CN1274728A - New human protein and its code sequence, preparation and application - Google Patents
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Abstract
The present invention provides one kind of new human gene necleotide sequence, more specifically, the cDNA sequence of human Py-CR, and the cDNA-encoded protein is the homologue of human pyrroline-5'-carboxylate reductase (P5CR) (EC1.5 1.2). The present invention also relates to the polypeptide encoded by the nucleotide sequence and the application and production process of the polynucleotide and polypeptide.
Description
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 Py-CR, this cDNA encoded protein be people's pyrroline-5 '-carboxylate reductase (pyrroline-5 '-carboxylate reductase, abbreviate " P5CR " as) (EC1.5.1.2) homologue.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.
Pyrroline-5 '-carboxylate reductase (pyrroline-5 '-carboxylate reductase, abbreviate " P5CR " as) (EC1.5.1.2) be distribution enzyme very widely, be prevalent in various prokaryotic organism and the eucaryon animals and plants.The catalysis of its single-minded ground rely on NAD (P) H, change pyrroline-5 '-carboxylic acid the reduction reaction of proline(Pro) into, and with NAD (P) H oxidation NAD (P)
+Its reaction generally can be expressed as:
Pyrroline-5 '-carboxylic acid+NAD (P) H → proline(Pro)+NAD (P)
+
In the different tissues of different organisms, P5CR is bringing into play different physiological functions.Need in a large number in the tissue of proline(Pro) inoblast, cartilage and other, the main effect of P5CR is to generate proline(Pro), and (Metabolism 1978,27:685-694) to participate in protein synthesis; And in red corpuscle, P5CR participates in reaction and produces NAD (P)
+As the coenzyme of glucose-6-phosphate dehydrogenase, thus the activation of stimulation hexose phosphate shunt (Biochem.Biophys.Res.Commun.1988,85:2036-2040); In the plant of environmental stresss such as tolerance arid or high salt, it produces proline(Pro) as a kind of effective osmoticum (Plant Cell.Physiol.1997,38 (10): 1095-1102); In addition, it also can be the attemperator of redox potential energy in the cell, so P5CR has important biological significance.
As far back as the eighties, people just separate the protein molecular that has obtained P5CR from different sourcess such as ox horn film, rat lens, HRBC (Biochim.Biophys.Acta 1982,717:215-219; Biochim.Biophys.Acta 1986,881:72-78; J.Biol.Chem.1989,264 (16): 9352-9358).Nineteen eighty-two, people such as Deutch obtain at first intestinal bacteria P5CR dna sequence dna (Nucleic Acid Res.1982,10:7701-7714).Nineteen ninety, people such as Delauney by function complementation experiment cloned soybean the P5CR gene (Mol.Gen.Genet.1990,221:299-305).1992, first people P5CR cDNA (J.Biol.Chem.1992.267 (2): 871-875) cloned in Dougherty group from the cDNA library in human liver cell strain source.After, people have found the gene of coding P5CR again in hot bacterium, fungi, insect and plant.
Yet up to now, Shang Weijian has the report consistent with sequence of the present invention.
An object of the present invention is to provide a kind of new polynucleotide, this polynucleotide encoding pyrroline-5 '-carboxylate reductase (pyrroline-5 '-carboxylate reductase, abbreviate " P5CR " as) newcomer of homologue, P5CR homologue member of the present invention is named as people Py-CR.
Another object of the present invention provides a kind of new people's P5CR homologue member, and this albumen is named as people Py-CR albumen.
A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's Py-CR polypeptide.
The present invention also provides this people's the Py-CR nucleotide sequence and the application of 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 Py-CR protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 136-1098 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 136-1098 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 136-1098 position.
