CN1631898A - Mitochondria traffic protein molecule for human marrow stromal cell and sequence encoding same and use thereof - Google Patents
Mitochondria traffic protein molecule for human marrow stromal cell and sequence encoding same and use thereof Download PDFInfo
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- CN1631898A CN1631898A CN 200310122654 CN200310122654A CN1631898A CN 1631898 A CN1631898 A CN 1631898A CN 200310122654 CN200310122654 CN 200310122654 CN 200310122654 A CN200310122654 A CN 200310122654A CN 1631898 A CN1631898 A CN 1631898A
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Abstract
The invention relates to a source of human stroma cell of bone marrow, which is Bone mearrow stromal cell-derived mitochondria carrier protein(HuBMSC-MCP). The invention supplies the method for loding polynucleotide of the protein element and generating the protein element by recombinant technology; besides, the application to code polynucleotide of the protein element is disclosed; it has proven that endocytosis of dendritic cell can be strengthened by it.
Description
Technical field
The invention belongs to biology field, specifically, the present invention relates to the polynucleotide of new coding human mitochondrion translocator molecule HuBMSC-MCP (human bone marrow stromal cell-derived mitochondriacarrier protein) polypeptide, 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 mitochondrial transport protein molecular.
Background technology
Marrow stromal cell is as the main component of hematopoieticmicroenviron-ment, to the differentiation of hemopoietic system with keep most important.Except with the direct effect of hemopoietic stem cell, they can also be by the differentiation of a large amount of cytokine modulating hemopoietic stem cell of secretion.To a certain extent, some leukemic generation and bone marrow microenvironment is relevant unusually.Marrow stromal cell also finds to have the various biological function at present except participating in the regulation and control to hematopoiesis, such as, by the secretion chemokine, somatomedin, cytokine modulating immune development and steady certainly; Marrow stromal cell has kept many differentiation potentials as mesenchymal cell, can be induced to differentiate into scleroblast, adipocyte, muscle cell, neuronal cell and myelin cell etc.; And under certain extraneous factor is induced, be divided into antigen presenting cell, stimulate the T cell activation.Therefore the biological function of studying the bone marrow substrate cell source associated molecule helps to inquire into leukemic pathogenesis, cytodifferentiation, tissue formation, immunoregulation and injury repairing, is the focus of current immunology research.
Inlaying many relevantly with its metabolic substd transhipment function associated on the mitochondrial inner membrane, the protein of structural similitude is collectively referred to as mitochondrial transport superfamily protein (mitochondrial carrier protein).The constructional feature of this superfamily molecule all contains three repeat regions for (1), and each zone comprises the amino acid about 100; (2) each zone comprises a sequence motifs (PX (D/E) Xh (K/R) X (R/K) X
20-30) (D/E) GX
4) a (K/R) GRG, wherein h represents hydrophobic amino acid, and a represents die aromatischen Aminosaeuren; (3) hydrophobicity analysis shows that each zone contains two transbilayer helixs, has a hydrophilic area to link to each other therebetween; (4) molecular weight is between 28-32kDa.This superfamily known member comprise: the albumen of ADP/ATP translocator, uncoupling protein, phosphate cotransporter albumen, ketoisocaproic/oxysuccinic acid translocator, citrate transporter albumen, dicarboxylic acid translocator, carnitine transporter, guanylic acid translocator and some unknown function, as MRS3 and the MRS4 albumen that from yeast saccharomyces cerevisiae, extracts, the sick albumen of Grave etc.
Specific immune response is by the antigen presenting cell capture antigen, bring out generation after through processing, after handling antigenic information being passed to T, bone-marrow-derived lymphocyte, therefore, antigenic processing treatment is the primary link of organism immune response with offering, and is directly connected to inducing of immuno-stimulating or immunological tolerance.
(dendritic cell is the strongest sole duty antigen presenting cell of function in the present known body DC) to dendritic cell, and it is mainly by two kinds of approach capture antigens: the huge endocytosis that gulps down drink and mannose receptor mediation of formation type.Effectively endocytosis is the key link of DC performance function.
Therefore, find that new mitochondrial transport protein molecular has important value for the mechanism of action of understanding immune cell etc.The mitochondrial transport protein molecular is the effect of performance important regulating and controlling in the generation of tumour, process of growth, and may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as antitumor, anti-inflammatory response, anti-infective and immunologic function adjusting and the immunotherapy.Therefore, this area presses for the mitochondrial transport protein molecular of the new human bone marrow substrate cell source of exploitation.
Summary of the invention
The mitochondrial transport albumen that the purpose of this invention is to provide a kind of new human bone marrow substrate cell source with and fragment, analogue and derivative.
