CN1238379C - New osteoclast formation inhibiting factor and its coding sequence and use - Google Patents

Abstract

The present invention provides a novel osteoclast formation inhibiting factor--OPG-372 protein, the DNA sequence for coding the OPG-372 protein, and a method for producing the OPG-372 protein by using a recombination technology. The OPG-372 protein is the variant of OPG. The present invention also discloses the use of the OPG-372 protein for treating various diseases, such as osteoporosis, hypercalcinemia, etc. The present invention also provides a medical composition containing the OPG-372 protein.

Description

New osteoclast forms supressor, its encoding sequence and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the dna sequence dna that new coding human osteoclast forms supressor-OPG-372, and the polypeptide of this dna sequence encoding.The invention still further relates to the purposes and the preparation of this dna sequence dna and polypeptide.Specifically, polypeptide of the present invention is that a kind of new osteoclast relevant with bone forms supressor, and it is a kind of OPG variant.

Senile osteoporosis has become the important diseases that the whole world is paid close attention to.The elderly is elderly woman particularly, because the minimizing gradually of estrogen secretion adds the adjusting of other factor in the bone microenvironment, causes the decline of scleroblast vigor suppression ratio osteoclast vigor faster.Therefore, after after a while, will show obvious osteoporosis symptom.Senile osteoporosis not only causes fracture easily, influences the elderly's life, and is difficult to treatment.

It at first is to be found by people such as Simonet that osteoclast forms supressor (osteoprotegerin or title osteoclastogenesis inhibitoryfactor are hereinafter to be referred as OPG), it is a kind of secretor type glycoprotein (W.S.Simonet et al. that suppresses osteoclast formation that has, Cell.Vol.89,309-319,1997).Subsequently, people such as Japanese scholar Yasuda has also reported this protein (Yasuda H.et al., Endocrinology, 1998 vol.139.No.3 p 1329-1337).Confirm that through further experiment osteopetrosis will appear in the mouse that changes the OPG gene, osteoporosis (Simonet, the same, 1997) then appears in OPG gene knock out mouse.In extracing the rat of ovary, OPG albumen can be treated owing to lacking the osteoporosis that oestrogenic hormon forms, and maybe can prevent owing to lacking the bone-loss that oestrogenic hormon causes (Simonet, the same, 1997).In addition, OPG significantly falls the blood calcium effect in vivo in addition, can make normal and extract parathyroid rat serum calcium concn obviously descend (Simonet, the same, 1997; Yamamoto M.et al., Endocrinology vol.139 No.9,4012-4015,1998).Up to the present, the mechanism of OPG adjusting bone density mainly is that OPG suppresses osteoclast formation (Simonet, 1997 on the one hand; Yasuda, 1998), OPG influences osteoclast and survives on the other hand, causes osteoclast apoptosis (Akatsut.et al., Biochemical and Biophysical Research Communication, 250.229-234,1998).Therefore, OPG has very big potential clinical value, and promptly OPG can prevent and treat elderly woman owing to lacking the osteoporosis that oestrogenic hormon causes, also can treat some hypercalcinemia.

Yet this area does not report that also osteoclast forms other variants of supressor before the present invention.Therefore, this area presses for the new osteoclast of exploitation and forms supressor.

The purpose of this invention is to provide a kind of new human osteoclast form supressor OPG-372 albumen with and fragment, analogue and derivative.

Another object of the present invention provides the dna sequence dna of these polypeptide of coding.

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

In a first aspect of the present invention, novel isolated OPG-372 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 dna sequence dna of isolating these polypeptide of coding is provided, this dna sequence dna 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) dna sequence dna of the above-mentioned people OPG-372 polypeptide of coding; (b) with dna sequence dna (a) complementary dna sequence dna.Preferably, this dna sequence encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of this dna sequence dna is be selected from down group a kind of: the sequence that (a) has 1-1119 position among the SEQ ID NO:1; (c) has the sequence of 1-1163 position among the SEQ ID NO:1.

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

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people OPG-372 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human OPG-372, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people OPG-372 protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people OPG-372 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1163 Nucleotide in the above-mentioned dna sequence dna.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people OPG-372 polypeptide active is provided, and the compound that suppresses people OPG-372 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 OPG-372 polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of OPG-372 in the test sample, it comprises: sample is contacted with the proteic specific antibody of OPG-372, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample OPG-372 albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people OPG-372 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 OPG-372 polypeptide active, and perhaps screening suppresses the antagonist of people OPG-372 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people OPG-372 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 OPG-372 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as osteoporosis, hypercalcemia.