In another aspect of this invention, provide a kind of isolating people Py-CR protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID 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 Py-CR protein-active, this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people Py-CR protein-active operationally is connected in expression regulation sequence, form people Py-CR protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 136-1098 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 Py-CR;
(c) under the condition that is fit to expressing human Py-CR protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people Py-CR protein-active.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1121 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 136-1098 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 Py-CR albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people PY-CR protein-active is as 136-1098 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 136-1098 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 136-1098 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 136-1098 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 136-1098 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people Py-CR identical function.These variant forms 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 " people Py-CR protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of people Py-CR protein-active.This term also comprises having and variant form people Py-CR 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 Py-CR 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 Py-CR DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people Py-CR polypeptide to obtain.The present invention also provides other polypeptide, as comprises people Py-CR polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people Py-CR polypeptide.Usually, this fragment have people Py-CR 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 Py-CR albumen or polypeptide.The difference of these analogues and natural human Py-CR 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 Py-CR 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
| 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 |
The present invention also comprises people Py-CR polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people Py-CR 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 Py-CR 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 Py-CR.
The present invention also comprises the method that detects people Py-CR 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 Py-CR 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 comprises that also people Py-CR DNA or the polypeptide of its fragment coding are had the grand antibody of specific many models and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people Py-CR gene product or fragment.Preferably, refer to that those can combine with people Py-CR 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 Py-CR, comprise that also those do not influence the antibody of people Py-CR protein function.The present invention also comprise those can with modify or without the people Py-CR 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 Py-CR gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human Py-CR or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people Py-CR function and the antibody that does not influence people Py-CR function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people Py-CR 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 Py-CR 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 Py-CR 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.
In one embodiment of the invention, the cDNA nucleotide sequence of people Py-CR is so to obtain, with people liver λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5 '-CAGAAGCTGCTGTTAGCTCGCCG-3 ' is a forward primer, oligonucleotide B:5 '-TCACAGAGGGGACAGATGCTGCC-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after amplified production checked order.
Pyrroline-5 '-carboxylate reductase is the catalysis person of the synthetic middle final step reaction of proline(Pro), is coenzyme with NADPH or NADH, promotes that pyrroline-5 '-carboxylic acid is reduced into proline(Pro), and simultaneous oxidation NAD (P) H is NAD (P)
+It has important biological function, in the endogenous generation of proline(Pro), regulate cellular oxidation-reduction potential energy, the activation hexose phosphate shunt, keep Premeabilisation of cells and press, participating in energy all has important effect from nitrogen-fixing plants to various physiological processs such as its endosymbiont conversion.The analogue of pyrroline-5 '-carboxylate reductase is also being brought into play important effect probably in similar approach.
In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people Py-CR of the present invention and people's pyrroline-5 '-carboxylate reductase (P5CR).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 Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The clone and the mensuration of the cDNA sequence of people Py-CR
1. primer amplification
With people liver λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5 '-CAGAAGCTGCTGTTAGCTCGCCG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide B:5 '-TCACAGAGGGGACAGATGCTGCC-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, 60 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 1.1kb.
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), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Use SequiTherm EXCEL
TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1121bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 136-1098 position Nucleotide.
Derive the aminoacid sequence of people Py-CR according to the full length cDNA sequence that obtains, totally 320 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 Py-CR 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 P5CR gene and the proteins encoded thereof of they and different sources as people P5CR (M77836), soybean P5CR (X16352) and Actinidia deliciosa P5CR (U92287), have homology widely.Be to find relatively that with PCGENE software it and the identity of people P5CR on protein level reach 84%, other has 6.6% amino acid similarity, therefore, people Py-CR albumen of the present invention can be classified P5CR albumen homologue, and can infer that it has the general utility functions of P5CR albumen homologue.
Pyrroline-5 '-carboxylate reductase distributes very extensive, in various organisms such as bacterium, fungi, insect, Mammals, plant expression is arranged all.It is the catalysis person of the synthetic middle final step reaction of proline(Pro), is coenzyme with NADPH or NADH, promotes that pyrroline-5 '-carboxylic acid is reduced into proline(Pro), and simultaneous oxidation NAD (P) H is NAD (P)
+It has important biological function, in the endogenous generation of proline(Pro), regulate cellular oxidation-reduction potential energy, the activation hexose phosphate shunt, keep Premeabilisation of cells and press, participating in energy all has important effect from nitrogen-fixing plants to various physiological processs such as its endosymbiont conversion.The P5CR of different sources is owing to play different physiological functions, and the substrate avidity that shows, coenzyme demand and product inhibition are different.