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.
In a first aspect of the present invention, novel isolated HuBMSC-MCP polypeptide is provided, this polypeptide is a human bone marrow substrate cell source, and 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 selected from down group: the polypeptide that (a) has SEQ ID NO:2 aminoacid sequence; (b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the function that strengthens the dendritic cell endocytosis by (a) polypeptides derived.More preferably, described polypeptide has the aminoacid sequence shown in the SEQ ID NO:2.
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 80% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people HuBMSC-MCP 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 192-1154 position among the SEQ ID NO:1; (b) has the sequence of 1-1433 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, preparation people HuBMSC-MCP is provided proteic method, this method comprises:
(a) under expression condition, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate people HuBMSC-MCP albumen.
In a fifth aspect of the present invention, provide and above-mentioned people HuBMSC-MCP polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 15-1433 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people HuBMSC-MCP polypeptide active is provided, and the compound that suppresses people HuBMSC-MCP 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 HuBMSC-MCP polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of HuBMSC-MCP in the test sample, it comprises: sample is contacted with the proteic specific antibody of HuBMSC-MCP, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample HuBMSC-MCP albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people HuBMSC-MCP polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: detect the described HuBMSC-MCP expression of polypeptides amount of coding in the cell sample.
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 HuBMSC-MCP polypeptide active, and perhaps screening suppresses the antagonist of people HuBMSC-MCP polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people HuBMSC-MCP 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 composition is provided, it contains people HuBMSC-MCP polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These compositions can be used for promoting the endocytosis of dendritic cell, aspect the illnesss such as treatment tumour, inflammation, neural system and cardiovascular diseases application prospect are being arranged.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
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 HuBMSC-MCP of the present invention.
Fig. 2 is the full length amino acid sequence of people HuBMSC-MCP of the present invention.
Fig. 3 is people HuBMSC-MCP of the present invention and the proteic amino acid sequence homology comparison diagram of other mitochondrial transports.The top sequence is people HuBMSC-MCP, and the below sequence is other mitochondrial transport albumen.Identical amino acid marks with black matrix between a plurality of sequences, and similar amino acid marks with grey body.
Fig. 4 is a people HuBMSC-MCP RT-PCR expression analysis of the present invention, and prompting HuBMSC-MCP is expressed in some tumour cell.
Fig. 5 is the interior location of the cell of people HuBMSC-MCP of the present invention, and prompting HuBMSC-MCP is distributed in the human mitochondrion.
After Fig. 6 shows that people HuBMSC-MCP of the present invention crosses expression, significantly strengthen the endocytosis activity of DC.
Embodiment
The inventor is through extensive and deep research, separated first bone marrow substrate cell source novel mitochondrial transport protein molecular (Bone marrow stromal cell-derived mitochondria carrier protein, HuBMSC-MCP).RT-PCR analysis revealed HuBMSC-MCP has expression in some tumour cell, this shows that HuBMSC-MCP is keeping the differentiation of some tumour cell.
In addition, human mitochondrion translocator molecule HuBMSC-MCP of the present invention and known family member have high homology, have mitochondrial transport superfamily protein member's entire infrastructure feature.This hint HuBMSC-MCP is the same with other family members to transport the substrate that a kind of and plastosome oxidative phosphorylation are correlated with, for the vital movement of cell provides energy.Laser Scanning Confocal Microscope is observed and is shown that HuBMSC-MCP and plastosome are positioned in the pseudopodium of MCF-7-MCF-7 altogether.And the formation of pseudopodium to be tumour cell pass through under the vascular endothelial cell that basilar membrane forms the notable feature that shifts, therefore, human mitochondrion translocator molecule HuBMSC-MCP of the present invention may shift relevantly with invasion by tumor cells, and being the formation of tumour cell pseudopodium and moving provides energy.Simultaneously, human mitochondrion translocator molecule HuBMSC-MCP of the present invention can significantly strengthen the endocytosis of DC.
Therefore, HuBMSC-MCP albumen or its relevant antagonist, agonist etc. can be diseases such as treating tumour provides new immunodiagnosis and targeted therapy approach, thereby has great application prospect.
In the present invention, term " HuBMSC-MCP albumen ", " HuBMSC-MCP polypeptide " or " mitochondrial transport protein molecular HuBMSC-MCP " are used interchangeably, and all refer to have the albumen or the polypeptide of human mitochondrion translocator molecule HuBMSC-MCP aminoacid sequence (SEQ ID NO:2).
