CN1373220A - Coding sequence of human tonB-dependent receptor protein and its preparing process and application - Google Patents

Abstract

A cDNA sequence of a novel human tonB-dependent receptor protein (hTDRP), the polypeptide coded by it, the process for preparing said polynucleotide sequence and said polypeptide, and their application are disclosed.

Description

Receptor protein encoding sequence, Preparation Method And The Use that human tonB relies on

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of the receptor protein that human novel tonB relies on and the polypeptide of this sequence encoding.The invention still further relates to the production method of described polynucleotide sequence and described polypeptide, and the purposes of these polynucleotide and described polypeptide.

Background technology

Material turnover cell must pass through cytolemma, and the special construction of cytolemma has determined the complexity difference of different substances by cell.Material has following several form by the transhipment of cytolemma:

(1) passive transport: comprise two kinds of forms of simple diffusion and facilitation diffusion, be mainly used in and transport small molecules.

(2) active transport comprises primary active transport and secondary active transport.Active transport is meant that the cell consumption energy is with the process of material by the side transhipment of the lower concentration one side direction high density of film.Modal ionic pump transhipment is the sodium pump (Na+-K+ pump) on the cytolemma.

(3) go out born of the same parents and endocytosis.Go out the process that born of the same parents are meant that some macromolecular substance or material agglomerate are discharged by cell, be mainly seen in the secretory activity of cell.Go into then some outer material agglomerate of phalangeal cell process of entering cell of born of the same parents.Because of being called receptor-mediated formula, the outer receptors bind of specific molecular and cytolemma and the endocytosis that causes at this place go into born of the same parents.

TonB is one of albumen of numerous participation material cross-cell membrane transhipments, and it relates to the need that rely on by acceptor can arrive extracellular process with matter transportation by transporting mode.The metabolism coupling connection of energy is provided in it and the cytoplasmic membrane, thereby provides energy (Braun V, Gunter K, Hantke K.1991, Biol Met for this transport process; 4 (1): 14-22).TonB is considered to have intensive to interact with cytoplasmic membrane and adventitia.In intestinal bacteria, tonB albumen and other adventitia acceptor interactions transport high-affinity and the specific substrate that needs energy toward film week space.These substrates or be difficult to, or concentration is very low by porous channel, under the condition of tonB disappearance, these materials and its receptors bind but do not transport.Studies show that colibacillary Tonb albumen and plasma membrane proton motive force coupling connection can activate the siderophore ion complex and adventitia (Higgs PI, Letain TE are passed through in the vitamin B12 transhipment, Merriam KK, Burke NS, Park H, Kang C, Postle K.2002, J Bacteriol; 184 (6): 1640-8).

The receptor protein that TonB relies on is extended familys.Studies show that the receptor protein that TonB relies on mainly participates in transporting cell grow necessary ion, especially iron ion.Having of discovered in recent years: btuB is used to transport the acceptor of cobalami; CirA is used to transport the acceptor of Colicine; FcuA, the acceptor of transhipment ferrichrome in Yersinia enterocolitica; FhuA is used to transport ferrichrome ionic acceptor; HemR transports the acceptor of protoferriheme in Yersinia enterocolitica; Or the like.Yet before the application, the newcomer of receptor protein family open or that delivered tonB dependence of the present invention also has no talent.

Summary of the invention

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of the receptor protein family that this polynucleotide encoding tonB relies on, the receptor protein that tonB of the present invention relies on be named as the receptor protein 1 that the novel tonB of people relies on (human tonB-dependent receptor proteins, hTDRP).

Another object of the present invention provides the receptor protein family member that a kind of new tonB relies on, and this albumen is named as hTDRP.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described hTDRP.

The present invention also provides the application of hTDRP gene order and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people hTDRP protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 166-378 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 166-378 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.More preferably, this sequence has among the SEQ ID NO.1 nucleotide sequence from the 166-378 position.

In another aspect of this invention, provide a kind of isolating hTDRP protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ IDNO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQID NO.2 polypeptide of sequence.

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

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

In another aspect of this invention, the method that provides a kind of generation to have the polypeptide of hTDRP protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hTDRP protein-active operationally is connected in expression regulation sequence, form the hTDRP protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 166-378 position among described nucleotide sequence and the SEQ ID NO.1;

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

(c) be fit to express under the condition of hTDRP protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hTDRP protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1969 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 166-378 position Nucleotide.

