CN1287173A - Human 3-phosphoserine transferase and its code sequence, preparation and application - Google Patents

Abstract

The present invention provides the cDNA sequence of human 3-phosphoserine transferase. The cDNA encoded protein is one homologue of 3-phosphoserine transferase in human body. The present invention also relates to the nucleotide sequence encoded polypeptide, the application of the polynucleotide and the polypeptide, and the production process of the polynucleotide and the polypeptide.

Description

People 3-phosphoserine transferring enzyme and encoding sequence thereof and method for making and purposes

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 people 3-phosphoserine transferring enzyme (abbreviating " people EPIP2 " as), this cDNA encoded protein is the homologue of 3-phosphoserine transferring enzyme in the people.The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

3-phosphoserine aminotransferase (EC 2.6.1.52) can single-minded catalysis phosphoserine and 2-oxoglutaric acid and 3-phosphohydroxypyruvic acid and L-glutamic acid between the reversible transamination.According to its sequence signature, 3-phosphoserine transferring enzyme belong to V class (Class V) transaminase family member ( Yeast1994,10:385-389).

1987, people such as Misrahi found that one induced by Progesterone in female rabbit uterus inner membrance of conceived 5 days, and be subjected to the new gene EPIP that antagonist RU486 suppresses fully (endometrial progesterone-inducedprotein, EPIP) ( Biochemistry1987,26 (13): 3975-3982).After, according to the sequence comparative result determine that it is mammiferous 3-phosphoserine aminotransferase ( Curr.Genet.1995,27:501-508).

In July, 1999, announced people's EPIP cDNA sequence among the GeneBank.

Research hints that the 3-phosphoserine aminotransferase is relevant with some diseases, and therefore, to research and develop people 3-phosphoserine aminotransferase significant for therapeutic purpose.

An object of the present invention is to provide a kind of new polynucleotide, a homologue of this polynucleotide encoding 3-phosphoserine transferring enzyme, 3-phosphoserine transferring enzyme homologue of the present invention is named as people EPIP2.

Another object of the present invention provides a kind of new 3-phosphoserine transferring enzyme homologue, and this albumen is named as people EPIP2 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people EPIP2 polypeptide.

The present invention also provides the application of this people EPIP2 nucleotide sequence 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 EPIP2 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 43-1155 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 43-1155 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 43-1155 position.

In another aspect of this invention, provide a kind of isolating people EPIP2 protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

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

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

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people EPIP2 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people EPIP2 protein-active operationally is connected in expression regulation sequence, form people EPIP2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 43-1155 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people EPIP2;

(c) under the condition that is fit to expressing human EPIP2 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people EPIP2 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1171 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 43-1155 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 " people EPIP2 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people EPIP2 protein-active is as 43-1155 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 43-1155 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 43-1155 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 43-1155 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 43-1155 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of natural people EPIP2 identical function.These variant forms comprise (but being not limited to): disappearance, insertion and/or the replacement of several (be generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) Nucleotide, and add several Nucleotide at 5 and/or 3 ends.Encoding sequence of the present invention can be DNA or RNA, can be strand or two strands.

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

In the present invention, term " people EPIP2 protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of natural human EPIP2 protein-active.This term also comprises having and variant form people EPIP2 albumen identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several 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 people EPIP2 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 EPIP2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people EPIP2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people EPIP2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people EPIP2 polypeptide.Usually, this fragment have people EPIP2 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 EPIP2 albumen or polypeptide.The difference of these analogues and natural human EPIP2 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 EPIP2 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

The present invention also comprises people EPIP2 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people EPIP2 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 people EPIP2 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 coding people EPIP2.