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating HuBMSC-MCP albumen or polypeptide " is meant that the HuBMSC-MCP polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying HuBMSC-MCP albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of people HuBMSC-MCP, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human HuBMSC-MCP albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " people HuBMSC-MCP polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of people HuBMSC-MCP protein-active.This term also comprises having and variant form people HuBMSC-MCP albumen identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or one or more (being generally in 20 of N-terminal interpolation, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or more amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people HuBMSC-MCP and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people HuBMSC-MCP DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people HuBMSC-MCP polypeptide to obtain.The present invention also provides other polypeptide, as comprises people HuBMSC-MCP polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people HuBMSC-MCP polypeptide.Usually, this fragment have people HuBMSC-MCP peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of people HuBMSC-MCP albumen or polypeptide.The difference of these analogues and natural human HuBMSC-MCP polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " people HuBMSC-MCP albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | ?Val |
| Arg(R) | Lys；Gln；Asn | ?Lys |
| Asn(N) | Gln；His；Lys；Arg | ?Gln |
| Asp(D) | Glu | ?Glu |
| Cys(C) | Ser | ?Ser |
| Gln(Q) | Asn | ?Asn |
| Glu(E) | Asp | ?Asp |
| Gly(G) | Pro；Ala | ?Ala |
| His(H) | Asn；Gln；Lys；Arg | ?Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | ?Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | ?Ile |
| Lys(K) | Arg；Gln；Asn | ?Arg |
| Met(M) | Leu；Phe；Ile | ?Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | ?Leu |
| Pro(P) | Ala | ?Ala |
| Ser(S) | Thr | ?Thr |
| Thr(T) | Ser | ?Ser |
| Trp(W) | Tyr；Phe | ?Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | ?Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | ?Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1% SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding HuBMSC-MCP.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People HuBMSC-MCP 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.Science1985 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 HuBMSC-MCP 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 HuBMSC-MCP polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people HuBMSC-MCP polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people HuBMSC-MCP polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J BioChem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people HuBMSC-MCP DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, aLaboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the 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 HuBMSC-MCP 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 HuBMSC-MCP protein function as pharmacological agent HuBMSC-MCP protein function.The peptide molecule that can suppress or stimulate people HuBMSC-MCP protein function that can be used for seeking therapeutic value with the recombinant human HuBMSC-MCP protein screening peptide library of expressing.
On the other hand, the present invention also comprises people HuBMSC-MCP 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 HuBMSC-MCP gene product or fragment.Preferably, refer to that those can combine with people HuBMSC-MCP 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 HuBMSC-MCP, comprise that also those do not influence the antibody of people HuBMSC-MCP protein function.The present invention also comprise those can with modify or without the people HuBMSC-MCP 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; 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 HuBMSC-MCP gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human HuBMSC-MCP 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 HuBMSC-MCP protein function and the antibody that does not influence people HuBMSC-MCP protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people HuBMSC-MCP 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 HuBMSC-MCP 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 HuBMSC-MCP can be used in the immunohistochemistry technology, detects the people HuBMSC-MCP albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people HuBMSC-MCP, inject in the body and can follow the tracks of its position and distribution.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people HuBMSC-MCP albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people HuBMSC-MCP 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 HuBMSC-MCP 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, and the exchange by disulfide linkage is incorporated into toxin on the antibody.
The production of polyclonal antibody can choose HuBMSC-MCP 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 HuBMSC-MCP albumen interactional material takes place, as part, 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 or its agonist and antagonist can be directly used in disease treatment, for example are used for the treatment of tumour aspect.In use, also can use the other treatment agent simultaneously.
The present invention also provides a kind of pharmaceutical composition, and it contains HuBMSC-MCP 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 HuBMSC-MCP 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 HuBMSC-MCP 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 HuBMSC-MCP of the proteic nothing expression of HuBMSC-MCP or unusual/non-activity.The HuBMSC-MCP albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic HuBMSC-MCP protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the HuBMSC-MCP transgenosis to cell.The method that structure carries the recombinant viral vector of HuBMSC-MCP gene is found in existing document (Sambrook, et al.).Recombinant human HuBMSC-MCP 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 HuBMSC-MCPmRNA 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 HuBMSC-MCP obtains.During screening, must carry out mark to people HuBMSC-MCP protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people HuBMSC-MCP protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people HuBMSC-MCP protein level that is detected in the test can be with laying down a definition the importance of people HuBMSC-MCP albumen in various diseases and be used to the disease of diagnosing HuBMSC-MCP albumen to work.
Whether having the proteic method of HuBMSC-MCP in a kind of detection test sample is to utilize the proteic specific antibody of HuBMSC-MCP to detect, and it comprises: sample is contacted with the HuBMSC-MCP protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample HuBMSC-MCP albumen.
