventional 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 fragments, and then connect and to obtain the very long fragment of sequence.

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

The fragment of albumen of the present invention is except available recombination method produces, and also available solid phase technique is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of AppliedBiosystems.Can distinguish each fragment of chemosynthesis albumen of the present invention, be connected to produce the molecule of total length with chemical method then.

The coded sequence of albumen of the present invention also can be used for the assignment of genes gene mapping.For example,, the cDNA clone is hybridized with the chromosome of metaphase, can carry out chromosome mapping exactly by fluorescence in situ hybridization technique (FISH).This technology can be used the cDNA that is as short as about 500bp; Also can use and grow to about 2000bp or longer cDNA.For this technology, can be referring to people such as Verma, Human Chromosomes:A Manual ofBasic Techniques, Pergamon Press, New York (1988).

In case sequence is located in certain exact position on the chromosome, the physical location of sequence on chromosome can be associated with the genetic map data.These genetic map data can obtain, for example by Mendel (Mendelian) people genetic database (can obtain on the net by Johns Hopkins University Welch MedicalLibrary).Then, come identified gene by linkage analysis and be positioned correlativity between the disease of same chromosomal region.

Then, be necessary to determine the cDNA between diseased individuals and the healthy individual or the difference of genome sequence aspect.Be not present in normal individual if a certain sudden change is present in part or all of diseased individuals, this sudden change may be exactly the paathogenic factor of this disease so.

Utilize albumen of the present invention,, can filter out with RTN4B interactional material takes place, as acceptor, inhibitor or antagonist etc. by various conventional screening techniques.

Albumen of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, but these materials are formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, 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, and comprising (but being not limited to): intramuscular, peritonaeum are interior, subcutaneous, intracutaneous or topical.

With human RTN 4 B protein of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.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.Human RTN 4 B protein of the present invention can be made into the injection form, for example is prepared by conventional method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, 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 human RTN 4 B protein polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to mammal, wherein should treat effective dose 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 method of administration, patient health situation, and these all are within the skilled practitioners skill.

In the present invention, the cDNA nucleotide sequence of human RTN 4 B is so to obtain, cDNA library (available from Clontech company) with the human liver tissue is a template, with a pair of oligonucleotides is that primer-A:5 ＇-AGCCATGGAAGACCTGGACCAG-3 ＇ is a forward primer, oligonucleotides B:5 ＇-GTCAAGGTTCGTTCTTCCCTGAC-3 ' is a reverse primer, carry out PCR, about 1400bp fragment smoothly effectively increases.Design two-way primer again, the amplification order-checking, we have obtained the cDNA order of a 1216bp length, see the full length cDNA sequence of SEQID NO:3.

This is detected in liver cancer tissue to experiment showed, the RTN4B gene transcription, does not express and have in cancer beside organism.This has illustrated that RTN4B is the gene of specifically expressing in a kind of liver cancer tissue, provides a kind of feasible effectively mark thereby for detection people liver whether canceration takes place.

In addition, because human RTN 4 B of the present invention has the natural acid sequence that is derived from the people, therefore, compare, estimate to have higher active and/or lower spinoff (for example the immunogenicity in human body lower or do not have) being applied to man-hour with the albumen of the same clan that derives from other species.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the C terminal amino acids sequence of people RTN4 of the present invention and RTN2, RTN3, NSP albumen.Wherein, identical amino acid marks with black shade.

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

Embodiment 1

The clone and the mensuration of the cDNA sequence of human RTN 4 B

1. primer amplification

CDNA library (available from Clontech company) with the human liver tissue is a template, with a pair of oligonucleotides is that primer-A:5 '-AGCCATGGAAGACCTGGACCAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotides B:5 '-GTCAAGGTTCGTTCTTCCCTGAC-3 ' (SEQ ID NO:2) is a reverse primer, carry out PCR, about 1400bp fragment smoothly effectively increases.

2.PCR the order-checking of product

With above-mentioned pcr amplification product AB and pGEM-T ^Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid 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 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 1216bp, detailed sequence is seen SEQID NO:3, and wherein open reading frame is positioned at 5-1126 position nucleotide.