In animal body, proline(Pro) is the synthetic proteic main raw material of collagen, therefore needing in a large number in the tissue of proline(Pro) as cartilage, bone, eye etc., the expression level of P5CR is high especially, its main effect is exactly to reduce pyrroline-5 '-carboxylic acid, and (Metabolism 1978,27:685-694) to generate proline(Pro), and have higher speed of reaction with NADH during for coenzyme, and the inhibition that is subjected to the product proline(Pro) is regulated.In addition, P5CR also has expression at red corpuscle, and particularly it also has function (Biochem.Biophys.Res.Commun.1981,101 (3): 1018-1025), point out its another vital role: produce NADP in the lymphoblast of the synthetic defective of proline(Pro)
+, regulate intracellular redox potential energy, activate glycometabolic another important channel---hexose phosphate shunt (HMP branch road).The HMP branch road all exists in various biologies, it can circulate and decompose hexose generation NADPH, for the anabolism of materials such as lipid acid, sterol provides necessary energy derive, be main breakdown of glucose approach in the tissues such as mammary gland, fatty tissue, adrenal cortex.And more total enzymes of HPM branch road and photosynthesis of plant and intermediate product concern very closely, and its existence in plant is more general.Pyrroline-5 '-carboxylic acid and proline(Pro) can be used as a pair of redox couple, provide a kind of redox potential energy shuttle mechanism (Curr.Top Cell Reg.1985,25:91-132).P5CR acts on this moment, shows not to be subjected to the product proline(Pro) and to be subjected to NADP
+Regulate with the inhibition of ATP.
Although also do not find the genetic diseases that P5CR causes unusually at present, but according to its important physical function, can predict the defective of P5CR, can cause illness (J.Biol.Chem.1992,267 (2): 871-875) such as cretinism, dyschondroplasia, cataract, dysgalactia, hemolytic anemia.In the cataractous mouse of a kind of congenital infiltration (Nakano mouse) lens, HMP branch road level always be lower than the control group level (Exp.Eye Res.1985,41:767-775).The defective of Py-CR may also can cause similar disease.
People Py-CR 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 Py-CR can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor Py-CR and the N end of people P5CR are exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor Py-CR, be used to screen other members (as other analogues of P5CR) of this family, perhaps be used for affinity purification associated protein (as other members of this family).
In addition, inventor Py-CR nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people Py-CR or the overexpression that suppresses people Py-CR.People Py-CR albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people Py-CR 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 Py-CR 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 Py-CR 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 Py-CR cDNA as inserting fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5 '-TCAGGTCGACATGAGCGTGGGCTTCATCG-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of SalI restriction enzyme, is 19 Nucleotide of the people Py-CR encoding sequence that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5 '-TTGGAAGCTTTTAGTCCTTCTTGCCTCCC-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people Py-CR of HindIII 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 SalI and HindIII 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 (Kan
r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people Py-CR 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 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 people Py-CR from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people Py-CR 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 34KDa.
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 Py-CR 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 Py-CR 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 Py-CR cDNA as inserting fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TCAGAAGCTTATGAGCGTGGGCTTCATCG-3′(SEQ?ID?NO.7)
This primer contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the people Py-CR encoding sequence that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5’-TTGGGAATTCTTAGTCCTTCTTGCCTCCC-3’(SEQ?ID?NO.8)
This primer contains the part encoding sequence of the restriction enzyme site of EcoRI restriction enzyme, translation termination and people Py-CR.
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 (fl 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 HindIII and EcoRI 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 has correctly inserted carrier with the cDNA fragment of XbaI enzyme cutting and sequence verification people Py-CR.
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 50mMTris HCl (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 50mMTris á HCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTris á HCl (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 34KDa.