The proteic polynucleotide of HuBMSC-MCP can be used for the diagnosis and the treatment of HuBMSC-MCP protein related diseases.Aspect diagnosis, the proteic polynucleotide of HuBMSC-MCP can be used for detecting the proteic expression of HuBMSC-MCP HuBMSC-MCP abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of HuBMSC-MCP as the HuBMSC-MCPDNA 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 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 HuBMSC-MCP albumen and also can detect the proteic transcription product of HuBMSC-MCP.
The sudden change that detects the HuBMSC-MCP gene also can be used for the disease of diagnosing HuBMSC-MCP albumen relevant.The form of HuBMSC-MCP protein mutation comprises that the point mutation compared with normal wild type HuBMSC-MCP 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 HuBMSC-MCP 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 polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human bone marrow substrate cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1433 bases, and its open reading frame is positioned at the 192-1154 position, and the coding total length is 321 amino acid whose people HuBMSC-MCP albumen (SEQ ID NO:2).This HuBMSC-MCP albumen belongs to mitochondrial transport protein family molecule, has certain homology with ADP/ATP translocator, uncoupling protein, phosphate cotransporter albumen, ketoisocaproic/aminoacid sequences such as oxysuccinic acid translocator, consistence can be up to more than 25% in plastosome translocator structural domain, and similarity then can reach 50%.RT-PCR analysis revealed HuBMSC-MCP expresses in kinds of tumor cells system.Therefore, HuBMSC-MCP albumen or its relevant antagonist, agonist etc. can be diseases such as treatment tumour, inflammation, neural system and cardiovascular diseases new immunodiagnosis and targeted therapy approach are provided, 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 HuBMSC-MCP cDNA
Extract the total RNA of human bone marrow substrate cell with Trizol reagent (Life Technologies company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (available from Gibco) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform DH5 α bacterium and form the cDNA plasmid library.Measure the sequence of random choose clone's 5 ' end 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 (as SEQ ID NO:1 and shown in Figure 1), the new protein (as SEQ IDNO:2 and shown in Figure 2) of encoding.This protein is named as human bone marrow substrate cell source mitochondrial transport protein molecular HuBMSC-MCP, its encoding gene called after human bone marrow substrate cell source mitochondrial transport protein molecular HuBMSC-MCP gene.
Sequence SEQ ID NO:1 total length is 1433bp, comprises 5 ' the end non-coding region of 191bp and 3 ' the end non-coding region of 276bp, and coding contains 321 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 35.4kD.
They are different with known for the BLAST analysis revealed, has certain homology with the aminoacid sequence of human mitochondrion translocator, consistence reaches 28%, and similarity reaches 50% (Fig. 3), belongs to mitochondrial transport albumen (mitochondrial carrier protein) family molecule.
Embodiment 2: carry out the cell expressing analysis of people HuBMSC-MCP with the RT-PCR method
Be in logarithmic phase CCL188 LoVo, prostate cancer cell line PC-3, cervical cancer tumer line HeLa, ovarian cancer cell line CaoV3, the isocellular cell total rna of breast cancer cell line MCF-7 with the extraction of Trizol reagent.Get 5 μ g cell total rnas and 1 μ g Oligo-dT
12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing
2O dilutes.The used primer of pcr amplification HuBMSC-MCP is as follows: adopted primer 5 '-GAG ACC AAC ATC CGT GAC-3 ' (SEQ ID NO:3) is arranged, antisense primer 5 ' CCTCGTCCTT ATGACTTC-3 ' (SEQ ID NO:4), simultaneously with beta-actin as positive control.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (Takara), the amplification parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, capable 1.5% agarose gel electrophoresis of PCR product is tentatively confirmed after 28 circulations.The dna sequence analysis result shows that the dna sequence dna of this PCR product and the 386-971 shown in the SEQ ID NO:1 are identical.
The result shows that HuBMSC-MCP mRNA has expression in most tumors cells such as MCF-7 breast cancer cell line, HeLa cervical cancer tumer line, do not see Table to reach (Fig. 4) in LoVo clone.
Embodiment 3: people HuBMSC-MCP is recombinant expressed
In this embodiment, Trizol reagent (Life Technologies company) extracts the total RNA of human bone marrow substrate cell as template, through after the reverse transcription, 5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people HuBMSC-MCP DNA as inserting fragment.
5 ' the end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-GCG?GTA?CCG?ACT?GAG?TAC?GGT?CTT?CTA?A-3’(SEQ?ID?NO：5)
This primer contains the restriction enzyme site of KpnI restriction enzyme, is the part encoding sequence of people HuBMSC-MCP after this restriction enzyme site;
3 ' end primer sequence is:
5’-CGG?GAT?CCC?ATG?GCG?ACG?GGC?GGC?CAG?C-3’(SEQ?ID?NO：6)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people HuBMSC-MCP of BamHI restriction enzyme.