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

In the present invention, term " hTDRP albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with hTDRP protein-active is as 166-378 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 166-378 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 166-378 position nucleotide sequence homology be low to moderate about 90% the degenerate sequence described sequence of SEQID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 166-378 position.Also term also comprise with SEQ ID NO.1 in from the nucleotide sequence of the homology of nucleotide sequence at least 90% of Nucleotide 166-378 position.

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

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

In the present invention, term " hTDRP protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hTDRP protein-active.This term also comprises having and variant form people hTDRP identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of hhTDRP1 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of hTDRP DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hTDRP polypeptide to obtain.The present invention also provides other polypeptide, as comprises hTDRP polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of hTDRP polypeptide.Usually, this fragment have the hTDRP 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 hTDRP albumen or polypeptide.The difference of these analogues and natural hTDRP 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.

The present invention also comprises the antisense sequences of hTDRP polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hTDRP in the cell.

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

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

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

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

On the other hand, the present invention also comprises hTDRP 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 hTDRP gene product or fragment.Preferably, refer to that those can combine with hTDRP 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 hTDRP, comprise that also those do not influence the antibody of hTDRP protein function.The present invention also comprise those can with modify or without the hTDRP 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 hTDRP gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hTDRP or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1969; People such as Kohler, Eur.J.Immunol.6:292,1969; People such as Hammerling, In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the hTDRP function and the antibody that does not influence the hTDRP function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hTDRP 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 hTDRP gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People hTDRP nucleotide sequence 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about 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.Certainly, by first synthetic a plurality of small segments, and then connect and obtain the very long fragment of sequence.

In the present invention, the cDNA nucleotide sequence of hTDRP is so to obtain: with people's testis λ gt10cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-AGCCAGGCTGCCCGGTTT TAT-3 ' is a forward primer, oligonucleotide A2:5 '-CTTGGAATGGCAAATGTCCTG-3 ' is a reverse primer, carries out PCR.The PCR condition of A1/A2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 66 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product obtains the fragment that length is about 900 Nucleotide.

Find to have the type sequence of the receptor protein of tonB dependence in the protein sequence of hTDRP of the present invention by the homology retrieval, this points out hTDRP of the present invention to belong to the receptor protein family that tonB relies on, and has the correlation function of this protein family.

The proteic main effect of TonB is and other adventitia acceptor interactions, with need can specific substrate transport toward film week space.These substrates or very difficult by porous channel, or concentration is very low.Under the condition of tonB disappearance, these materials and its receptors bind but do not transport.The receptor protein that TonB relies on is extended familys.Studies show that the receptor protein that TonB relies on mainly participates in transporting cell grow necessary ion, especially iron ion.

As from the foregoing, albumen involved in the present invention may relate to human nutrition material, toxin or drug absorption, especially one of important albumen of iron ion absorption.Utilize hTDRP of the present invention, can diagnose and treat for TDRP disappearance or the undue disease that causes of expressing.Nucleotide coding sequence (seeing SEQ ID NO 1 for details) and the proteins encoded (seeing SEQ ID NO 2 for details) thereof of hTDRP of the present invention are made medicament or test kit, can be used for prevention, detect and even alleviate the ionic absorption undesirable condition; Encoding sequence by hTDRP of the present invention obtains it and instead makes the hTDRP protein antibodies with sequence or by aminoacid sequence, can absorb some ionic play retardation.

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hTDRP

1. primer amplification

Going into gt10cDNA library (available from Clontech company) with people's testis is template, with a pair of oligonucleotide is primer---A1:5 '-AGCCAGGC TGCCCGGTTT TAT-3 ' is a forward primer, oligonucleotide A2:5 '-CTTGGAATGGC AAATGTCCTG-3 ' is a reverse primer, carries out PCR.The PCR condition of A1/A2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 66 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product obtains the fragment that length is about 900 Nucleotide.

