CN1299827A - New human CD20 homolgous molecule and its code sequence and use - Google Patents

Abstract

The present invention relates to a novel human cell surface protein molecule -CD20LM protein, it and human CD20 have homology. Said invention provides the polynucleotide for coding said protein and method for producing this protein by means of recombination technology. Said invention also discloses the application of polynucleotide for coding this novel human CD20LM. Said invention also discloses the characteristics of high expression of this protein on the surface of lymphoma B. Said inventino also discloses an antagonist resisting said protein molecule and its therapeutic action, specially it can be used for diagnosing and curing lymphoma B.

Description

New human CD 20 homolgous molecule, its encoding sequence and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the proteic polynucleotide of new coding immunocyte membrane molecule-CD20LM (CD20-like molecular abbreviates " CD20LM " as), and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of new cell surface molecule relevant with treatment with the B Lymphoma Diagnosis.

The research of immunocyte membrane molecule all has crucial meaning for the essence of understanding immunne response in depth and diagnosis, prevention and the treatment of clinical some disease.The CD molecule has significance in various biological phenomenas and disease development process.T cell differentiation antigen and corresponding monoclonal antibody thereof are widely used in basis and clinical immunology research, and the CD monoclonal antibody can be used for: 1. the state of machines body immunity function; 2. leukemia, lymphoma are diagnosed and immunophenotyping, for treatment provides foundation; 3. Zhi Bei immunotoxin can be treated tumour or is used for control of bone marrow transplantation, graft-rejection etc.Therefore, the discovery of new CD molecule all has profound significance on basis and clinical immunology.

People CD20 molecule is that a kind of molecular weight is the cytolemma glycoprotein of 33-37KD, and its assignment of genes gene mapping is in o.11 karyomit(e), is about to be 16Kb.CD20 is expressed in the B cell specifically, and its major function is to play Ca ²⁺The effect of passage.Be expressed in this phenomenon of B cell specifically at CD20, people place hope on the CD20 molecule as treatment B lymphadenomatous target molecule, found that the certain high expression level CD20 of people B lymphoma molecule.American scholar has prepared anti-CD-20 monoclonal antibody (called after Rituximab) subsequently, and enters clinical treatment in November, 1997 by the FDA approval.

Rituximab combines the immune globulin variable region of mouse and human IgG1's constant region and the chimeric antibody that forms with recombinant technology, can mediate the cell killing that cytotoxicity that complement relies on and antibody rely on.The lymphoma cell strain of some anti-chemotherapeutics is to the Rituximab sensitivity, and can promote the apoptosis of lymphoma cell.Rituximab is that first of FDA approval is used for the mab treatment scheme of tumour, also is first single medicine treatment plan that is used for lymphoma treating.Use this scheme lymphoma patient intractable to 166 or recurrence and treat, total effective rate is 48%, and wherein 6% reaches fully and to alleviate, and 42% reaches part alleviates, and meta working lipe is 13.2 months.In the serum of treatment back the sustainable 3-6 of Rituximab level month, the ultimate effect that the retardance tumour of some months is dwindled appearred in some patient.After 60 are used Rituximab treatment effectively again in the case of recurrence, reuse this medicine still have 40% efficient (Grillo-Lopez et al.SeminOncol.1999,26:66).

At present, carrying out combined utilization Rituximab and Interferon, rabbit, chemotherapy (CHOP scheme) and radiotherapy (application of radiation ¹³¹I or ⁹⁰Y mark CD20 antibody carries out guidance quality treatment) etc. methods of treatment research (Press.SeminOncol, 1999,26:58), obtained result preferably.Wherein in the lymphadenomatous treatment of 38 slow progress types, Rutuximab and CHOP scheme combined utilization are efficient to be 100%, and meta got nowhere lifetime above 2.5 years.In 31 moderate malignant lymphomas and CHOP scheme combined utilization efficient be 96%.Simultaneously, also there is the investigator to use Rituximab to acute B Lymphocytic leukemia (Vartholomatos et al.ActaHaematol.1999,102:94), hairy cell leukemia (Hagberg.Med Oncol.1999,16:221), the relevant lymphoma of AIDS and the nervus centralis lymphoma report for the treatment of.