With the PCR product purification that obtains after KpnI and BamHI enzyme cut and recombinate according to a conventional method with plasmid pRsetB (Invitrogen company) 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).Plasmid transformation escherichia coli BL21 with the people HuBMSC-MCPcDNA of correct sequence.Positive colony is cut evaluation with KpnI and BamHI enzyme, the capable 1.0% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted designed HuBMSC-MCP encoding sequence.
Choosing the positive BL21 clone who expresses HuBMSC-MCP is inoculated in the 100mlLB substratum, 37 ℃ of 300rpm shaking culture 12-15hr, the LB substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 1M IPTG to 1mM 37 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na
2HPO
4, 1.8mM KH
2PO
4PH7.3) resuspended, add 20%Triton X-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 the 2mlHis-Sepharose chromatography column, behind 1 * PBS thorough washing, adding 500ul elution buffer room temperature left standstill after 30 minutes collects elutriant, repeat wash-out 2-3 time, obtain people HuBMSC-MCP albumen.Molecular weight conforms to predictor.
Embodiment 4: anti-people HuBMSC-MCP production of antibodies
The recombinant protein people HuBMSC-MCP that obtains among the embodiment 3 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 HuBMSC-MCP gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
The structure of embodiment 5:HuBMSC-MCP eucaryon fusion expression vector
In this embodiment, Trizol reagent (Life Technologies company) extracts the total RNA of human bone marrow substrate cell as template, through after the reverse transcription, 5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people HuBMSC-MCP DNA as inserting fragment.The PCR reaction parameter be 95 ℃ 15 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds, 20 circulations were extended 10 minutes for back 72 ℃,
Total length HuBMSC-MCP upstream primer is 5 '-GCG GTA CCG ACT GAG TAC GGT CTT CTA A-3 ' (SEQ ID NO:5), downstream primer is 5 '-CGG GAT CCC ATG GCG ACG GGC GGC CAG C-3 ' (SEQ ID NO:6), product coding total length HuBMSC-MCP albumen.
The PCR product purification and is recombinated according to a conventional method with pEGFP-N1 carrier (Clontech company) and is converted into competence bacterium DH5 α after KpnI and BamHI enzyme are cut.Picking white clone identifies, also order-checking of purifying.Confirm through order-checking, inserted designed HuBMSC-MCP encoding sequence.
Localized immunofluorescence analysis in the embodiment 6:HuBMSC-MCP albuminous cell
Utilize LipofectAMINE reagent (Invitrogen) to carry out the gene transfection of eukaryotic cell 293T with HuBMSC-MCP total length fusion expression vector plasmid DNA constructed among the embodiment 5.The transient transfection cell after transfection 48 hours with trysinization, be laid on the sterility cover slide, under Laser Scanning Confocal Microscope, observe.Organoid is detection and localization altogether, treat cell attachment after, dye with plastosome, endoplasmic reticulum and Golgi's organs, lysosome specificity dyestuff respectively earlier, concentration is 1 μ M, 37 ℃, 15 minutes.After washing with PBS, observe under the Laser Scanning Confocal Microscope.
The result shows: the fluorescent signal of HuBMSC-MCP transfectional cell all is dispersed in and is distributed in the endochylema; Organoid is the positioning experiment results suggest altogether, and HuBMSC-MCP is distributed in the plastosome (Fig. 5).
Embodiment 7: people HuBMSC-MCP significantly strengthens the endocytosis activity of DC
Collect 5 days dendritic cell of vitro culture, wash twice with the RPMI1640 of serum-free, and accent cell concn to 1 * 10
7Cells/ml.Get 300 μ l cell suspensions and mix, add in the electric shock cup of 0.2-cm, with BTX ECM-830 electroporation apparatus, 505V, 99 μ s, 2 electricimpulses, midfeather 10sec with the HuBMSC-MCP mRNA of 20 μ g in-vitro transcription.24h behind the electroporation gets 5 * 10
5DC and 0.1mg/ml FITC-dextran are hatched 60min altogether at 4 ℃ or 37 ℃ respectively.After ice PBS washes, flow cytometry analysis.
The result shows, behind the people HuBMSC-MCP transfection DC, significantly strengthens the endocytosis activity (A of Fig. 6 and B are respectively the DC after contrast and the transfection) of DC.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Immunology Inst., No.2 Military Medical Univ.