Derive the amino acid sequence of human RTN 4 B according to the full length cDNA sequence that obtains, totally 373 amino acid residues, its amino acid sequence following (SEQ ID NO:4).

MEDLDQSPLV?SSSDSPPRPQ?PAFKYQFVRE?PEDEEEEEEE?EEEDEDEDLE?50

ELEVLERKPA?AGLSAAPVPT?APAAGAPLMD?FGNDFVPPAP?RGPLPAAPPV?100

APERQPSQDP?SPVSSTVPAP?SPLSAAAVSP?SKLPEDDEPP?ARPPPPPPAS?150

VSPQAEPVWT?PPAPAPAAPP?STPAAPKRRG?SSGSVVVDLL?YWRDIKKTGV?200

VFGASLFLLL?SLTVFSIVSV?TAYIALALLS?VTISFRIYKG?VIQAIQKSDE?250

GHPFRAYLES?EVAISEELVQ?KYSNSALGHV?NCTIKELRRL?FLVDDLVDSL?300

KFAVLMWVFT?YVGALFNGLT?LLILALISLF?SVPVIYERHQ?AQIDHYLGLA?350

NKNVKDAMAK?IQAKIPGLKR?KAE??????????????????????????????373

373 amino acid (not comprising terminator codon)

Embodiment 2

Homology relatively

Full length cDNA sequence and encoding proteins thereof with human RTN 4 B of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that, it is correct that the sequence of relatively supporting homologous protein compares, the C-end and the NSPC of RTN4B derivation albumen on amino acid levels, RTN2, RTN3 all have higher homology, its 186-373 amino acid and NSPC, RTN2, same rate such as RTN3 reaches 66%, 50% and 60% respectively, likelihood reaches 82%, 70% and 80% (Fig. 1) respectively.Simultaneously behind the PRODOM software analysis, also find two independently transmembrane domains territories at the C-of RTN4B end, be respectively 119-235 amino acids GVVFGASLFLLLSLTVFSIVSVTAYIALALLSVTISF, with 302-336 amino acids FAVLMWVFTYVGALFNGLTLLILALISLFSVPVIY, show that this albumen playing the part of important role in contact albumen and endoplasmic reticulum.This specific character also exists in other members of RTN family.But the N-of RTN4B and other RTN of people holds no tangible homology (＜40%).But these zones have some characteristics similar with RTN2 to RTN1, and for example, this area discover is rich in alkaline amino acid residue, proline and serine.In addition, in these zones, also found a large amount of protein phosphorylation sites.

Human RTN 4 B 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 with other albumen, such as producing fusion with immunoglobulin (Ig).In addition, inventor RTN4B can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of the N of inventor RTN4B end with the RTN4B of mouse exchanged, to produce the albumen that new activity is higher or have new features.

Human RTN 4 B of the present invention also can be used for screening antagonist that suppresses protein active of the present invention or the activator that strengthens protein active of the present invention etc.

At the antibody of inventor RTN4B, 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 RTN4B nucleic acid (coded sequence or antisense sequences) can be introduced into cell, with expression that improves human RTN 4 B or the overexpression that suppresses human RTN 4 B.Human RTN 4 B protein of the present invention or its active peptides fragment can be applied to patient, with treatment or alleviate because of human RTN 4 B disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people RTN4 in Escherichia coli

In this embodiment, be template with pcr amplification product A/B among the embodiment 1, the cDNA sequence of coding human RTN 4 B is used corresponding to 5 ＇ of this dna sequence dna and the PCR Oligonucleolide primers of 3 ＇ end increased, obtain human RTN 4 B cDNA as inserting fragment.

The 5 ＇ Oligonucleolide primers sequences of using in the PCR reaction are:

5 ＇-GACTGGATCCATGGAAGACCTGGACCAGT-3 ' (SEQ ID NO:5), this primer contains the restriction enzyme site of BamH I restriction enzyme, is 19 nucleotide of the human RTN 4 B coded sequence that begun by initiation codon after this restriction enzyme site;

3 ＇ end primer sequence is:

5 ＇-TTGGAAGCTT TCATTCAGCTTTGCGCTTC-3 ＇ (SEQ ID NO:6), this primer contains the part coded sequence of restriction enzyme site, translation termination and the human RTN 4 B of Hind III restriction enzyme.