It is good that Rituximab carries out the tolerance of clinical treatment, illustrates that this methods of treatment and means have very big prospect.But the lymphoma patient of some somatotypes and other tumours are still arranged to this kind treatment unsatisfactory curative effect, this prompting still has very large space to the research of lymphoma cell or other surface moleculars of other tumour cells and antibody thereof.

Therefore, this area presses for the new new cell surface molecule of exploitation, especially high expression level and or specific expressed surface molecular in tumour cell such as lymphoma cell.

The purpose of this invention is to provide a kind of new people CD molecule-CD20LM polypeptide with and fragment, analogue and derivative.This new CD molecule and CD20 homology, and therefore high expression level can be used as a kind of new B lymphoma treating target molecule in the B lymphoma.At the proteic specific antibody of CD20LM (especially monoclonal antibody), can be used for lymphadenomatous diagnosis of B and treatment, so CD20LM albumen is a kind of immune molecule that has critical function and have very big application prospect.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

Another object of the present invention provides the pharmaceutical composition that contains CD20LM albumen or its antagonist.

In a first aspect of the present invention, novel isolated CD20LM polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people CD20LM polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 97-774 position among the SEQ ID NO:1; (b) has the sequence of 1-1289 position among the SEQ ID NO:1.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people CD20LM protein-active, this method comprises: (a) under the proteic condition of suitable expressing human CD20LM, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people CD20LM protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people CD20LM polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1289 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people CD20LM polypeptide active is provided, and the compound that suppresses people CD20LM polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people CD20LM polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of CD20LM in the test sample, it comprises: sample is contacted with the proteic specific antibody of CD20LM, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CD20LM albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people CD20LM polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people CD20LM polypeptide active, and perhaps screening suppresses the antagonist of people CD20LM polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people CD20LM of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people CD20LM polypeptide of the present invention or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as cancer such as B lymphoma.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is the cDNA sequence of people CD20LM of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is represented with capitalization

Fig. 2 is the proteic full length amino acid sequence of people CD20LM of the present invention.

Fig. 3 is people CD20LM albumen of the present invention and the proteic amino acid sequence homology comparison diagram of people CD20.The below sequence is people CD20LM, and the top sequence is a people CD20 albumen.Identical amino acid marks with " | " between two sequences, and similar amino acid marks with ": " or ". ".

Fig. 4 is the hydrophobicity graphic representation of the Kyte-doolitte hydrophobicity analysis of people CD20LM of the present invention.

As used herein, " separation " refers to that material separates (if natural thing from its primal environment Matter, primal environment namely are natural surroundingses). Such as the polynucleotide under the native state in the active somatic cell and polypeptide be Do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist Separately, then for separation and purification.

As used herein, " CD20LM albumen or the polypeptide of separation " refers to that the CD20LM polypeptide is substantially free of Natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use standard Purified technology of protein purifying CD20LM albumen. Basically pure polypeptide is on non-reduced polyacrylamide gel Can produce single master tape. The purity of CD20LM polypeptide can be used amino acid sequence analysis.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. This Bright polypeptide can be the product of natural purifying, or the product of chemical synthesis, or uses recombinant technique from protokaryon Or produce in the eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). According to The host that the recombinant production scheme is used, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analog of people CD20LM albumen. As used herein, term " fragment ", " derivative " and " analog " refer to basically keep natural human CD20LM egg of the present invention The biological function of Bai Xiangtong or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can be (ⅰ) Have one or more conservative or non-conservation amino acid residue (preferred conservative amino acid residue) is substituted many Peptide, and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ⅱ) one The polypeptide that has substituted radical in individual or a plurality of amino acid residues, or (ⅲ) mature polypeptide and another compound (such as Prolong the compound of polypeptide half-life, for example polyethylene glycol) merge formed polypeptide, or (ⅳ) additional amino acid Sequence is fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for this polypeptide of purifying Sequence or proteinogen sequence, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, this A little fragments, derivative and analog belong to the known scope of those skilled in the art.