＜120〉the mitochondrial transport protein molecular of human bone marrow substrate cell source and encoding sequence and purposes
<130>037861
<160>6
<170>PatentIn?version?3.1
<210>1
<211>1433
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
<221>CDS
<222>(192)..(1154)
<223>
<400>1
ggcgccggcg?gccacgccgc?ggaaggcgcg?ggccgagcag?agccgggcgt?tggagcccgc???60
gcgcgcatgg?aggcgttgcc?ggcagccctc?tgagggcagc?ggggagacaa?gacccggcga??120
cctcgcgcat?ccctcgagcc?gccacgcgct?ctcgccaccg?ggcggcgacg?ggccgcggag??180
ccggcgcggc?c?atg?gcg?acg?ggc?ggc?cag?cag?aag?gag?aac?acg?ctg?ctt???230
Met?Ala?Thr?Gly?Gly?Gln?Gln?Lys?Glu?Asn?Thr?Leu?Leu
1???????????????5???????????????????10
cac?ctc?ttc?gcc?ggc?ggg?tgt?gga?ggc?aca?gtt?ggt?gct?att?ttc?act????278
His?Leu?Phe?Ala?Gly?Gly?Cys?Gly?Gly?Thr?Val?Gly?Ala?Ile?Phe?Thr
15??????????????????20??????????????????25
tgt?cca?cta?gaa?gtc?att?aag?aca?cgg?ttg?cag?tct?tca?aga?tta?gct????326
Cys?Pro?Leu?Glu?Val?Ile?Lys?Thr?Arg?Leu?Gln?Ser?Ser?Arg?Leu?Ala
30??????????????????35??????????????????40??????????????????45
ctc?cgg?aca?gtc?tac?tat?cct?cag?gtt?cat?ctg?ggg?acc?att?agt?gga????374
Leu?Arg?Thr?Val?Tyr?Tyr?Pro?Gln?Val?His?Leu?Gly?Thr?Ile?Ser?Gly
50??????????????????55??????????????????60
gct?gga?atg?gtg?aga?cca?aca?tcc?gtg?aca?cct?gga?ctc?ttt?cag?gtt????422
Ala?Gly?Met?Val?Arg?Pro?Thr?Ser?Val?Thr?Pro?Gly?Leu?Phe?Gln?Val
65??????????????????70??????????????????75
ctg?aag?tcg?atc?ttg?gag?aaa?gag?gga?cca?aag?tca?ctt?ttt?aga?ggc????470
Leu?Lys?Ser?Ile?Leu?Glu?Lys?Glu?Gly?Pro?Lys?Ser?Leu?Phe?Arg?Gly
80??????????????????85??????????????????90
ttg?ggt?cca?aat?ttg?gtt?gga?gtt?gca?cca?tca?agg?gct?gta?tac?ttt????518
Leu?Gly?Pro?Asn?Leu?Val?Gly?Val?Ala?Pro?Ser?Arg?Ala?Val?Tyr?Phe
95??????????????????100?????????????????105
gca?tgt?tac?tcc?aaa?gcc?aaa?gag?caa?ttt?aat?ggc?att?ttc?gtg?cct????566
Ala?Cys?Tyr?Ser?Lys?Ala?Lys?Glu?Gln?Phe?Asn?Gly?Ile?Phe?Val?Pro
110?????????????????115?????????????????120?????????????????125
aac?agc?aat?att?gtg?cat?att?ttc?tca?gct?ggc?tct?gca?gct?ttt?atc????614
Asn?Ser?Asn?Ile?Val?His?Ile?Phe?Ser?Ala?Gly?Ser?Ala?Ala?Phe?Ile
130?????????????????135?????????????????140
aca?aat?tcc?tta?atg?aat?cct?ata?tgg?atg?gtt?aaa?acc?cga?atg?cag????662
Thr?Asn?Ser?Leu?Met?Asn?Pro?Ile?Trp?Met?Val?Lys?Thr?Arg?Met?Gln
145?????????????????150?????????????????155
cta?gaa?cag?aaa?gtg?agg?ggc?tct?aag?cag?atg?aat?aca?ctc?cag?tgt????710
Leu?Glu?Gln?Lys?Val?Arg?Gly?Ser?Lys?Gln?Met?Asn?Thr?Leu?Gln?Cys
160?????????????????165?????????????????170
gct?cgt?tac?gtt?tac?cag?acc?gaa?ggc?att?cgt?ggc?ttc?tat?aga?gga????758
Ala?Arg?Tyr?Val?Tyr?Gln?Thr?Glu?Gly?Ile?Arg?Gly?Phe?Tyr?Arg?Gly
175?????????????????180?????????????????185
tta?act?gcc?tcg?tat?gct?gga?att?tcc?gaa?act?ata?atc?tgc?ttt?gct????806
Leu?Thr?Ala?Ser?Tyr?Ala?Gly?Ile?Ser?Glu?Thr?Ile?Ile?Cys?Phe?Ala
190?????????????????195?????????????????200?????????????????205
att?tat?gaa?agt?tta?aag?aag?tat?ctg?aaa?gaa?gct?cca?tta?gcc?tct????854
Ile?Tyr?Glu?Ser?Leu?Lys?Lys?Tyr?Leu?Lys?Glu?Ala?Pro?Leu?Ala?Ser
210?????????????????215?????????????????220
tct?gca?aat?ggg?act?gag?aaa?aat?tcc?aca?agt?ttt?ttt?gga?ctt?atg????902
Ser?Ala?Asn?Gly?Thr?Glu?Lys?Asn?Ser?Thr?Ser?Phe?Phe?Gly?Leu?Met