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 Py-CR 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: (i) applicant: Fudan University is denomination of invention (ii): a kind of new people's albumen and encoding sequence thereof, and (iii) sequence number of method for making and purposes: information (i) sequence signature of 8 (2) SEQ ID NO.1
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:CAGAAGCTGC TGTTAGCTCG CCG 23 (2) SEQ ID NO.2
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2:TCACAGAGGG GACAGATGCT GCC 23 (2) SEQ ID NO.3: (i) sequence signature:
(A) length: 1121bp
(B) type: nucleic acid
(C) chain: strand
( D ) ： ( ii ) ：cDNA ( iii ) ：SEQ ID NO.3：CAGAAGCTGC TGTTAGCTCG CCGGTCCTCG GACGCCGCCC GTTCGCCCCT GCGCTGTCCG 60CCCTTCCCCT AGCGTTACTT CCGGTCCCTC GCTGAGGGGG TTCGTGCGGC TCCCAGGAGG 120CGTGAACCGC GGACCATGAG CGTGGGCTTC ATCGGGGCCG GCCAGCTGGC TAATGCTCTG 180GCGCGGGGCT TCACGGCCGC AGGCATCCTG TCGGCTCACA AGATAATAGC CAGCTCCCCA 240GAAATGAACC TGCCCACGGT GTCCGCGCTC AGGAAGATGG GTGTGAACCT GACACGCAGC 300AACAAGGAGA CGGTGAAGCA CAGCGACGTC CTGTTTCTGG CTGTGAAGCA CATTATCATC 360CCCTTCATCC TGGATGAGAT TGGGGCCGAC GTGCAAGCCA GACACATCGT GGTCTCCTGT 420GCGGCTGGTG TCACCATCAG CTCTGTGGAG AAGAAGCTGA TGGCATTCCA GCCAGCCCCC 480AAAGTGATTC GCTGCATGAC CAACACACCT GTGGTAGTGC AGGAAGGCGC TACAGTGTAC 540GCCACGGGCA CCCATGCCCT GGTGGAGGAT GGGCAGCTCC TGGAGCAGCT CATGAGCAGC 600GTGGGCTTCT GCACTGAGGT GGAAGAGGAC CTCATCGATG CCGTCACGGG GCTCAGTGGC 660AGCGGGCCTG CCTATGCATT CATGGCTCTG GACGCATTGG CTGATGGTGG GGTGAAGATG 720GGTTTGCCAC GGCGCCTGGC AATCCAACTC GGGGCCCAGG CTTTGCTGGG AGCTGCCAAG 780ATGCTGCTGG ACTCGGAGCA GCATCCATGC CAGCTTAAGG ACAATGTCTG CTCCCCTGGG 840GGAGCCACCA TCCACGCCCT GCACTTTCTA GAGAGTGGGG GCTTCCGCTC TCTGCTCATC 900AATGCAGTTG AGGCCTCCTG TATCCGAACA CGAGAGCTAC AGTCCATGGC CGACCAAGAA 960AAGATCTCCC CAGCTGCCCT TAAGAAGACC CTCTTAGACA GAGTGAAGCT GGAATCCCCC 1020ACAGTCTCCA CACTGACCCC CTCCAGCCCA GGGAAGCTCC TCACAAGAAG CCTGGCCCTG 1080GGAGGCAAGA AGGACTAAGG CAGCATCTGT CCCCTCTGTG A ( 2 ) SEQ ID NO.4： ( i ) ：
(A) length: 320 amino acid
(B) type: amino acid
(D) topological framework: linearity is molecule type (ii): polypeptide
( xi ) ：SEQ ID NO.4：Met Ser Val Gly Phe Ile Gly Ala Gly Gln Leu Ala Asn Ala Leu 15Ala Arg Gly Phe Thr Ala Ala Gly Ile Leu Ser Ala His Lys Ile 30Ile Ala Ser Ser Pro Glu Met Asn Leu Pro Thr Val Ser Ala Leu 45Arg Lys Met Gly Val Asn Leu Thr Arg Ser Asn Lys Glu Thr Val 60Lys His Ser Asp Val Leu Phe Leu Ala Val Lys His Ile Ile Ile 75Pro Phe Ile Leu Asp Glu Ile Gly Ala Asp Val Gln Ala Arg His 90Ile Val Val Ser Cys Ala Ala Gly Val Thr Ile Ser Ser Val Glu 105Lys Lys Leu Met Ala Phe Gln Pro Ala Pro Lys Val Ile Arg Cys 120Met Thr Asn Thr Pro Val Val Val Gln Glu Gly Ala Thr Val Tyr 135Ala Thr Gly Thr His Ala Leu Val Glu Asp Gly Gln Leu Leu Glu 150Gln Leu Met Ser Ser Val Gly Phe Cys Thr Glu Val Glu Glu Asp 165Leu Ile Asp Ala Val Thr Gly Leu Ser Gly Ser Gly Pro Ala Tyr 180Ala Phe Met Ala Leu Asp Ala Leu Ala Asp Gly Gly Val Lys Met 195Gly Leu Pro Arg Arg Leu Ala Ile Gln Leu Gly