The present invention also comprises the method that detects people EPIP2 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 people EPIP2 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

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

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

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 people EPIP2 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 EPIP2 gene product or fragment.Preferably, refer to that those can combine with people EPIP2 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 EPIP2, comprise that also those do not influence the antibody of people EPIP2 protein function.The present invention also comprise those can with modify or without the people EPIP2 gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people EPIP2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human EPIP2 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 EPIP2 function and the antibody that does not influence people EPIP2 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people EPIP2 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 EPIP2 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 EPIP2 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 come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.Then can be with in various dna moleculars (as carrier) and the cell in this dna sequence dna introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

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

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

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

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

Utilize albumen of the present invention,, can filter out with EPIP2 interactional material takes place, as acceptor, inhibitor or antagonist etc. by various conventional screening methods.

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

With people EPIP2 albumen of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.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.People EPIP2 albumen 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 people EPIP2 protein polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Because people EPIP2 of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

In one embodiment of the invention, the cDNA nucleotide sequence of people EPIP2 is so to obtain, with people's placenta λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5-TTCGGTCCTCCTTGGCTGACTC-3 ' is a forward primer, oligonucleotide B:5 '-CCTGGTTAAGATGTGTTCATAGC-3 is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after amplified production checked order.

People EPIP2 of the present invention is a kind of mammiferous 3-phosphoserine aminotransferase (EC 2.6.1.52), can single-minded catalysis phosphoserine and 2-oxoglutaric acid and 3-phosphohydroxypyruvic acid and L-glutamic acid between the reversible transamination, the generation of endogenous Serine is played an important role.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of people EPIP2 of the present invention and rabbit EPIP (rbEPIP) aminoacid sequence (M17099).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 2 is the homology comparison diagram of people EPIP2 of the present invention and people's phosphoserine aminotransferase (hPSA) aminoacid sequence (AF113132).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

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

Embodiment 1

The clone and the mensuration of the cDNA sequence of people EPIP2

1. primer amplification

With people's placenta λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is that primer-A:5-TTCGGTCCTCCTTGGCTGACTC-3 (SEQ ID NO:1) is a forward primer, oligonucleotide B:5-CCTGGTTAAGATGTGTTCATAGC-3 (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A/B be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 1.2kp.

2.PCR the order-checking of product

Above-mentioned pcr amplification product A/B is connected with pGEM-T carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), (Epicentre Technologies) checks order to extractive plasmid with SequiTherm EXCELTMDNA sequencing kit, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1171bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 43-1155 position Nucleotide.

Derive the aminoacid sequence of people EPIP2 according to the full length cDNA sequence that obtains, totally 370 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people EPIP2 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that the 3-phosphoserine aminotransferase of they and different sources has significant homology.Show that with the PCGENE software analysis homology of people EPIP2 albumen of the present invention and rabbit EPIP (M17099) encoded protein is 93.5%, similarity is 97.1% (Fig. 1); With people's phosphoserine aminotransferase hPSA (AF113132) encoded protein basically identical, just 46 amino acid whose differences (Fig. 2) are arranged at the C end, this may be the result of different montage modes when transcribing.

In addition, people EPIP2 albumen of the present invention contains the pyridoxal phosphate binding sequence of V class (Class V) transaminase family member unanimity:

[L/I/V/F/Y/C/H/T][D/G/H][L/I/V/M/F/T/A/C][L/I/V/M/F/T/A]X ₂[G/S/T/A/C][G/S/T/A][H/Q/R]KX _4，6GX[G/S/A/T]X[L/I/V/M/F/Y/S/A/C]，

Wherein Methionin directly and the pyridoxal phosphate combination, square brackets are represented in the amino acid any one drawn together, X in the sequence _n, m represents any n or m amino acid, sequence corresponding among the present invention is ₁₉₁FGVIFAGAQKNVGSAGVTVV ₂₁₀

Albumen of the present invention also and the 3-phosphoserine aminotransferase that contains different plant species all contain one section characteristic sequence: LKGHRX ₂GGXR ( Yeast1994,10:385-389), X in the formula ₂Represent any 2 amino acid, sequence corresponding among the present invention is respectively ₃₃₂LKGHRSVGGIR ₃₄₂Therefore can determine the 3-phosphoserine aminotransferase that new albumen of the present invention is the people, and have the general utility functions of 3-phosphoserine aminotransferase.