225?????????????????230?????????????????235
gca?gct?gct?gct?ctt?tct?aag?ggc?tgt?gcc?tcc?tgc?att?gct?tat?cca????950
Ala?Ala?Ala?Ala?Leu?Ser?Lys?Gly?Cys?Ala?Ser?Cys?Ile?Ala?Tyr?Pro
240?????????????????245?????????????????250
cac?gaa?gtc?ata?agg?acg?agg?ctc?cgg?gaa?gag?ggc?acc?aag?tac?aag????998
His?Glu?Val?Ile?Arg?Thr?Arg?Leu?Arg?Glu?Glu?Gly?Thr?Lys?Tyr?Lys
255?????????????????260?????????????????265
tct?ttt?gtc?cag?acg?gcg?cgc?ctg?gtg?ttc?cgg?gaa?gaa?ggc?tac?ctt????1046
Ser?Phe?Val?Gln?Thr?Ala?Arg?Leu?Val?Phe?Arg?Glu?Glu?Gly?Tyr?Leu
270?????????????????275?????????????????280?????????????????285
gcc?ttt?tat?aga?gga?ctg?ttt?gcc?cag?ctt?atc?cgg?cag?atc?cca?aat????1094
Ala?Phe?Tyr?Arg?Gly?Leu?Phe?Ala?Gln?Leu?Ile?Arg?Gln?Ile?Pro?Asn
290?????????????????295?????????????????300
act?gcc?att?gtg?ttg?tct?act?tat?gag?tta?att?gtg?tac?ctg?tta?gaa????1142
Thr?Ala?Ile?Val?Leu?Ser?Thr?Tyr?Glu?Leu?Ile?Val?Tyr?Leu?Leu?Glu
305?????????????????310?????????????????315
gac?cgt?act?cag?taacaggccg?gaaaattgtg?ctctagaaga?ataaaactga????????1194
Asp?Arg?Thr?Gln
320
aaaactctag?agaatttttt?ttccccattg?atgtttagaa?agtttgagac?tgaaacagga??1254
aaggccataa?aatatctggt?tcatatcacc?tgttggacat?ttccttttgg?attcatgctt??1314
tctggaaggt?ttaaattcat?taacgttaat?agttaattat?aacttttttt?ttaacttaag??1374
aggattcagg?gttaagcccc?aactaaatta?aatcatgcta?tttaatttaa?gtataaaaa???1433
<210>2
<211>321
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met?Ala?Thr?Gly?Gly?Gln?Gln?Lys?Glu?Asn?Thr?Leu?Leu?His?Leu?Phe
1???????????????5???????????????????10??????????????????15
Ala?Gly?Gly?Cys?Gly?Gly?Thr?Val?Gly?Ala?Ile?Phe?Thr?Cys?Pro?Leu
20??????????????????25??????????????????30
Glu?Val?Ile?Lys?Thr?Arg?Leu?Gln?Ser?Ser?Arg?Leu?Ala?Leu?Arg?Thr
35??????????????????40??????????????????45
Val?Tyr?Tyr?Pro?Gln?Val?His?Leu?Gly?Thr?Ile?Ser?Gly?Ala?Gly?Met
50??????????????????55??????????????????60
Val?Arg?Pro?Thr?Ser?Val?Thr?Pro?Gly?Leu?Phe?Gln?Val?Leu?Lys?Ser
65??????????????????70??????????????????75??????????????????80
Ile?Leu?Glu?Lys?Glu?Gly?Pro?Lys?Ser?Leu?Phe?Arg?Gly?Leu?Gly?Pro
85??????????????????90??????????????????95
Asn?Leu?Val?Gly?Val?Ala?Pro?Ser?Arg?Ala?Val?Tyr?Phe?Ala?Cys?Tyr
100?????????????????105?????????????????110
Ser?Lys?Ala?Lys?Glu?Gln?Phe?Asn?Gly?Ile?Phe?Val?Pro?Asn?Ser?Asn
115?????????????????120?????????????????125
Ile?Val?His?Ile?Phe?Ser?Ala?Gly?Ser?Ala?Ala?Phe?Ile?Thr?Asn?Ser
130?????????????????135?????????????????140
Leu?Met?Asn?Pro?Ile?Trp?Met?Val?Lys?Thr?Arg?Met?Gln?Leu?Glu?Gln
145?????????????????150?????????????????155?????????????????160
Lys?Val?Arg?Gly?Ser?Lys?Gln?Met?Asn?Thr?Leu?Gln?Cys?Ala?Arg?Tyr
165?????????????????170?????????????????175
Val?Tyr?Gln?Thr?Glu?Gly?Ile?Arg?Gly?Phe?Tyr?Arg?Gly?Leu?Thr?Ala
180?????????????????185?????????????????190
Ser?Tyr?Ala?Gly?Ile?Ser?Glu?Thr?Ile?Ile?Cys?Phe?Ala?Ile?Tyr?Glu
195?????????????????200?????????????????205
Ser?Leu?Lys?Lys?Tyr?Leu?Lys?Glu?Ala?Pro?Leu?Ala?Ser?Ser?Ala?Asn
210?????????????????215?????????????????220
Gly?Thr?Glu?Lys?Asn?Ser?Thr?Ser?Phe?Phe?Gly?Leu?Met?Ala?Ala?Ala
225?????????????????230?????????????????235?????????????????240
Ala?Leu?Ser?Lys?Gly?Cys?Ala?Ser?Cys?Ile?Ala?Tyr?Pro?His?Glu?Val