In the present invention, term " people CD20LM polypeptide " refers to have the SEQ ID of people CD20LM protein active The polypeptide of NO.2 sequence. This term also comprise have with people CD20LM albumen identical function, SEQID NO. The variant form of 2 sequences. These variant forms comprise (but being not limited to): several (are generally 1-50, Good ground 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, with And C end and/or N terminal add one or several (being generally in 20, preferably is in 10, More preferably be in 5) amino acid. For example, in the art, the amino acid close or similar with performance carries out During replacement, usually can not change the function of protein. Again such as, C end and/or N terminal add one or Several amino acid also can not change the function of protein usually. This term also comprises the activity of people CD20LM albumen Fragment and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural sudden change Body, induced mutation body, under high or low stringency condition can with the DNA institute of people CD20LM DNA hybridization The albumen of coding and the polypeptide or the albumen that utilize the antiserum acquisition of anti-humen CD 20 LM polypeptide. The present invention also Other polypeptide are provided, as have comprised the fusion of people CD20LM polypeptide or its fragment. Except total length almost Outside the polypeptide, the present invention has also comprised the soluble fragments of people CD20LM polypeptide. Usually, this fragment has the people The CD20LM peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, Goodly at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 Individual continuous amino acid.

Invention also provides the analog of people CD20LM albumen or polypeptide. These analogs and natural human CD20LM The difference of polypeptide can be the difference on the amino acid sequence, and can be affect poor on the modified forms of sequence yet Different, perhaps have both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can pass through Various technology obtain, and as by radiation or be exposed to mutagens and produce random mutagenesis, also can pass through the direct mutagenesis method Or the biological technology of other known moleculars. Analog also comprise have be different from the amino acid whose residue of natural L-(as D-amino acid) analog, and have that non-natural exists or synthetic amino acid (such as β, gamma-amino acid) Analog. Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(usually the not changing primary structure) form of modification comprises: chemically derived form such as the second of the polypeptide that body is interior or external Acidylate or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further add work step Carry out glycosylation modified in rapid and polypeptide that produce. This modification can by polypeptide is exposed to carry out glycosylated Enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorylation amino The sequence of acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Thereby also comprising being modified improves Its anti-proteolysis performance or optimized the polypeptide of solubility property.

In the present invention, " people CD20LM albumen conservative variation polypeptide " refers to the amino acid with SEQ ID NO:2 Sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5,3 amino acid at the most best Replaced by similar performance or close amino acid and form polypeptide. These conservative variation polypeptide are preferably according to table 1 Carry out amino acid substitution 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

Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, base Because of group DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA compiles Code chain or noncoding strand. The coding region sequence of encoding mature polypeptide can with the code area shown in the SEQ ID NO:1 The variant of the identical or degeneracy of sequence. As used herein, " variant of degeneracy " refers to compile in the present invention Code has the protein of SEQ ID NO:2, but with the differentiated nuclear of coding region sequence shown in the SEQ ID NO:1 Acid sequence.

The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coded sequence of an encoding mature polypeptide; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional additional volume The code sequence) and non-coding sequence.

Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can be The polynucleotides that also comprise additional code and/or non-coding sequence.