The L-Serine is the intermediate product of the key of many important pathways metabolisms.Except the vital role in the building-up process of glycine, L-halfcystine, it also can change into L-homocysteine and L-methionine(Met).It is the essential or main precursor molecule of many materials, as the phosphoric acid fat of sphingophospholipid such as taurine, cystathionine, sphingosine and complicated sphingolipid, some kind, porphyrin, purine, thymus pyrimidine etc.Although a carbon-based group can derive from the oxidation Demethylation of Histidine metabolism and N-methylsarcosine and sarkosine, topmost carbon-based group source be Serine and glycine ( Arch.Biochem.Biophys.1985,237 (1): 186-196).

The L-Serine is a non-essential amino acid, exists active route of synthesis in many mammalian tissues.Although the external source ratio it be unclear that in the L-Serine, and is verified at least at brain, endogenous L-Serine synthesizes and is absolutely necessary because blood circulation can not provide abundant external source L-Serine ( J.Neurochem.1970,17:1461-1475).In addition, have and report that in malignant tissue the enzyme level in the L-Serine route of synthesis improves, reflected high demand a carbon-based group, with satisfy the synthetic needs of its DNA ( Biochem.Trans.1979,7:1048-1050).The L-Serine can have carbohydrate synthetic by two approach.Article one, be called as " phosphorylation approach ": the 3-phoshoglyceric acid that is generated by breakdown of glucose produces the 3-phosphohydroxypyruvic acid after dehydrogenation, generates the 3-phosphoserine by transamination again, and last hydrolysis dephosphorization acid groups produces the L-Serine.Another approach is called " non-phosphorylating approach ": generate the L-Serine by the D-2-phosphorylglyceric acid at last through D-R-Glyceric acid, oxypyroracemic acid.Under physiological condition, the general backward reaction of non-phosphorylating approach becomes the approach that utilizes of a L-Serine, but not route of synthesis ( Biochem.J.1980,190:451-455; Arch.Biochem.Biophys.1985,237 (1): 186-196).The 3-phosphoserine aminotransferase only participates in the phosphorylation approach, and the transamination during for the generation Serine has vital role.

3-phosphoserine aminotransferase (EC2.6.1.52) can single-minded catalysis phosphoserine and 2-oxoglutaric acid and 3-phosphohydroxypyruvic acid and L-glutamic acid between the reversible transamination.This enzyme depends on the coenzyme pyridoxal phosphate as the time spent, and pyridoxal phosphate and transaminase are combined closely, be amino intermediate transfer body ( Yeast1994,10:385-389).

Once had report Mammals Serine synthetic phosphorylation approach be subjected to steroid hormone regulation and control ( Principles in Biochemistry:general aspects1983 Mc.Grow-Hill Book Co., New York), can infer that thus endometrial Serine biosynthesizing is essential to embryo's nutritional needs.

People EPIP2 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor EPIP2 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor EPIP2 and the N end of rabbit EPIP are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor EPIP2, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor EPIP2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people EPIP2 or the overexpression that suppresses people EPIP2.People EPIP2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people EPIP2 disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people EPIP2 in intestinal bacteria

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people EPIP2 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.5 and 6) of 3 ' end increase, obtain people EPIP2 cDNA as inserting fragment.

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

5′-TCAGGGATCCATGGACGCCCCCAGGCAGG-3′(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people EPIP2 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5-TTGGGTCGACTCATAGCTGATGCATCTCC-3(SEQ?ID?NO.6)，

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people EPIP2 of Sal I restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding 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 BamH I and Sal I 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 lac I repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people EPIP2 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 into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lac I repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation culture, the collecting cell lysate also is diluted in it in 6M Guanidinium hydrochloride.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people EPIP2 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people EPIP2 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.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.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 40KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 4

The expression of people EPIP2 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people EPIP2 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.7 and 8) of 3 ' end increase, obtain people EPIP2 cDNA as inserting fragment.