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 gel electrophoresis figure of OPG-372 gene cDNA.

Fig. 2 has shown the colibacillus expression plasmid pET-OPG-372 that contains the OPG-372 gene.

Fig. 3 has shown the methanol yeast expression plasmid pPIC-OPG-372 that contains the OPG-372 gene.

Fig. 4 is that the SDS-PAGE of methanol yeast expression product analyzes collection of illustrative plates, and wherein swimming lane 1 is a molecular weight marker, swimming lane 7,6, and 5 are respectively 3,4,5 days sample, and swimming lane 4-2 is the sample of other two mono-clonal bacterial strains.

Fig. 5 A and 5B have shown insect baculovirus transferring plasmid pBac-OPG-372 and the pBacHT-OPG-372 that contains the OPG-372 gene.

Fig. 6 is that the SDS-PAGE of Tn-5B1-4 cell expression product analyzes collection of illustrative plates, and wherein swimming lane 1 is a molecular weight marker, and swimming lane 2 and 3 is non-reduced expression sample, and the indicator binary forms; Swimming lane 5 and 5 is for going back raw sample; Swimming lane 6 is not integrated the metainfective expression sample of Bacmid of OPG gene for Tn-5B1-4.

Fig. 7 has shown the Western engram analysis result that expression product carries out with anti-His antibody in the Tn-5B1-4 cell to recombinant baculovirus BacHTOPG-372.

Fig. 8 has shown that OPG-372 and His-OPG-372 are at the intravital blood droping calcium activity of mouse.

In the present invention, term " OPG-372 albumen ", " OPG-372 polypeptide " or " osteoclast formation Inhibiting factor OPG-372 " be used interchangeably, all refer to have human osteoclast and form inhibiting factor OPG-372 ammonia Albumen or the polypeptide of base acid sequence (SEQ ID NO:2). They comprise the broken bone that contains or do not contain initial methionine Cell forms inhibiting factor OPG-372.

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, " OPG-372 albumen or the polypeptide of separation " refers to that the OPG-372 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 OPG-372 albumen. Basically pure polypeptide is on non-reduced polyacrylamide gel Can produce single master tape. The purity of OPG-372 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 OPG-372 albumen. As used herein, term " fragment ", " derivative " and " analog " refer to basically keep natural human OPG-372 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 OPG-372 polypeptide " refers to have the SEQ ID of people OPG-372 protein active The polypeptide of NO.2 sequence. This term also comprise have with people OPG-372 albumen identical function, SEQ ID 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 OPG-372 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 OPG-372DNA hybridization The albumen of coding and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people OPG-372 polypeptide. The present invention also Provide other polypeptide, as the fusion that comprises people OPG-372 polypeptide or its fragment is (such as SEQ ID NO:3 Shown fusion). Except the polypeptide of total length almost, what the present invention had also comprised people OPG-372 polypeptide can The dissolubility fragment. Usually, this fragment have people OPG-372 peptide sequence at least about 20 continuous amino acids, logical Often at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 Continuous amino acid is best at least about 100 continuous amino acids.

Invention also provides the analog of people OPG-372 albumen or polypeptide. These analogs and natural human OPG-372 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 OPG-372 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

In one embodiment, provide the OPG-372 fusion of N end with the His-Tag structure, it is little The blood calcium effect of falling in the mouse body descends to some extent. In another embodiment, provide C end 5kD left and right sides fragment to be fallen The OPG-372 active fragment of separating, the degraded of this 5kD fragment is to almost not impact of blood droping calcium activity.

Dna sequence dna of the present invention can be dna form or rna form. Dna form comprise cDNA, Genomic 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 dna sequence dna of the mature polypeptide of coding SEQ ID NO:2 comprises: the code sequence of an encoding mature polypeptide Row; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional attached Add coded sequence) and non-coding sequence.

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

The invention still further relates to the varient of above-mentioned dna sequence dna, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of this dna sequence dna can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of a dna sequence dna, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the dna sequence dna of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile dna sequence dna of dna sequence dna of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile dna sequence encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding OPG-372.