The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention Polypeptide or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural generation etc. The variant that position variant or non-natural take place. These nucleotide diversity bodies comprise replacement variant, deletion mutation body With the insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, and it may Replacement, disappearance or the insertion of one or more nucleotides, but can be from the merit of the polypeptide that changes in fact its coding Energy.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotides of at least 80% homogeny more preferably. The present invention be more particularly directed under stringent condition and the present invention The interfertile polynucleotides of described polynucleotides. In the present invention, " stringent condition " refers to: (1) is at low ion Hybridization under intensity and the higher temperature and wash-out, such as 0.2 * SSC, 0.1%SDS, 60 ℃; Or (2) hybridization adds the time Denaturant is arranged, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) are only two Homogeny between the bar sequence is at least more than 90%, is more preferably 95% and just hybridizes when above. And, can mix The polypeptide of the polynucleotide encoding of handing over and the mature polypeptide shown in the SEQ ID NO:2 have identical biological function with Active.

The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization. As used herein, " nucleic acid fragment " Length contains 15 nucleotides at least, better is at least 30 nucleotides, is more preferably at least 50 nucleotides, Well more than at least 100 nucleotides. The amplification technique (such as PCR) that nucleic acid fragment can be used for nucleic acid with determine and/ Or the polynucleotide of separation coding CD20LM albumen.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

People CD20LM Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

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

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

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

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or CD20LM albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the CD20LM polypeptide of reorganization.In general following steps are arranged:

(1). with the proteic polynucleotide of coding people CD20LM of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, people CD20LM albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people CD20LM encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be handled with the CaCl2 method in exponential growth after date results, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The people CD20LM albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism CD20LM protein function as pharmacological agent CD20LM protein function.The peptide molecule that can suppress or stimulate people CD20LM protein function that can be used for seeking therapeutic value with the recombinant human CD20LM protein screening peptide library of expressing.

On the other hand, the present invention also comprises people CD20LM DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people CD20LM gene product or fragment.Preferably, refer to that those can combine with people CD20LM 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 CD20LM, comprise that also those do not influence the antibody of people CD20LM protein function.The present invention also comprise those can with modify or without the people CD20LM 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 CD20LM gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human CD20LM albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people CD20LM protein function and the antibody that does not influence people CD20LM protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people CD20LM gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people CD20LM gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-humen CD 20 LM can be used in the immunohistochemistry technology, detects the people CD20LM albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people CD20LM, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people CD20LM albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people CD20LM or activity.

Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people CD20LM albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attack the amino of antibody,, toxin is incorporated on the antibody by the exchange of disulfide linkage, this hybrid antibody can be used for killing the cell (for example tumour cell, as B lymphoma cell etc.) of people CD20LM protein positive.

The production of polyclonal antibody can choose CD20LM albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Utilize albumen of the present invention,, can filter out with CD20LM albumen interactional material takes place, as acceptor, inhibitor or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intravenously, intracutaneous or topical.

Polypeptide of the present invention or its antibody can be directly used in disease treatment, for example, and malignant tumour, B lymphoma etc.When using CD20LM albumen of the present invention or its antibody, also can use other treatment agent, for example Interferon, rabbit, Rituximab etc. simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains CD20LM polypeptide of the present invention or its antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that CD20LM albumen or its antagonist with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of people CD20LM also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of CD20LM of the proteic nothing expression of CD20LM or unusual/non-activity.The CD20LM albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic CD20LM protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the CD20LM transgenosis to cell.The method that structure carries the recombinant viral vector of CD20LM gene is found in existing document (Sambrook, et al.).Recombinant human CD20LM gene can be packaged in the liposome in addition, and then is transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people CD20LM mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people CD20LM obtains.During screening, must carry out mark to people CD20LM protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people CD20LM protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people CD20LM protein level that is detected in the test can be with laying down a definition the importance of people CD20LM albumen in various diseases and be used to the disease of diagnosing CD20LM albumen to work.

Whether having the proteic method of CD20LM in a kind of detection test sample is to utilize the proteic specific antibody of CD20LM to detect, and it comprises: sample is contacted with the CD20LM protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CD20LM albumen.

