CN1259574A - New human phosphatide transferase, its code sequence, prepn. and use thereof - Google Patents
New human phosphatide transferase, its code sequence, prepn. and use thereof Download PDFInfo
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Abstract
The present invention relates to new human phosphtidase PLS2. The present invention provides cDNA coding sequence of said new phosphatidase, polypeptide of said sequencial code, and method of utilizing recomibination technology to produce said new human phosphatidase. The present invention also provides the application of said new human phosphatidase and its coding sequence.
Description
The present invention relates to the genetically engineered field.The present invention relates to a kind of new polynucleotide particularly, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.Polypeptide of the present invention is accredited as a kind of new human phosphatide transferase by deduction.
The distribution of the phosphatide on the cytoplasmic membrane is normally asymmetric, phosphatidylcholine and sphingomyelin generally are distributed in the outside of duplicature, and amino phosphatide, phosphatidylserine and phosphatidic acid second ammonia alcohol are confined to tenuigenin one side (Schrait A.J.et al Biochim.Biophys.Acta 1071:313-329,1991).Phospholipid molecule is general rare flip-flop movement (flip-flop) in plasma membrane, but Ca in the cell that physiological activities such as cell activation, cell injury, apoptosis cause
2+Level rises and can cause two-way move (WilliamsonP.et al Biochemistry 31:6355-6360,1992) of phospholipid molecule in the plasma membrane.The flip-flop movement of phospholipid molecule in the film both sides can promote the gathering and the activation of key enzyme in blood coagulation system, the complement system, and quickens the removing (Bevers E.M.et al Blood Rev.5:146-154,1991) of damaging cells and apoptotic cell.
Phosphatide transferase (phospholipid scramblase abbreviates " PLS " as) is newfoundly in recent years can mediate Ca
2+A protein of dependent form phospholipid molecule flip-flop movement.1996, people such as Basse separate the film integral protein that has obtained an about 37KD from people's red corpuscle, this albumen has the function (Basse F.et al J.Biol.Chem.271 (29): 17205-17210,1996) of mediation artificial liposome phospholipid molecule upset; 1997, people such as this laboratory Zhou cloned this proteic cDNA sequence (Zhou Q.et al J.Biol.Chem.272 (29): 18240-18244,1997); 1998, people such as Zhou cloned the cDNA sequence (Zhou Q.et al Biochemistry 37:2356-2360,1998) of the homologue of this albumen in mouse again.
Yet, new human phosphatide transferase involved in the present invention or its sequence were not disclosed before the application.
An object of the present invention is to provide a kind of new polynucleotide, new phosphatide transferase of this polynucleotide encoding, the new human phosphatide transferase of the present invention is named as people PLS2.
Another object of the present invention provides a kind of new human phosphatide transferase albumen, and this enzyme is named as people PLS2.
A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new human phosphatide transferase.
The invention still further relates to the application of this human phosphatide transferase 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 PLS2 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 118-849 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 118-849 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 118-849 position.
In another aspect of this invention, provide a kind of isolating people PLS2 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 PLS2 protein-active, this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people PLS2 protein-active operationally is connected in expression regulation sequence, form people PLS2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 118-849 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 PLS2;
(c) under the condition that is fit to expressing human PLS2 protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people PLS2 protein-active.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 892 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 118-849 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 PLS2 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people PLS2 protein-active is as 118-849 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 118-849 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 118-849 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 118-849 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 118-849 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people PLS2 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 PLS2 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people PLS2 protein-active.This term also comprises having and variant form human phosphatide transferase PLS2 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 PLS2 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 PLS2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people PLS2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people PLS2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of people PLS2 polypeptide.Usually, this fragment have people PLS2 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 PLS2 albumen or polypeptide.The difference of these analogues and natural human PLS2 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 can also comprise 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 PLS2 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.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; Nor-leucine | Leu |
| Leu(L) | Nor-leucine; 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; Nor-leucine | Leu |
The present invention also comprises people PLS2 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people PLS2 in the cell.
The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people PLS2 nucleotide 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 PLS2.
The present invention also comprises the method that detects people PLS2 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 can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of people PLS2 polypeptide.Primer length is generally 20-50 Nucleotide.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.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 PLS2 DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people PLS2 gene product or fragment.Preferably, refer to that those can combine with people PLS2 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 PLS2, comprise that also those do not influence the antibody of people PLS2 protein function.The present invention also comprise those can with modify or without the people PLS2 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 PLS2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human PLS2 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 PLS2 function and the antibody that does not influence people PLS2 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people PLS2 gene product, obtains by immunological technique.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 PLS2 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 PLS2 nucleotide sequence full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can SEQ ID NO.3 sequence disclosed according to the present invention, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of pressing the currently known methods preparation 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.Certainly, 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, polynucleotide total length of the present invention is 892 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 118-849 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A1:5 '-TACAGTCCACAGCAACCCAGTAC-3 ' and reverse primer B1:5 '-AGTGCTGACTGTAAGCCCAATCC-3 ' carry out PCR, obtain the purpose fragment of 892bp.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.