Ala Gln Ala Leu 210Leu Gly Ala Ala Lys Met Leu Leu Asp Ser Glu Gln His Pro Cys 225Gln Leu Lys Asp Asn Val Cys Ser Pro Gly Gly Ala Thr Ile His 240Ala Leu His Phe Leu Glu Ser Gly Gly Phe Arg Ser Leu Leu Ile 255Asn Ala Val Glu Ala Ser Cys Ile Arg Thr Arg Glu Leu Gln Ser 270Met Ala Asp Gln Glu Lys Ile Ser Pro Ala Ala Leu Lys Lys Thr 285Leu Leu Asp Arg Val Lys Leu Glu Ser Pro Thr Val Ser Thr Leu 300Thr Pro Ser Ser Pro Gly Lys Leu Leu Thr Arg Ser Leu Ala Leu 315Gly Gly Lys Lys Asp ( 2 ) SEQ ID NO.5 ( i )
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5:TCAGGTCGAC ATGAGCGTGG GCTTCATCG 29 (2) SEQ ID NO.6
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.6:TTGGAAGCTT TTAGTCCTTC TTGCCTCCC 29 (2) SEQ ID NO.7
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TCAGAAGCTT ATGAGCGTGG GCTTCATCG 29 (2) SEQ ID NO.8
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:TTGGGAATTC TTAGTCCTTC TTGCCTCCC 29
Claims (10)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people Py-CR protein-active,
Show at least 70% homology from the nucleotides sequence of Nucleotide 136-1098 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 136-1098 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 136-1098 position.
4. isolating people Py-CR protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 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.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 Py-CR protein-active, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people Py-CR protein-active operationally is connected in expression regulation sequence, form people Py-CR protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 136-1098 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 Py-CR;
(c) under the condition that is fit to expressing human Py-CR protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people Py-CR protein-active.
9. energy and the described people Py-CR 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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102844435A (en) * | 2010-02-22 | 2012-12-26 | 库尔纳公司 | Treatment of pyrroline-5-carboxylate reductase 1 (pycr1) related diseases by inhibition of natural antisense transcript to pycr1 |
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1999
- 1999-05-25 CN CN 99107071 patent/CN1274728A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102844435A (en) * | 2010-02-22 | 2012-12-26 | 库尔纳公司 | Treatment of pyrroline-5-carboxylate reductase 1 (pycr1) related diseases by inhibition of natural antisense transcript to pycr1 |
| JP2013534512A (en) * | 2010-02-22 | 2013-09-05 | カッパーアールエヌエー，インコーポレイテッド | Treatment of pyrroline-5-carboxylate reductase 1 (PYCR1) related diseases by inhibition of natural antisense transcripts against PYCR1 |
| US8962586B2 (en) | 2010-02-22 | 2015-02-24 | Curna, Inc. | Treatment of pyrroline-5-carboxylate reductase 1 (PYCR1) related diseases by inhibition of natural antisense transcript to PYCR1 |
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