2.PCR the order-checking of product

Will be with above-mentioned amplified production and pGEM-T ^Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprepPlasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiThermEXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, obtains full length cDNA sequence at last, is total to 1969bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 166-378 position Nucleotide.Derive the aminoacid sequence of hTDRP according to the full length cDNA sequence that obtains, totally 70 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

Structural analysis

Full length cDNA sequence and proteins encoded thereof with hTDRP carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database.Found that the type sequence that has the receptor protein that tonB relies in the protein sequence of hTDRP, this points out hTDRP of the present invention to belong to the receptor protein family that tonB relies on, and has the correlation function of this protein family.

The conservative significant sequence of the strictness of the receptor protein family that TonB relies on is: [LYGSTANEQ]-x (3)-[GSTAENQ]-x-[PGE]-R-x-[LIVFYWA]-x-[LIVMFTA]-[STAGNQ]-[LIVMFYGTA]-x-[LIVMFYWGTADQ]-the x-F[notes: X is an arbitrary amino acid in this sequence, numerals such as " 3 " is an amino acid number, and " [GSTAENQ] " expression is optional amino acid from these 7 amino acid].In intestinal bacteria, tonB albumen and other adventitia acceptor interactions, with need can specific substrate transport toward film week space.These substrates or very difficult by porous channel, or concentration is very low.Under the condition of tonB disappearance, these materials and its receptors bind but do not transport.

When TonB albumen participates in matter transportation, need and special acceptor interaction, the receptor protein that TonB relies on is extended familys.Studies show that the receptor protein that TonB relies on mainly participates in transporting cell grow necessary ion, especially iron ion.Drug absorption also is identical approach with the poisoning use.Therefore, albumen involved in the present invention may relate to one of important albumen of human nutrition material, toxin or drug absorption.By to further investigation of the present invention, one finds the relation of it and many clinical cases surely, and final for addressing these problems developing new thinking and method.

People hTDRP of the present invention is used for further functional study except can be used as receptor protein family a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor hTDRP can also merge with other members of this family or exchange fragment, to produce new albumen.

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

For example, inventor hTDRP nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people hTDRP or the overexpression that suppresses people hTDRP.People hTDRP albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people hTDRP disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of hTDRP in intestinal bacteria

In this embodiment, use the PCR Oligonucleolide primers corresponding to 5 ' and 3 ' end of the cDNA sequence of coding hTDRP to increase, the cDNA that obtains hTDRP is as inserting fragment.

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

5′-TCAAGGATCCATGGA?GTCTCACACT?GTCA-3′

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is the nearly N-terminal partial nucleotide sequence of hTDRP encoding sequence;

3 ' end primer sequence is:

5’-AGTCGTCGACGCCCA?AATGCCACAC?ACAA-3

This primer contains the restriction enzyme site of SalI restriction enzyme, the partial nucleotide sequence of translation termination and hTDRP.

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

With BamHI and SalI digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, and the cDNA fragment of sequence verification hTDRP has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when reaching 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved hTDRP from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hTDRP from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 4

The expression of hTDRP in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, use PCR Oligonucleolide primers to increase, obtain hTDRP cDNA as inserting fragment corresponding to 5 ' and 3 ' end of the cDNA sequence of coding hTDRP.

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

5′-TCAAGTCGACATGGA?GTCTCACACT?GTCA-3′

This primer contains the restriction enzyme site of SalI restriction enzyme, is the nearly N-terminal partial nucleotide sequence of hTDRP encoding sequence after this restriction enzyme site;

3 ' end primer sequence is:

5’-AGTCGGATCCGCCCA?AATGCCACAC?ACAA-3’

This primer contains the partial nucleotide sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hTDRP.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3 (Invitrogen company), this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With BamHI and SalI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, and the cDNA fragment of sequence verification hTDRP has correctly been inserted carrier.

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

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, specific as follows.Recombinant protein 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 hTDRP gene translation product with it.