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

With BamH I and Hind III digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting potpourri subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multicopy, and it is expressed lac I repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB double dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification human RTN 4 B has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB fluid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 dilution rate dilution, be inoculated into then in the large volume nutrient culture media, cultured cell grows to 600 optical density (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lac I repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued cultured cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation culture, the collecting cell lysate also is diluted in it in 6M guanidine hydrochloride.After the clarification, by containing under the condition that 6-His label albumen combines closely making, with the human RTN 4 B of nickel-chelate column chromatography purifying dissolving from solution.With 6M guanidine hydrochloride (pH5.0) wash-out human RTN 4 B from post.Available several method is the sex change protein precipitation from guanidine hydrochloride.Perhaps, use the dialysis step to remove guanidine hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear guanidine hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used guanidine 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 (Mr) of expressing protein is about 40318.

In addition, hold the amino acid of each 10 amino acid length to check order to N end and the C that reaches albumen, find consistent with the sequence of SEQ ID NO:4 with conventional method.

Embodiment 4

The expression of human RTN 4 B in eukaryotic (Chinese hamster ovary celI strain)

In this embodiment, be template with pcr amplification product A/B among the embodiment 1, the cDNA sequence of coding human RTN 4 B is used corresponding to 5 ＇ of this dna sequence dna and the PCR Oligonucleolide primers of 3 ＇ end increased, obtain human RTN 4 B cDNA as inserting fragment.

The 5 ＇ Oligonucleolide primers sequences of using in the PCR reaction are:

5＇-TCAGAAGCTT?ATGGAAGACCTGGACCAGT-3＇(SEQ?ID?NO:7)

This primer contains the restriction enzyme site of Hind III restriction enzyme, is 19 nucleotide of the human RTN 4 B coded sequence that begun by initiation codon after this restriction enzyme site;

3 ＇ end primer sequence is:

5＇-TTGGGGATCC?TCATTCAGCTTTGCGCTTC-3＇(SEQ?ID?NO:8)

This primer contains the part coded sequence of the restriction enzyme site of BamH I restriction enzyme, translation termination and human RTN 4 B.

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 antibiotic resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promoter, a viral promotors (P-CMV), a Sp6 promoter, a SV40 promoter, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With Hind III and BamH I digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting potpourri Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB double dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB fluid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, the cDNA fragment of sequence verification human RTN 4 B has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTris.HCl (pH7.6) solution that contains 0.05%Triton is equilibrium liquid and eluent, uses the active peak of collecting above-mentioned albumen through the Superdex of pre-equilibration G-75 post.Using the DEAE-Sepharose post of 50mMTris.HCl (pH8.0) balance again, is that eluent carries out gradient elution with 50mMTris.HCl (pH8.0) solution that contains 0-1M NaCl, collects the active peak of above-mentioned albumen.Be that dislysate 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 (Mr) of expressing protein is about 40318.

In addition, hold the amino acid of each 10 amino acid length to check order to N end and the C that reaches albumen, find consistent with the sequence of SEQ ID NO:4 with conventional method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out intraperitoneal injection to mouse.After 14 days,, mouse is carried out intraperitoneal 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 idiosyncrasy that obtains is active to be assessed in the ability of external precipitation human RTN 4 B gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.

The expression pattern analysis of embodiment 6:RTN4B in health adult tissue

Multiple Northern (MTNTM) blotting membrane (16 kinds of tissues) of organizing is available from Clontech company, increase from people's skeletal muscle cDNA library (Clontech) with primer A1 (5 ＇-CGAACCTTGACGTTGCAGTGCAG-3 ') and A2 (5 ＇-GAAAACAGATGGTAGTGCTCCTAG-3 ＇) and to obtain the fragment of 618bp, with this fragment is probe, usefulness α- ³²P-dATP (DuPont company) carries out random primer labelling to it, condition be 37 ℃ 1 hour, MegaprimeDNA Mk system instructions (Amersham) is seen in concrete operations.The user manual operation is pressed in Northern hybridization.Key step is as follows: and (1) preparation MTN hybridization solution (final concentration is as follows: 5XSSPE, and 10Xdenhardt ' s, 100 μ g/mlCTDNA, 50% formamide, 2%SDS), 68 ℃ of preheating hybridization solutions, thoroughly dissolution precipitation thing.(2) scald film, pour a little boiled 0.05%SDS solution on film, concussion, cooling back are surveyed signal (3) nylon membrane are placed in the hybrid pipe, add prehybridization solution, probe adds in the hybrid pipe after (4) sex change in 12 hours of 42 ℃ of prehybridizations, and film is washed in 42 ℃ of hybridization 24 hours (5), 42 ℃, use 2XSSC, 0.05%SDS solution washes twice, and each 20 minutes (6) carry out radioautograph with the X-ray sheet.