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

5′-TCAGGGATCCATGGACGCCCCCAGGCAGG-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of BamH I restriction enzyme, is 19 Nucleotide of the people EPIP2 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5-TTGGGTCGACTCATAGCTGATGCATCTCC-3(SEQ?ID?NO.8)

This primer contains the part encoding sequence of the restriction enzyme site of EcoR I restriction enzyme, translation termination and people EPIP2.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding 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 BamH I and EcoR I 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, the cDNA fragment of sequence verification people EPIP2 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (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 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (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.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 40KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 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, 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 EPIP2 gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information: (ⅱ) denomination of invention: people 3-phosphoserine transferring enzyme and encoding sequence thereof and method for making and purposes (ⅲ) sequence number: information (ⅰ) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 22 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.1:TTCGGTCCTC CTTGGCTGAC TC 22 (2) SEQ ID NO.2

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:CCTGGTTAAG ATGTGTTCAT AGC 23 (2) SEQ ID NO.3: (ⅰ) sequence signature:

(A) length: 1171bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO.3： 1 TTCGGTCCTC CTTGGCTGAC TCACCGCCCT CGCCGCCGCA CCATGGACGC CCCCAGGCAG 61 GTGGTCAACT TTGGGCCTGG TCCCGCCAAG CTGCCGCACT CAGTGTTGTT AGAGATACAA121 AAGGAATTAT TAGACTACAA AGGAGTTGGC ATTAGTGTTC TTGAAATGAG TCACAGGTCA181 TCAGATTTTG CCAAGATTAT TAACAATACA GAGAATCTTG TGCGGGAATT GCTAGCTGTT241 CCAGACAACT ATAAGGTGAT TTTTCTGCAA GGAGGTGGGT GCGGCCAGTT CAGTGCTGTC301 CCCTTAAACC TCATTGGCTT GAAAGCAGGA AGGTGTGCGG ACTATGTGGT GACAGGAGCT361 TGGTCAGCTA AGGCCGCAGA AGAAGCCAAG AAGTTTGGGA CTATAAATAT CGTTCACCCT421 AAACTTGGGA GTTATACAAA AATTCCAGAT CCAAGCACCT GGAACCTCAA CCCAGATGCC481 TCCTACGTGT ATTATTGCGC AAATGAGACG GTGCATGGTG TGGAGTTTGA CTTTATACCC541 GATGTCAAGG GAGCAGTACT GGTTTGTGAC ATGTCCTCAA ACTTCCTGTC CAAGCCAGTG601 GATGTTTCCA AGTTTGGTGT GATTTTTGCT GGTGCCCAGA AGAATGTTGG CTCTGCTGGG661 GTCACCGTGG TGATTGTCCG TGATGACCTG CTGGGGTTTG CCCTCCGAGA GTGCCCCTCG721 GTCCTGGAAT ACAAGGTGCA GGCTGGAAAC AGCTCCTTGT ACAACACGCC TCCATGTTTC781 AGCATCTACG TCATGGGCTT GGTTCTGGAG TGGATTAAAA ACAATGGAGG TGCCGCGGCC841 ATGGAGAAGC TTAGCTCCAT CAAATCTCAA ACAATTTATG AGATTATTGA TAATTCTCAA901 GGATTCTACG TTTGTCCAGT GGAGCCCCAA AATAGAAGCA AGATGAATAT TCCATTCCGC961 ATTGGCAATG CCAAAGGAGA TGATGCTTTA GAAAAAAGAT TTCTTGATAA AGCTCTTGAA1021 CTCAATATGT TGTCCTTGAA AGGGCATAGG TCTGTGGGAG GCATCCGGGC CTCTCTGTAT1081 AATGCTGTCA CAATTGAAGA CGTTCAGAAG CTGGCCGCCT TCATGAAAAA ATTTTTGGAG1141 ATGCATCAGC TATGAACACA TCCTAACCAG G ( 2 ) SEQ ID NO.4： ( ⅰ ) ：