SEQUENCE LISTING＜110〉Fudan University＜120〉human tonB the receptor protein coded sequence, Preparation Method And The Use＜130 that rely on〉13＜160〉2＜170〉Patentln version 3.1＜210〉1＜211〉1015＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(166) .. (378)＜223〉＜400〉1ggagccaggc tgcccggttt tatagccctg cttgacctgt gtgatccgag aataatcttg 60tttaatctcc acaagtttga ggactaaact gggtgccaca gagaattaag taactggtcg 120aggttgtatc agtaagaagg gagcacggaa cagattcaag ctcag atg gag tct cac 177

Met?Glu?Ser?His

1act?gtc?acc?cgg?gct?gga?atg?cag?tgg?tgt?gat?ctt?ggc?tca?cct?ccc 225Thr?Val?Thr?Arg?Ala?Gly?Met?Gln?Trp?Cys?Asp?Leu?Gly?Ser?Pro?Pro5 10 15 20agg?ttc?aag?cga?ttc?tgc?tgc?ctc?tgc?ctc?ccg?agc?agc?tgg?gat?tcc 273Arg?Phe?Lys?Arg?Phe?Cys?Cys?Leu?Cys?Leu?Pro?Ser?Ser?Trp?Asp?Ser

25 30 35agg?gag?cct?ggt?tgg?act?tca?ggc?ccc?tgt?gag?gct?gac?tct?gta?gtc 321Arg?Glu?Pro?Gly?Trp?Thr?Ser?Gly?Pro?Cys?Glu?Ala?Asp?Ser?Val?Val

40 45 50ctg?gag?aaa?ctc?aac?agc?cag?gaa?ctt?agg?acc?att?tgt?gtg?tgg?cat 369Leu?Glu?Lys?Leu?Asn?Ser?Gln?Glu?Leu?Arg?Thr?Ile?Cys?Val?Trp?His

55 60 65ttg?ggc?taa?ccagactggt?ggcttctaaa?gaagatggaa?agcatcacgt 418Leu?Gly

70ttcctgtatt?taaattattt?ggtgatctca?atactggttg?ttagccaaag?tacacaatgg 478agacagccta?tttgctctac?tacttttgat?gtagaaatat?tactaagctt?gagctgatta 538atagaactgt?tacacatttc?tgtgctcccc?ttactggtgg?catgaaataa?ttcttttaaa 598acctgccatg?tgttcacatt?taaaactaaa?agtaatttag?attacatgga?tttgagaagc 658ccatgttgac?acgtaattgc?caacagatgg?gctttcaaat?gtaatgaagt?gaaaatggtc 718agtaccccat?gtgttggatt?ttgcaatttt?ccaaagtaag?ctgtcatttt?tggcatgtca 778tgctagttaa?gaacacaatg?tatttatagc?tcaattatac?attttctgat?gaatttggca 838tggcattttg?tttttccatc?agtcacttaa?gacaggacat?ttgccattcc?aagaatgaat 898taacactctt?ctaagtgatc?tataaactga?gatgtaaaat?aaattacaaa?ttgggactgt 958tttagttttt?caagcaaagg?gtgtttaaat?aaagcttgaa?atcataaacc?ttaaaaa 1015<210>2<211>70<212>PRT<213>Homo?sapiens<400>2Met?Glu?Ser?His?Thr?Val?Thr?Arg?Ala?Gly?Met?Gln?Trp?Cys?Asp?Leu1 5 10 15Gly?Ser?Pro?Pro?Arg?Phe?Lys?Arg?Phe?Cys?Cys?Leu?Cys?Leu?Pro?Ser

20 25 30Ser?Trp?Asp?Ser?Arg?Glu?Pro?Gly?Trp?Thr?Ser?Gly?Pro?Cys?Glu?Ala

35 40 45Asp?Ser?Val?Val?Leu?Glu?Lys?Leu?Asn?Ser?Gln?Glu?Leu?Arg?Thr?Ile

50 55 60Cys?Val?Trp?His?Leu?Gly65 70

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hTDRP protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 166-378 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 166-378 position.

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

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has the sequence of Nucleotide 166-378 position among the SEQ ID NO.1.

4. isolating hTDRP protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

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

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

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

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

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

(a) nucleotide sequence that coding is had a polypeptide of hTDRP protein-active operationally is connected in expression regulation sequence, form the hTDRP protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 166-378 position among described nucleotide sequence and the SEQ ID NO.1;

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

(c) be fit to express under the condition of hTDRP protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hTDRP protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 166-378 position among the SEQ ID NO.1.

12. energy and the described hTDRP protein polypeptide of claim 4 specificity bonded antibody.

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

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