The result

The Northern results of hybridization of 16 kinds of tissues demonstrates three hybrid belts, long is respectively 5.0kb, 2.5kb and 1.8kb, corresponding be RTN4A respectively, RTN4B and RTN4C.The RTN4B gene is the transcript that wide spectrum is expressed, thymus gland, prostate, placenta, ovary, small intestine, large intestine, peripheral blood leucocyte, placenta, lung, skeletal muscle, kidney and etc. expression is all arranged in 15 kinds of tissues, expression is variant slightly.It should be noted that RTN4B does not see that in hepatic tissue expression is arranged.

The expression of RTN4A is very limited, only is detected in brain, expresses extremely low in testis.

In RTN4C 4 kinds in 16 kinds of tissues expression being arranged, is respectively brain, liver, skeletal muscle and kidney, and expression is the highest in skeletal muscle, and is minimum in the liver.

The expression analysis of embodiment 7:RTN4B in tumor cell line

(1). choose 13 kinds of human tumor cell lines, be respectively:

A375 (cytochrome oncocyte), BEL7402 (hepatoma carcinoma cell), CNE (nasopharyngeal carcinoma cell), DAMI (monocyte), HEPB3 (hepatoma carcinoma cell), K562 (erythroleukemia cell), L-02 (variation liver cell), SH-77 (small cell lung cancer cell), LTEPA2 (lung adenocarcinoma cell), NCIH446 (small cell lung cancer cell), NCIH460 (maxicell lung carcinoma cell), RAJI (lymphocytic cancer cell), T24 (transitional cell bladder carcinoma cell line)

Cultured cell:

In the nutrient solution that contains 10% calf serum and 5%CO2,37 ℃ of cultivation (nutrient solution RPMI1640 (Life

Technologies) prescription is as follows: 25mM HEPES, 2mM L-glutamine, 16mM NaHCO3), cultivate

Go down to posterity when converging sheet 80%.

(2) extracting RNA:

Method is as follows: (adopting the TRIZOL reagent of LIFE TECHNOLOGIES company)

The cell line homogenize

Add 1mlTRIZOL reagent (every 10cm2 double dish area adds 1mlTRIZOL) directly dissolved cell strain in double dish.

Phase-splitting

Cultivated the good sample of homogenize 5 minutes for 20 ℃.The TRIZOL reagent of every 1ml adds the chloroform of 0.2ml, and concussion and temperature were bathed 2 to 3 minutes, and the back is less than 10, centrifugal 15 minutes of 000xg at 2-8 ℃.RNA is positioned at water at this moment.

The RNA precipitation

Shift water to and newly manage, by mixing isopropyl alcohol (the TRIZOl 0.5ml of every 1ml) RNA is precipitated from aqueous phase, bathed 10 minutes 15-30 ℃ of temperature, the back is less than 10, centrifugal 10 minutes of 000xg at 2-8 ℃.RNA is deposited in the pipe end.

Washing RNA

Ethanol with 75% (every 1mlTROZOL adds the ethanol of 1ml at least) washing once mixes the back at 2-8 ℃, is less than 7, centrifugal 5 minutes of 500xg.

Again dissolve RNA

Air drying RNA with 0.5%SDS solution dissolving RNA, bathed 10 minutes 55-60 ℃ of temperature then.

(3) Northern hybridization:

Method is the same with embodiment 6 with probe.