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

People OPG-372 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 dna sequence dna of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or OPG-372 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 OPG-372 polypeptide of reorganization.In general following steps are arranged:

(1). with the dna sequence dna (or varient) of coding of the present invention people OPG-372 polypeptide, or with the recombinant expression vector that contains this dna sequence dna proper host cell that transforms or transduce;

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

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

A kind of preferred production methods is, earlier transform for example intestinal bacteria by the insect baculovirus donor plasmid that will contain the OPG-372 gene, extracting recombinant baculovirus DNA (reorganization Bacmid), thereby obtain to contain the recombinant baculovirus of OPG-372 gene, transfection insect cell (for example cabbage looper cell Tn-5B1-4) again is to produce OPG-372 albumen of the present invention.

Among the present invention, people OPG-372DNA sequence nucleotide 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 OPG-372 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

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

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

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

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

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

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

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

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the 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 OPG-372 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 OPG-372 protein function as pharmacological agent OPG-372 protein function.The peptide molecule that can suppress or stimulate people OPG-372 protein function that can be used for seeking therapeutic value with the recombinant human OPG-372 protein screening peptide library of expressing.

On the other hand, the present invention also comprises people OPG-372DNA 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 OPG-372 gene product or fragment.Preferably, refer to that those can combine with people OPG-372 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 OPG-372, comprise that also those do not influence the antibody of people OPG-372 protein function.The present invention also comprise those can with modify or without the people OPG-372 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 OPG-372 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human OPG-372 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 prepare (see people such as Kohler, Nature256; 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 OPG-372 protein function and the antibody that does not influence people OPG-372 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people OPG-372 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 OPG-372 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-people OPG-372 can be used in the immunohistochemistry technology, detects the people OPG-372 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people OPG-372, 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 OPG-372 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people OPG-372 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 OPG-372 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people OPG-372 protein positive.

The production of polyclonal antibody can choose OPG-372 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 OPG-372 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, agonist, 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, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of osteoporosis, hypercalcemia aspect.When using OPG-372 albumen of the present invention, also can use the other treatment agent simultaneously, as OPG, oestrogenic hormon etc.

The present invention also provides a kind of pharmaceutical composition, and it contains OPG-372 polypeptide of the present invention or its agonist, 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 the OPG-372 albumen of safe and effective amount or its antagonist, agonist are 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 OPG-372 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 OPG-372 of the proteic nothing expression of OPG-372 or unusual/non-activity.The OPG-372 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic OPG-372 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the OPG-372 transgenosis to cell.The method that structure carries the recombinant viral vector of OPG-372 gene is found in existing document (Sambrook, et al.).Recombinant human OPG-372 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 OPG-372mRNA 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 OPG-372 obtains.During screening, must carry out mark to people OPG-372 protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people OPG-372 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people OPG-372 protein level that is detected in the test can be with laying down a definition the importance of people OPG-372 albumen in various diseases and be used to the disease of diagnosing OPG-372 albumen to work.

Whether having the proteic method of OPG-372 in a kind of detection test sample is to utilize the proteic specific antibody of OPG-372 to detect, and it comprises: sample is contacted with the OPG-372 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample OPG-372 albumen.

The proteic polynucleotide of OPG-372 can be used for the diagnosis and the treatment of OPG-372 protein related diseases.Aspect diagnosis, the proteic polynucleotide of OPG-372 can be used for detecting the proteic expression of OPG-372 OPG-372 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of OPG-372 as the OPG-372DNA sequence.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 dna sequence dna 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 OPG-372 albumen and also can detect the proteic transcription product of OPG-372.

The sudden change that detects the OPG-372 gene also can be used for the disease of diagnosing OPG-372 albumen relevant.The form of OPG-372 protein mutation comprises that the point mutation compared with normal wild type OPG-372DNA sequence, 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 OPG-372 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 separated DNA sequence is provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Dna sequence dna of the present invention is isolated from people's normal liver cell.Its sequence is shown in SEQ ID NO:1, and the dna sequence dna sequence total length that it comprises is 1163 bases, and its open reading frame is positioned at the 1-1119 position, and the coding total length is 373 amino acid whose people OPG-372 albumen (SEQ ID NO:2).Having removed the length behind the initial methionine is 372 amino acid, so be called OPG-372 albumen.