The proteic polynucleotide of CD20LM can be used for the diagnosis and the treatment of CD20LM protein related diseases.Aspect diagnosis, the proteic polynucleotide of CD20LM can be used for detecting the proteic expression of CD20LM CD20LM abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CD20LM as the CD20LM dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CD20LM albumen and also can detect the proteic transcription product of CD20LM.

The sudden change that detects the CD20LM gene also can be used for the disease of diagnosing CD20LM albumen relevant.The form of CD20LM protein mutation comprises that the point mutation compared with normal wild type CD20LM dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of CD20LM prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance inMan (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the mature polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1289 bases, and its open reading frame is positioned at the 97-774 position, coding 180 amino acid whose people CD20LM albumen of total length (SEQ IDNO:2).

CD20LM albumen of the present invention has certain homology (28% consistence being arranged, 37% similarity) at amino acid levels and people CD20 albumen.The more important thing is, CD20LM albumen high expression level in B lymphoma cells such as Raji, Daudi, so it can be used as the target molecule of attacking or kill cancer cells such as B lymphoma cell, thereby have great application prospect.

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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of people CD20LM cDNA

Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is inserted into cDNA fragment orientation on the multiple clone site of carrier with SuperScript II clone's test kit (available from Gibco), transforms DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as people CD20LM, and its encoding gene name is people CD20LM gene.

Sequence SEQ ID NO:1 total length is 1289bp, comprises 3 ' end non-coding region of 5 of 96bp ' end non-coding region and 514bp.The coding region is the 97-774 position, and coding contains 225 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is 24kD.

They are different with known for the BLAST analysis revealed, at protein level and people CD20 to a certain degree homology (Fig. 3) are arranged, and supposition may be new class CD20 molecule, called after CD20LM.

Embodiment 2: with the encoding sequence of RT-PCR method human cloning CD20LM

Be in logarithmic phase B lymphoma cell strain Raji cell total rna with Trizol (Gibco company) extraction, get 6 μ g cell total rnas and 0.5 μ g Oligo-dT _12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, adds 80 μ l ddH2O after reaction finishes and dilutes.The used primer of pcr amplification is as follows: have adopted primer 5 '-AGAAACCGTTGATGGGACTGAGAAACCAGA-3 ' (SEQ ID NO:3), antisense primer 5 ' GATTCAACAATACATTTTTAATATAGCTTT-3 ' (SEQ ID NO:4).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4 μ M primer, 0.2 μ M dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.Detect the amplified production of about 1300bp.

The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 1-1274 position shown in the SEQ ID NO:1 are identical.

CD20LM albumen is carried out Kyte-doolitte hydrophobicity analysis (Fig. 4), show that it is a membranin.

The Northern engram analysis of embodiment 3 CD20LM

Carry out the Northern trace by following ordinary method: filter membrane to be checked places the hybridization solution of 10ml through 68 ℃ of preheatings, in hybrid heater (Bellco) in 68 ℃ of prehybridizations 30 minutes; The cDNA probe that mark is good was in 95～100 ℃ of sex change 2～5 minutes, and (cDNA probe final concentration is 2～10ng/ml or 1～2 * 10 to put rapid cooling back adding hybridization solution on ice ⁶Cpm/ml), fully mixing was hybridized 2 hours in 68 ℃.After hybridization finishes, filter membrane with 2 * SSC, the drip washing of 0.05%SDS room temperature for several times, the washing lotion several is changed in continuing vibration flushing 30～40 minutes therebetween.Use 0.1 * SSC, 0.1%SDS in 50 ℃ of vibration flushings 20～40 minutes subsequently.Last filter membrane wrapped up with plastic fresh-keeping membrane, in-70 ℃ of exposure X line films 24～48 hours.

Northern blot hybridization result shows: certain expression is arranged in liver, spleen, peripheral blood, the high expression level of high level is especially arranged at B lymphoma cell strains such as Raji, Daudi.