The Northern results of hybridization of 13 kinds of tumor cell lines shows that the RTN4B gene is the wide spectrum expressing gene in various JEG-3, in 13 kinds of cell lines such as cytochrome knurl, liver cancer, nasopharyngeal carcinoma, monocyte, erythroleukemia, variation liver, small-cell carcinoma of the lung, adenocarcinoma of lung, maxicell lung cancer, small-cell carcinoma of the lung, lymph cancer, carcinoma of urinary bladder expression is arranged all, expression is variant slightly.But the expression that compares RTN4B in people's normal structure and tumor cell line, visible evident difference.In normal person's liver, only detect the transcript of 1.8kb, and in BEL7402 and L-02 cell line, detect 1.8 and the transcript of 2.5kb simultaneously, in HepG2, only detect the fragment of 2.5kb.

The expression analysis of embodiment 8:RTN4B in liver cancer tissue and cancer beside organism

The extracting of total tissue RNA:

The homogenize tissue: every 50-100mg tissue adds 1ml TRIZOL reagent, with glass homogenizer homogenize tissue.

Following method is identical with extracting RNA among the embodiment 7 with step.

Northern hybridization:

Method is the same with embodiment 6 with probe.Difference only is that the extractive process of the kind of its tissue and RNA thereof is different with embodiment 6.

The result:

The Northern results of hybridization of liver cancer tissue and cancer beside organism shows the RTN4B gene transcription, and this is detected in liver cancer tissue, does not express and have in cancer beside organism.This has illustrated that RTN4B is the gene of specifically expressing in a kind of liver cancer tissue, provides a kind of feasible effectively mark thereby for detection people liver whether canceration takes place.

Embodiment 9

The preparation of kit

Kit 1:

It is right that it contains the primer of (1) specific amplification human RTN 4 B, and operation instruction.This kit also can contain or not contain the required reagent that the mRNA reverse transcription is become cDNA.Wherein, the sequence of this Auele Specific Primer is derived from the sequence of SEQ ID NO:3, and length is 15-35.The cDNA that reverse transcription is become carries out specific amplification, whether to contain the nucleotide sequence of RTN4B in the test sample.This kit also can contain and carries out other required reagent of PCR reaction, for example damping fluid etc.

In addition, preferably, at least one used primer has been crossed over two extrons, because will not produce amplified production corresponding to the genome sequence of RTN4B this moment.

Kit 2:

This kit contains the specific antibody (for example polyclonal antibody of preparation among the embodiment 5, the perhaps monoclonal antibody of the anti-RTN4B of the hybridoma technology generation of usefulness standard) at human RTN 4 B, and operation instruction.This kit is used for direct test sample and whether has RTN4B albumen.Form immune complex by specific immune response earlier, detect immune complex with routine techniques then.

Kit 3:

This kit contains can be specifically and the probe of the mRNA hybridization of human RTN 4 B.It also can contain or not contain hybridization buffer.This kit comes whether to exist in the test sample nucleic acid molecules of RTN4B by the hybridization reaction between nucleic acid molecules and the RTN4B specific probe.

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:

(ⅱ) denomination of invention: human RTN 4 B protein and coded sequence, and method for making and purposes

(ⅲ) sequence number: the information of 8 (2) SEQ ID NO:1

(ⅰ) sequence signature

(A) length: 22 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: oligonucleotides

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:1:AGCCATGGAA GACCTGGACC AG 22 (2) SEQ ID NO:2

(ⅱ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: oligonucleotides

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2GTCAAGGTTC GTTCTTCCCT GAC 23 (2) SEQ ID NO:3:

(ⅰ) sequence signature:

(A) length: 1216bp

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO:3AGCCATGGAA GACCTGGACC AGTCTCCTCT GGTCTCGTCC TCGGACAGCC CACCCCGGCC 60GCAGCCCGCG TTCAAGTACC AGTTCGTGAG GGAGCCCGAG GACGAGGAGG AAGAAGAGGA 120GGAGGAAGAG GAGGACGAGG ACGAAGACCT GGAGGAGCTG GAGGTGCTGG AGAGGAAGCC 180CGCCGCCGGG CTGTCCGCGG CCCCAGTGCC CACCGCCCCT GCCGCCGGCG CGCCCCTGAT 240GGACTTCGGA AATGACTTCG TGCCGCCGGC GCCCCGGGGA CCCCTGCCGG CCGCTCCCCC 300CGTCGCCCCG GAGCGGCAGC CGTCTTGGGA CCCGAGCCCG GTGTCGTCGA CCGTGCCCGC 360GCCATCCCCG CTGTCTGCTG CCGCAGTCTC GCCCTCCAAG CTCCCTGAGG ACGACGAGCC 420TCCGGCCCGG CCTCCCCCTC CTCCCCCGGC CAGCGTGAGC CCCCAGGCAG AGCCCGTGTG 480GACCCCGCCA GCCCCGGCTC CCGCCGCGCC CCCCTCCACC CCGGCCGCGC CCAAGCGCAG 540GGGCTCCTCG GGCTCAGTGG TTGTTGACCT CCTGTACTGG AGAGACATTA AGAAGACTGG 600AGTGGTGTTT GGTGCCAGCC TATTCCTGCT GCTTTCATTG ACAGTATTCA GCATTGTGAG 660CGTAACAGCC TACATTGCCT TGGCCCTGCT CTCTGTGACC ATCAGCTTTA GGATATACAA 720GGGTGTGATC CAAGCTATCC AGAAATCAGA TGAAGGCCAC CCATTCAGGG CATATCTGGA 780ATCTGAAGTT GCTATATCTG AGGAGTTGGT TCAGAAGTAC AGTAATTCTG CTCTTGGTCA 840TGTGAACTGC ACGATAAAGG AACTCAGGCG CCTCTTCTTA GTTGATGATT TAGTTGATTC 900TCTGAAGTTT GCAGTGTTGA TGTGGGTATT TACCTATGTT GGTGCCTTGT TTAATGGTCT 960GACACTACTG ATTTTGGCTC TCATTTCACT CTTCAGTGTT CCTGTTATTT ATGAACGGCA 1020TCAGGCACAG ATAGATCATT ATCTAGGACT TGCAAATAAG AATGTTAAAG ATGCTATGGC 1080TAAAATCCAA GCAAAAATCC CTGGATTGAA GCGCAAAGCT GAATGAAAAC GCCCAAAATA 1140ATTAGTAGGA GTTCATCTTT AAAGGGGATA TTCATTTGAT TATACGGGGG AGGGTCAGGG 1200AAGAACGAAC CTTGAC 1216 ( 2 ) SEQ ID NO:4：

(ⅰ) sequence signature:

(A) length: 373 amino acid

(B) type: amino acid

(D) topological structure: linearity

(ⅱ) molecule type: polypeptide

( ⅹⅰ ) ：SEQ ID NO:4Met Glu Asp Leu Asp Gln Ser Pro Leu Val Ser Ser Ser Asp Ser 15Pro Pro Arg Pro Gln Pro Ala Phe Lys Tyr Gln Phe Val Arg Glu 30Pro Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu 45Asp Glu Asp Leu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala 60Ala Gly Leu Ser Ala Ala Pro Val Pro Thr Ala Pro Ala Ala Gly 75Ala Pro Leu Met Asp Phe Gly Asn Asp Phe Val Pro Pro Ala Pro 90Arg Gly Pro Leu Pro Ala Ala Pro Pro Val Ala Pro Glu Arg Gln 105Pro Ser Trp Asp Pro Ser Pro Val Ser Ser Thr Val Pro Ala Pro 120Ser Pro Leu Ser Ala Ala Ala Val Ser Pro Ser Lys Leu Pro Glu 135Asp Asp Glu Pro Pro Ala Arg Pro Pro Pro Pro Pro Pro Ala Ser 150Val Ser Pro Gln Ala Glu Pro Val Trp Thr Pro Pro Ala Pro Ala 165Pro Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro Lys Arg Arg Gly 180Ser Ser Gly Ser Val Val Val Asp Leu Leu Tyr Trp Arg Asp Ile 195Lys Lys Thr Gly Val Val Phe Gly Ala Ser Leu Phe Leu Leu Leu 210Ser Leu Thr Val Phe Ser Ile Val Ser Val Thr Ala Tyr Ile Ala 225Leu Ala Leu Leu Ser Val Thr Ile Ser Phe Arg Ile Tyr Lys Gly 240Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly His Pro Phe Arg 255Ala Tyr Leu Glu Ser Glu Val Ala Ile Ser Glu Glu Leu Val Gln 270Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Cys Thr Ile Lys 285Glu Leu Arg Arg Leu Phe Leu Val Asp Asp Leu Val Asp Ser Leu 300Lys Phe Ala Val Leu Met Trp Val Phe Thr Tyr Val Gly Ala Leu 315Phe Asn Gly Leu Thr Leu Leu Ile Leu Ala Leu Ile Ser Leu Phe 330Ser Val Pro Val Ile Tyr Glu Arg His Gln Ala Gln Ile Asp His 345Tyr Leu Gly Leu Ala Asn Lys Asn Val Lys Asp Ala Met Ala Lys 360Ile Gln Ala Lys Ile Pro Gly Leu Lys Arg Lys Ala Glu 373 ( 2 ) SEQ ID NO:5