Subsequently, therefrom also cloned the OPG-372 gene among the total RNA of compatriots' placenta, proved that the OPG-372 gene is not is owing to organize differences to produce.To 3 ' genome sequence analysis revealed of two normal Chinese's peripheral blood lymphocytes and the tire kidney cell line OPG of Canadian gene, the generation of OPG-372 is not to be because due to people's difference between species and the rna level processing yet.This shows that OPG-372 is the variant that a kind of extensive existence, normal human osteoclast form supressor.OPG-372 provides new treatment approach for diseases such as treatment osteoporosis, hypercalcemias, thereby has 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:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The structure of the clone of OPG-372 gene and escherichia coli cloning plasmid pSK-OPG-372

In order to obtain the OPG mature polypeptide coding sequence, according to the OPG gene order of external report, a pair of primer OPGPRI1 and OPGPRI2 have been synthesized in design, and its nucleotide sequence is respectively:

OPGPRI1：5’-GGATCCATGAAACGTTTCCTCCAAAGTACC-3’(SEQ?ID?NO：3)

OPGPRI2：5’-ACCTCGAGGCCATTTCCAGTTATAAGCAGC-3’(SEQ?ID?NO：4)

Wherein, OPFPRI1 is the encoding sequence of OPG maturation protein N end; OPGPRI2 is the complementary sequence of the non-coding region of OPG C end encoding sequence and terminator codon back.

With OPGPRI1 and OPGPRI2 is primer, with extractive total RNA from Chinese's liver cell line LO2 and Chinese's placenta is that the template synthetic first chain cDNA is a template, pcr amplification OPG gene order is introduced BamHI and XhoI restriction enzyme site respectively at its 5 ' end and 3 ' end.The PCR condition is: 94 ℃ of 1min, and 52 ℃ of 1min, 72 ℃ of 2min, totally 35 circulations, first 94 ℃ of sex change 5min that circulate, last circulation is extended 10min for 72 ℃.PCR product electrophoresis result is seen Fig. 1.At about 1200bk place one amplified band is arranged.

The PCR product is cloned in the EcoRV site of pSK (+) behind the low melting-point agarose purifying, makes up plasmid pSK-OPG-372.Obtained the nucleotide sequence shown in the SEQ ID NO:1 through order-checking, the new protein (shown in SEQ ID NO:2) of encoding.

Analysis revealed, this new proteic gene has been compared a base difference with the OPG gene of external report, but this base changes terminator codon of generation, make this variant proteins lack 8 amino acid, so let us be called the OPG-372 gene with this variant gene than the external protein C end of reporting.

Further test shows that the sequence of being cloned is consistent from Chinese's liver cell line LO2 and placenta.The analytical results that the genome sequence of the OPG gene 3 ' end of cloning from people from two China peripheral blood lymphocyte and Canadian tire kidney cell line is carried out shows that the sequence of three's gained is consistent with the cDNA sequence.Therefore, thinking that the OPG-372 variant gene is not owing to organize differences, neither process owing to rna level, also be not that people's difference between species causes, but one has the ubiquity gene.

Embodiment 2

The structure that contains the escherichia coli expression pET-OPG-372 of OPG-372 gene

With pSK-OPG-372 plasmid BamHI and XhoI double digestion, isolate the OPG-372 gene on the plasmid, and be connected with prior pET-28a (+) plasmid through BamHI and XhoI double digestion, make up and form the colibacillus expression plasmid pET-OPG-372 (Fig. 2) that contains the OPG-372 gene.

Embodiment 3

The structure that contains the methanol yeast expression plasmid pPIC-OPG-372 of OPG-372 gene

The escherichia coli cloning plasmid pSK-OPG-372 that will contain the OPG-372 gene cuts (EcoRI is the restriction enzyme site on the pSK carrier) with BamHI and EcoRI enzyme, separate the OPG-372 gene fragment with low melting-point agarose, and be connected with the pPIC3.5K that the EcoRI enzyme is cut through BamHI in advance, make up the methanol yeast expression plasmid pPIC-OPG-372 (Fig. 3) contain the OPG-372 gene.