Embodiment 4 people CD20LM are recombinant expressed

In this embodiment, be template with the pcr amplification product among the embodiment 2, increase with the PCR Oligonucleolide primers of 5 ' and 3 following ' end of sequence, obtain people CD20LM DNA as inserting fragment.

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

5’-cg?gga?tcc?ATGACATCACAACCTGTtcc-3’(SEQ?ID?NO：5)

This primer contains the restriction enzyme site of BamH I restriction enzyme, is the part Nucleotide of the encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-cg?gga?tcc?TTAAGTGAAGCCGGCCA-3’(SEQ?ID?NO.6)，

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people CD20LM of BamH I restriction enzyme.

CD20LM cDNA PCR product purification after BamH I enzyme cut and recombinate according to a conventional method with plasmid pUC18 again and be converted into the competence bacillus coli DH 5 alpha, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The CD20LM cDNA BamH I endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia company), forms carrier pGEX-2T-CD20LM and be converted into bacillus coli DH 5 alpha.Positive colony is cut the evaluation direction of insertion with EcoR I enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete CD20LM encoding sequence.

Choosing the positive DH5 α clone who expresses CD20LM is inoculated in 100ml 2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mMIPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7 mM KCl, 10.1mM Na ₂HPO ₄, 1.8mM KH ₂PO ₄PH7.3) resuspended, add 20%TritonX-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10 mM gsh, 50 mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeats wash-out 2-3 time, and obtain N and hold the people CD20LM fusion rotein that contains GST, obtain CD20LM albumen after thrombin (Sigma) enzyme cuts except that GST, molecular weight is about 24kD.

Carry out the n terminal amino acid sequential analysis with the Edams hydrolysis method, confirm that its N terminal sequence conforms to the N terminal sequence shown in the SEQ ID NO:2.

Embodiment 5: anti-humen CD 20 LM production of antibodies

The recombinant protein c D20LM that obtains among the embodiment 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people CD20LM gene translation product with it.Found that antibody can combine 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:

(ⅱ) denomination of invention: new human CD 20 homolgous molecule, its encoding sequence and purposes

(ⅲ) sequence number: the information of 6 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 1289bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO：1：AGAAACCGTT GATGGGACTG AGAAACCAGA GTTAAAACCT CTTTGGAGCT TCTGAGGACT 60CAGCTGGAAC CAACGGGCAC AGTTGGCAAC ACCATCATGA CATCACAACC TGTTCCCAAT 120GAGACCATCA TAGTGCTCCC ATCAAATGTC ATCAACTTCT CCCAAGCAGA GAAACCCGAA 180CCCACCAACC AGGGGCAGGA TAGCCTGAAG AAACATCTAC ACGCAGAAAT CAAAGTTATT 240GGGACTATCC AGATCTTGTG TGGCATGATG GTATTGAGCT TGGGGATCAT TTTGGCATCT 300GCTTCCTTCT CTCCAAATTT TACCCAAGTG ACTTCTACAC TGTTGAACTC TGCTTACCCA 360TTCATAGGAC CCTTTTTTTT TATCATCTCT GGCTCTCTAT CAATCGCCAC AGAGAAAAGG 420TTGACCAAGC TTTTGGTGCA TAGCAGCCTG GTTGGAAGCA TTCTGAGTGC TCTGTCTGCC 480CTGGTGGGTT TCATTATCCT GTCTGTCAAA CAGGCCACCT TAAATCCTGC CTCACTGCAG 540TGTGAGTTGG ACAAAAATAA TATACCAACA AGAAGTTATG TTTCTTACTT TTATCATGAT 600TCACTTTATA CCACGGACTG CTATACAGCC AAAGCCAGTC TGGCTGGATC TCTCTCTCTG 660ATGCTGATTT GCACTCTGCT GGAATTCTGC CTAGCTGTGC TCACTGCTGT GCTGCGGTGG 720AAACAGGCTT ACTCTGACTT CCCTGGGGTG AGTGTGCTGG CCGGCTTCAC TTAACCTTGC 780CTAGTGTATC TTATCCCTGC ACTGTGTTGA GTATGTCACC AAGAGTGGTA GAAGGAACAA 840CCAGCCAATC ACGAGATCAC ATGGGAGGGC ATTTGCATTG TGATGGAAGA CAGAGAAGAA 900AAGCAGATGG CAATTGAGTA GCTGATAAGC TGAAAATTCA CTGGATATGA AAATAGTTAA 960TCATGAGAAA TCAACTGATT CAATCTTCCT ATTTTGTCAG CGAAGGGAAT GAGACTCTGG 1020GAAGTTAAAT GACTGGCCTG GCATTATGCT ATGAGTTTGT GCCTTTGCTG AGGACACTAG 1080AACCTGGCTT GCCTCCCTTA TAAGCAGAAA CAATTTCTGC CACAACCACT AGTCTCTTTA 1140ATAGTATTGA CTTGGTAAAG GGCATTTACA CACGTAACTG GATCCAGTGA ATGTCTTATG 1200CTCTGCATTT GCCCCTGGTG ATCTTAAAAT TCGTTTGCCT TTTTAAAGCT ATATTAAAAA 1260TGTATTGTTG AATCAAAAAA AAAAAAAAA 1289 ( 2 ) SEQ ID NO：2： ( ⅰ ) ：