(A) length: 370 amino acid

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO.4：1 Met Asp Ala Pro Arg Gln Val Val Asn Phe Gly Pro Gly Pro Ala 16 Lys Leu Pro His Ser Val Leu Leu Glu Ile Gln Lys Glu Leu Leu 31 Asp Tyr Lys Gly Val Gly Ile Ser Val Leu Glu Met Ser His Arg 46 Ser Ser Asp Phe Ala Lys Ile Ile Asn Asn Thr Glu Asn Leu Val 61 Arg Glu Leu Leu Ala Val Pro Asp Asn Tyr Lys Val Ile Phe Leu 76 Gln Gly Gly Gly Cys Gly Gln Phe Ser Ala Val Pro Leu Asn Leu 91 Ile Gly Leu Lys Ala Gly Arg Cys Ala Asp Tyr Val Val Thr Gly106 Ala Trp Ser Ala Lys Ala Ala Glu Glu Ala Lys Lys Phe Gly Thr121 Ile Asn Ile Val His Pro Lys Leu Gly Ser Tyr Thr Lys Ile Pro136 Asp Pro Ser Thr Trp Asn Leu Asn Pro Asp Ala Ser Tyr Val Tyr151 Tyr Cys Ala Asn Glu Thr Val His Gly Val Glu Phe Asp Phe Ile166 Pro Asp Val Lys Gly Ala Val Leu Val Cys Asp Met Ser Ser Asn181 Phe Leu Ser Lys Pro Val Asp Val Ser Lys Phe Gly Val Ile Phe196 Ala Gly Ala Gln Lys Asn Val Gly Ser Ala Gly Val Thr Val Val211 Ile Val Arg Asp Asp Leu Leu Gly Phe Ala Leu Arg Glu Cys Pro226 Ser Val Leu Glu Tyr Lys Val Gln Ala Gly Asn Ser Ser Leu Tyr241 Asn Thr Pro Pro Cys Phe Ser Ile Tyr Val Met Gly Leu Val Leu256 Glu Trp Ile Lys Asn Asn Gly Gly Ala Ala Ala Met Glu Lys Leu271 Ser Ser Ile Lys Ser Gln Thr Ile Tyr Glu Ile Ile Asp Asn Ser286 Gln Gly Phe Tyr Val Cys Pro Val Glu Pro Gln Asn Arg Ser Lys301 Met Asn Ile Pro Phe Arg Ile Gly Asn Ala Lys Gly Asp Asp Ala316 Leu Glu Lys Arg Phe Leu Asp Lys Ala Leu Glu Leu Asn Met Leu33l Ser Leu Lys Gly His Arg Ser Val Gly Gly Ile Arg Ala Ser Leu346 Tyr Asn Ala Val Thr Ile Glu Asp Val Gln Lys Leu Ala Ala Phe361 Met Lys Lys Phe Leu Glu Met His Gln Leu ( 2 ) SEQ ID NO.5 ( ⅰ )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.5:TCAGGGATCC ATGGACGCCC CCAGGCAGG 29 (2) SEQ ID NO.6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.6:TTGGGTCGAC TCATAGCTGA TGCATCTCC 29 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO.7:TCAGGGATCC ATGGACGCCC CCAGGCAGG 29 (2) SEQ ID NO.8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: SEQ ID NO.8:TTGGGTCGAC TCATAGCTGA TGCATCTCC 29

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people EPIP2 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 43-1155 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 43-1155 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.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 43-1155 position.

4. an isolating people EPIP2 protein 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.4 aminoacid sequence.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 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. a generation has the method for the polypeptide of people EPIP2 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people EPIP2 protein-active operationally is connected in expression regulation sequence, form people EPIP2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 43-1155 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people EPIP2;

(c) under the condition that is fit to expressing human EPIP2 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people EPIP2 protein-active.

9. energy and the described people EPIP2 of claim 4 protein polypeptide specificity bonded antibody.

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

Images