Embodiment 4

The expression of OPG-372 gene in methanol yeast

With pPIC-OPG-372 plasmid SalI linearization for enzyme restriction, the electricity consumption transform mode transforms methanol yeast GS115, and the positive bacterium colony of screening His on the MD flat board is containing screening OPG-372 gene multi-copy strains on the YEPD flat board of 2mg/mlG418 with it again.With these bacterium colonies overnight incubation in the YEPD substratum, its genomic dna of back extracting carries out PCR with OPGPRI1 and OPGPRI2 primer and detects, and the result shows that most of bacterium colonies are positive colony.With positive colony overnight incubation in the BMGY substratum, get its bacterium then, be diluted to A600=1 with BMMY, continue to cultivate, add methyl alcohol to 0.5% every day, after three days, get its cell every day and carry out the SDS-PAGE analysis with 0.5mm granulated glass sphere broken wall.

The result shows that the OPG-372 gene is expressed the albumen that produces two kinds of forms in methanol yeast, and a kind of is not glycosylated 43kD protein, and its molecular weight is close with the theoretical calculate value, and another kind is the glycosylated protein (see figure 4) of 70kD.

Embodiment 5

Contain the insect baculovirus donor plasmid pBac-OPG-372 of OPG-372 gene and the structure of pBacHT-OPG-372

With pSK-OPG-372 BamHI and XhoI double digestion, reclaim the OPG-372 gene fragment with low melting-point agarose, and be connected with pFastBacHTb through the pFastBacl of BamHI and XhoI double digestion in advance, make up insect baculovirus donor plasmid pBac-OPG-372 and the pBacHT-OPG-372 (Fig. 5) contain the OPG-372 gene.

Embodiment 6

Contain the acquisition of the recombinant baculovirus of OPG-372 gene

Get insect baculovirus donor plasmid pBac-OPG-372 and pBacHT-OPG-372 that 1 μ l contains the OPG-372 gene, transformed into escherichia coli DH10Bac screens the bacterium colony that contains recombinant baculovirus DNA (Bacmid) behind swivel base with penbritin, kantlex, gentamicin and four kinds of microbiotic of tsiklomitsin and blue hickie respectively.Recheck through same flat board, extracting recombinant baculovirus DNA (Bacmid) then gets the TC-100 substratum that 5 μ l recombinant baculovirus DNA (Bacmid) add 100 μ l serum-frees and is mixed again.Getting the TC-100 substratum that 6 μ l Lipofectin add 100 μ l serum-frees is mixed.Both are mixed, and room temperature was placed 15 minutes, and the TC-100 substratum that adds 800 μ l serum-frees is mixed.With the cabbage looper Tn-5B1-4 cell TC-100 substratum washed twice of serum-free of cultivating in advance in 35mm Dish, and dropwise add transferring plasmid and Lipofectin mixture, cultivated 3 days, and collected supernatant and carry out virus amplification for 27 ℃.Can obtain a large amount of the recombinant baculovirus BacOPG-372 and the BacHTOPG-372 that contain the OPG-372 gene.

Recombinant baculovirus BacHTOPG-372 is named as three-spotted phytometra nuclear polyhedrosis virus (the Autographa california nuclear polyhedrosis virus) BacHTOPG-372 of reorganization, and be deposited in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms on November 6th, 2000, China, Beijing), preserving number is CGMCC No.0505.

Embodiment 7

The expression of OPG-372 gene in cabbage looper cell Tn-5B1-4

The recombinant baculovirus BacOPG-372 that contains the OPG-372 gene and the BacHTOPG-372 that get amplification infect cabbage looper cell Tn-5B1-4, collecting cell after 3 days, add isopyknic 2 * protein sample-loading buffer after adding an amount of TE damping fluid, get 20 μ l and carry out the SDS-PAGE analysis, with Coomassie brilliant blue R250 dyeing.

The result shows that recombinant baculovirus BacOPG-372 and BacHTOPG-372 infection cabbage looper cell Tn-5B1-4 all can give expression to the protein (see figure 6) about 39kD, and about the theoretical little 5kD of its molecular weight ratio, reason may be to be degraded in cell.With anti-His antibody BacHTOPG-372 expressing viral product is carried out the Western engram analysis, the result shows that the 5kD fragment that is degraded is in C end (see figure 7).

Embodiment 8

The proteic blood droping calcium activity analysis of OPG-372 that Tn-5B1-4 expresses

In this embodiment, also the OPG-372 albumen and the His-OPG-372 of cabbage looper Tn-5B1-4 cell expressing carried out activation analysis.