(A) length: 225 amino acid

(B) type: amino acid

(D) topological structure: linear (ⅱ) molecule type: polypeptide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:2:MTSQPVPNET IIVLPSNVIN FSQAEKPEPT NQGQDSLKKH LHAEIKVIGT IQILCGMMVL 60SLGIILASAS FSPNFTQVTS TLLNSAYPFI GPFFFIISGS LSIATEKRLT KLLVHSSLVG 120SILSALSALV GFIILSVKQA TLNPASLQCE LDKNNIPTRS YVSYFYHDSL YTTDCYTAKA 180SLAGSLSLML ICTLLEFCLA VLTAVLRWKQ AYSDFPGVSV LAGFT 225 (2) SEQ ID NO:3

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:3:AGAAACCGTT GATGGGACTG AGAAACCAGA 30 (2) SEQ ID NO:4

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:GATTCAACAA TACATTTTTA ATATAGCTTT 30 (2) SEQ ID NO:5

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ IDNO:5:CGGGATCCAT GACATCACAA CCTGTTCC 28 (2) SEQID NO:6

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: SEQ ID NO:6:CGGATCCTT AAGTGAAGCC GGCCA 25

Claims (14)

1. an isolating people CD20LM polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 97-774 position among the SEQ ID NO:1;

(b) has the sequence of 1-1289 position among the SEQ ID NO:1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. preparation method with polypeptide of people CD20LM protein-active is characterized in that this method comprises:

(a) under the proteic condition of suitable expressing human CD20LM, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people CD20LM protein-active.

9. energy and the described people CD20LM of claim 1 protein-specific bonded antibody.

10. nucleic acid molecule, it contains a successive 10-1289 Nucleotide in the described polynucleotide of claim 3.

11. whether there is the proteic method of CD20LM in the test sample, it is characterized in that, comprising:

The described antibody of sample and claim 9 is contacted,

Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CD20LM albumen.

12. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people CD20LM protein-active, and perhaps screening suppresses the antagonist of people CD20LM protein-active or is used to the peptide finger print identification.

13. the purposes of a nucleic acid molecule as claimed in claim 10 is characterized in that, it is used as primer and is used for pcr amplification reaction, perhaps is used for hybridization as probe, perhaps is used to make gene chip or microarray.

14. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 or the described antibody of claim 9 and the pharmaceutically acceptable carrier of safe and effective amount.

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