(ⅰ) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: oligonucleotides

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5:GACTGGATCC ATGGAAGACC TGGACCAGT 29 (2) SEQ ID NO:6

(ⅰ) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: oligonucleotides

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:TTGGAAGCTT TCATTCAGCT TTGCGCTTC 29 (2) SEQ ID NO:7

(ⅰ) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: oligonucleotides

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:7TCAGAAGCTT ATGGAAGACC TGGACCAGT 29 (2) SEQ ID NO:8

(ⅰ) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ⅱ) molecule type: oligonucleotides

(ⅹ ⅰ) sequence description: SEQ ID NO:8TTGGGGATCC TCATTCAGCT TTGCGCTTC 29

Claims (15)

1. one kind is detected the method whether liver cell canceration takes place, and it is characterized in that, this method comprises:

Detect the transcript or the RTN4B albumen that whether there are RTN4B in the liver cell,

Exist the transcript of RTN4B or RTN4B albumen just to show that canceration has taken place this liver cell.

2. the method for claim 1 is characterized in that, this method is by detecting in the liver cell whether have RTN4B albumen, and it comprises step:

The hepatoma carcinoma cell sample is contacted with the specific antibody of anti-RTN4B;

Detect whether to have formed immune complex, form immune complex and just represent that canceration has taken place this liver cell.

3. the method for claim 1 is characterized in that, this method is the transcript that whether has RTN4B in the liver cell by detecting, and it comprises step:

Use the RTN4B Auele Specific Primer, with this hepatocellular mRNA of RT-PCR method amplification,

Whether have estimate RTN4B amplified production, exist amplified production just to represent that canceration has taken place this liver cell if detecting.

4. the method for claim 1 is characterized in that, this method is the transcript that whether has RTN4B in the liver cell by detecting, and it comprises step:

Use the RTN4B specific probe, with this hepatocellular cDNA sample hybridization,

Detect whether the hybridization product is arranged, exist the hybridization product just to represent that canceration has taken place this liver cell.

5. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of human RTN 4 B protein activity, a described nucleotide sequence coded polypeptide, and this polypeptide has the sequence shown in the SEQ IDNO:4.

6. the human RTN 4 B protein polypeptide of a separation is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragment or its reactive derivative with SEQ ID NO:4 amino acid sequence.

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

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

9. a generation has the method for the polypeptide of human RTN 4 B protein activity, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of human RTN 4 B protein activity operationally is connected in expression regulation sequence, form the human RTN 4 B protein expression vector, show at least 70% homology from the nucleotides sequence of nucleotide 5-1126 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 recombinant cell of human RTN 4 B protein;

(c) under the condition that is fit to expressing human RTN4B polypeptide, the recombinant cell in the incubation step (b);

(d) isolate polypeptide with human RTN 4 B protein activity.

10. antibody that energy combines with the described human RTN 4 B protein polypeptid specificity of claim 2.

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

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

13. a kit that detects liver cancer is characterized in that it contains

(1) primer of specific amplification human RTN 4 B is right,

(2) reagent of Ren Xuan reverse transcription mRNA.

14. a kit that detects liver cancer is characterized in that, it contains the specific antibody at human RTN 4 B.

15. a kit that detects liver cancer is characterized in that, it contains can be specifically and the probe of the mRNA hybridization of human RTN 4 B.

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