Recombinant baculovirus BacOPG-372 and BacHTOPG-372 through the Tn-5B1-4 cell amplification infect the Tn-5B1-4 cell, and collecting cell after 3 days is suspended among the PBS (pH7.4).Make cytoclasis through multigelation, collect supernatant liquor behind the high speed centrifugation, with 0.22 μ m micro-pore-film filtration degerming.With non-reorganization Bacmid infect the Tn-5B1-4 cell through same processing as negative control.4 ℃ of preservations are standby.The full cell pyrolysis liquid of recombinant baculovirus expression of taking a morsel carries out SDS-PAGE to be analyzed, and measures the per-cent that OPG-372 and His-OPG-372 albumen account for the cell soluble proteins, and full cell is separated the mensuration that liquid carries out gross protein.The OPG dosage injection of pressing 6mg/kg is through the Balb/C of 24h hunger mouse, and the non-reorganization Bacmid that injects identical total protein quality expresses the negative contrast of full cell pyrolysis liquid.SCT with 40ng/ dosage injects mouse as positive control.Take mouse blood (normal mouse and positive control are all got blood when 2h) behind 2h and the 4h, collect its serum and carry out calcium ion concn mensuration.Get the OCPC test solution and (take by weighing OCPC 32.5mg, 8-HQ 1.1g, add water 250ml, add concentrated hydrochloric acid 7.5ml, fully water complements to 500ml after the stirring and dissolving), diethylamine test solution (the 21ml diethylamine adds water to 500ml), methyl alcohol be made into the calcium colouring reagents in 1.5: 1.5: 1.0 ratio.Get 50 μ l serum and add 3ml calcium colouring reagents, measure the 570nm absorbance value.With 2.5,5,7.5,10,12.5, the lime carbonate standardized solution of 15.0mg/100ml makes the calcium ion concn typical curve.Calculate the calcium ion concn in the serum.

The result shows that as shown in Figure 8 OPG-372 has the blood calcium effect of significantly falling in the normal mouse body, and the blood calcium effect does not then almost fall in the His-OPG-372 of N end band His-Taq structure.This shows that the correct folding of the N end structure of OPG has a very important role to its activity, and the fragment that its C end is degraded about 5kD then influences its activity hardly.

Embodiment 9

The generation of anti-OPG-372 protein antibodies

The recombinant human OPG-372 albumen that obtains among the embodiment 7 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.With the albumen of 50-100mg/0.2ml emulsification, mouse is carried out peritoneal injection.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100mg/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 OPG-372 protein gene translation product with it.

Found that the antibody of acquisition can combine with albumen of the present invention specifically.

Industrial applicability

OPG-372 is a new OPG variant (OPG-372) gene, and this gene is compared with external report, at nucleosides Only differ a base on the acid sequence, but because this base mutation forms a terminator codon, make this One protein is at 8 amino acid of C end disappearance. The inventor carries out height with the OPG-372 gene in insect cell Efficient expression has higher expression than expressing abroad in Chinese hamster ovary cell (CHO), and operation Relatively simple. OPG is the endogenous protein that can produce through gene engineering method, with chemical synthesis Calcitonin (CT) is compared, and has safety, an advantage such as side effect is little and production cost is low, and OPG is in advance Anti-and treatment osteoporosis aspect is better than CT.

All quote in this application as a reference at all documents that the present invention mentions, just as each piece document quilt Quote separately as a reference such. Should be understood that in addition after having read above-mentioned instruction content of the present invention, this Those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application institute equally Attached claims limited range.

Sequence table

(1) general information:

(ii) denomination of invention: new osteoclast forms supressor, its encoding sequence and purposes

(iii) sequence number: 4

(2) information of SEQ ID NO:1:

(i) sequence signature:

(A) length: 1163bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: DNA

(xi) sequence description: SEQ ID NO:1:

ATGGAAACGT?TTCCTCCAAA?GTACCTTCAT?TATGACGAAG?AAACCTCTCA?TCAGCTGTTG 60

TGTGACAAAT?GTCCTCCTGG?TACCTACCTA?AAACAACACT?GTACAGCAAA?GTGGAAGACC 120

GTGTGCGCCC?CTTGCCCTGA?CCACTACTAC?ACAGACAGCT?GGCACACCAG?TGACGAGTGT 180

CTATACTGCA?GCCCCGTGTG?CAAGGAGCTG?CAGTACTCAA?GCAGGGAGTG?CAATCGCACC 240

CACAACCGCG?TGTGCGAATG?CAAGGAAGGG?CGCTACCTTG?AGATAGAGTT?CTGCTTGAAA 300

CATAGGAGCT?GCCCTCCTGG?ATTTGGAGTG?GTGCAAGCTG?GAACCCCAGA?GCGAAATACA 360

GTTTGCAAAA?GATGTCCAGA?TGGGTTCTTC?TCAAATGAGA?CGTCATCTAA?AGCACCCTGT 420

AGAAAACACA?CAAATTGCAG?TGTCTTTGGT?CTCCTGCTAA?CTCAGAAAGG?AAATGCAACA 480

CACGACAACA?TATGTTCCGG?AAACAGTGAA?TCAACTCAAA?AATGTGGAAT?AGATGTTACC 540

CTGTGTGAGG?AGGCATTCTT?CAGGTTTGCT?GTTCCTACAA?AGTTTACGCC?TAACTGGCTT 600

AGTGTCTTGG?TAGACAATTT?GCCTGGCACC?AAAGTAAACG?CAGAGAGTGT?AGAGAGGATA 660

AAACGGCAAC?ACAGCTCACA?AGAACAGACT?TTCCAGCTGC?TGAAGTTATG?GAAACATCAA 720

AACAAAGACC?AAGATATAGT?CAAGAAGATC?ATCCAAGATA?TTGACCTCTG?TGAAAACAGC 780

GTGCAGCGGC?ACATTGGACA?TGCTAACCTC?ACCTTCGAGC?AGCTTCGTAG?CTTGATGGAA 840

AGCTTACCGG?GAAAGAAAGT?GGGAGCAGAA?GACATTGAAA?AAACAATAAA?GGCATGCAAA 900

CCCAGTGACC?AGATCCTGAA?GCTGCTCAGT?TTGTGGCGAA?TAAAAAATGG?CGACCAAGAC 960

ACCTTGAAGG?GCCTAATGCA?CGCACTAAAG?CACTCAAAGA?CGTACCACTT?TCCCAAAACT 1020

GTCACTCAGA?GTCTAAAGAA?GACCATCAGG?TTCCTTCACA?GCTTCACAAT?GTACAAATTG 1080

TATCAGAAGT?TATTTTTAGA?AATGATAGGT?AACCAGGTCT?AATCAGTAAA?AATAAGCTGC 1140

TTATAACTGG?AAATGGCCTC?GAG 1163

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 373 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:2:

METFPPKYLH?YDEETSHQLL?CDKCPPGTYL?KQHCTAKWKT?VCAPCPDHYY 50

TDSWHTSDEC?LYCSPVCKEL?QYVKQECNRT?HNRVCECKEG?RYLEIEFCLK 100

HRSCPPGFGV?VQAGTPERNT?VCKRCPDGFF?SNETSSKAPC?RKHTNCSVFG 150

LLLTQKGNAT?HDNICSGNSE?STQKCGIDVT?LCEEAFFRFA?VPTKFTPNWL 200

SVLVDNLPGT?KVNAESVERI?KWQHSSQEQT?FQLLKLWKHQ?NKDQDIVKKI 250

IQDIDLCENS?VQWHIGHANL?TFEQLRSLME?SLPGKKVGAE?DIEKTIKACK 300

PSDQILKLLS?LWRIKNGDQD?TLKGLMHALK?HSKTYHFPKT?VTQSLKKTIR 350

FLHSFTMYKL?YQKLFLEMIG?NQV 373

(2) information of SEQ ID NO:3

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:3:

GGATCCATGA?AACGTTTCCT?CCAAAGTACC 30

(2) information of SEQ ID NO:4

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:4:

ACCTCGAGGC?CATTTCCAGT?TATAAGCAGC 30

Claims (3)

1. the purposes of an isolating people OPG-372 polypeptide, described OPG-372 polypeptide has the aminoacid sequence shown in the SEQ ID NO:2, it is characterized in that, and described OPG-372 polypeptide is used to prepare the medicine that falls blood calcium.

2. purposes as claimed in claim 1, it is characterized in that described OPG-372 polypeptide is that three-spotted phytometra nuclear polyhedrosis virus (Autographa california nuclear polyhedrosis virus) the BacHTOPG-372 CGMCC No.0505 of reorganization produces.

3. purposes as claimed in claim 2 is characterized in that, described OPG-372 polypeptide is by producing with described three-spotted phytometra nuclear polyhedrosis virus infected insect host cell.

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