245?????????????????250?????????????????255
Ile?Arg?Thr?Arg?Leu?Arg?Glu?Glu?Gly?Thr?Lys?Tyr?Lys?Ser?Phe?Val
260?????????????????265?????????????????270
Gln?Thr?Ala?Arg?Leu?Val?Phe?Arg?Glu?Glu?Gly?Tyr?Leu?Ala?Phe?Tyr
275?????????????????280?????????????????285
Arg?Gly?Leu?Phe?Ala?Gln?Leu?Ile?Arg?Gln?Ile?Pro?Asn?Thr?Ala?Ile
290?????????????????295?????????????????300
Val?Leu?Ser?Thr?Tyr?Glu?Leu?Ile?Val?Tyr?Leu?Leu?Glu?Asp?Arg?Thr
305?????????????????310?????????????????315?????????????????320
Gln
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(18)
＜223〉primer
<400>3
gagaccaaca?tccgtgac???????????????????????????????????????????????????18
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(18)
＜223〉primer
<400>4
cctcgtcctt?atgacttc???????????????????????????????????????????????????18
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer
<400>5
gcggtaccga?ctgagtacgg?tcttctaa????????????????????????????????????????28
<210>6
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(28)
＜223〉primer
<400>6
cgggatccca?tggcgacggg?cggccagc????????????????????????????????????????28
Claims (10)
1. an isolating people HuBMSC-MCP 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 selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the function that strengthens the dendritic cell endocytosis by (a) polypeptides derived.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.
5. 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 192-1154 position among the SEQ ID NO:1;
(b) has the sequence of 1-1433 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. proteic preparation method of people HuBMSC-MCP is characterized in that this method comprises:
(a) under expression condition, cultivate the described host cell of claim 7;
(b) from culture, isolate people HuBMSC-MCP albumen.
9. energy and the described people HuBMSC-MCP of claim 1 polypeptid specificity bonded antibody.
10. a composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101226543A CN100443501C (en) | 2003-12-24 | 2003-12-24 | Mitochondria traffic protein molecule for human marrow stromal cell and sequence encoding same and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101226543A CN100443501C (en) | 2003-12-24 | 2003-12-24 | Mitochondria traffic protein molecule for human marrow stromal cell and sequence encoding same and use thereof |
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| CN100443501C CN100443501C (en) | 2008-12-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109777807A (en) * | 2018-12-11 | 2019-05-21 | 福建农林大学 | A method of the localization and expression foreign protein in mitochondria |
-
2003
- 2003-12-24 CN CNB2003101226543A patent/CN100443501C/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109777807A (en) * | 2018-12-11 | 2019-05-21 | 福建农林大学 | A method of the localization and expression foreign protein in mitochondria |
| CN109777807B (en) * | 2018-12-11 | 2020-09-15 | 福建农林大学 | Method for positioning and expressing foreign protein in mitochondria |
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| CN100443501C (en) | 2008-12-17 |
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