According to homology result relatively, the phosphatide transferase of nucleotide sequence of the present invention and encoded protein matter sequence and different sources has shown significant homology, therefore, this shows that it is a new homologue of phosphatide transferase, and has the more proteic critical functions of phosphatide transferase.
Phospholipid molecule on the cytoplasmic membrane is asymmetrically distributed (Schrait A.J.et al Biochim.Biophys.Acta 1071:313-329,1991).Ca in the cell
2+Level rises can cause quick, the two-way upset (Williamson P.et al Biochemistry 31:6355-6360,1992) of phospholipid molecule on the lipid bilayer cytolemma.Phosphatide transferase is a wide expression on the cytolemma of various cells, can mediate Ca
2+The phospholipid molecule of dependent form is two-way mobile protein molecular (Basse F.et al J.Biol.Chem.271 (29): 17205-17210,1996) on cytolemma.The phospholipid molecule of phosphatide transferase mediation is in the distribution again of the double-deck molecule of cell membrane lipid, to blood vessel hemostatic mechanism and cell purge mechanism play an important role (Zhou Q.et al J.Biol.Chem.272 (29): 18240-18244,1997).Common and the phosphatidylserine response Ca of the expression level of phosphatide transferase
2+Level rises, and turn to the degree corresponding (Zhou J.et al J.Biol.Chem.273 (12): 6603-6606,1998) in the cytolemma outside.The homologue of people and mouse relatively be presented at this proteic kytoplasm partly contain one conservative, to the very similar Ca of known EF chiral structure sequence
2+In conjunction with territory (Zhou Q.et al Biochemistry37:2356-2360,1998), this structure and Ca
2+Regulate the activity relevant (Zhou J.et alBiochemistry 37:6361-6366,1998) of this enzyme.Scott syndromes (Scott Syndrome) relevant with the afunction of phosphatide transferase (Zhou Q.et alJ.Biol.Chem.272 (29): 18240-18244,1997).There is fabulous identical people's leukemia to generate that genes involved MmTRA1b may generate with leukemia and the differentiation relevant (Kasukabe T.et al Biochem Biophys.Res.Commun.249 (2): 449-455,1998) of monocytic leukemia cell with the phosphatide transferase sequence.
In the accompanying drawings,
Fig. 1 is the homology comparison diagram of the nucleotide sequence of people PLS2 of the present invention and mouse phosphatide transferase (MPLS).Wherein, identical Nucleotide marks with " | ".
Fig. 2 is the homology comparison diagram of the aminoacid sequence of people PLS2 of the present invention and mouse phosphatide transferase (MPLS).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. 3 is the homology comparison diagram of the nucleotide sequence of people PLS2 of the present invention and human phosphatide transferase (HPLS).Wherein, identical Nucleotide marks with " | ".
Fig. 4 is the homology comparison diagram of the aminoacid sequence of people PLS2 of the present invention and human phosphatide transferase (HPLS).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. 5 is the homology comparison diagram that people PLS2 of the present invention and people's leukemia generates the aminoacid sequence of genes involved MmTRA1b.
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 normal condition, such as people 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 PLS2
1. primer amplification
With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-TACAGTCCACAGCAACCCAGTAC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-AGTGCTGACTGTAAGCCCAATCC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR fragment that electrophoresis detection obtains is about the purpose fragment of 900bp.
2.PCR the order-checking of product
With pcr amplification product and the pGEM-T that as above obtains
Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use 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 892bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 118-849 position Nucleotide.
Derive the aminoacid sequence of people PLS2 according to the full length cDNA sequence that obtains, totally 243 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.
Embodiment 2
Homology comparison and functional study
Full length cDNA sequence and proteins encoded thereof with people PLS2 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST software, found that with phosphatide transferase to have shown higher homology.Use the PCGENE software analysis, the phosphatide transferase (MPLS) of it and mouse (gi|2935162|gb|AF015790|AF015790) has 70.3% identity (Fig. 1) on nucleic acid level, 48.6% identity is arranged on protein level and 13.6% amino acid similarity (Fig. 2) is arranged in addition; And for example, it and people's phosphatide transferase (HPLS) (gi|2282600|gb|AF008445|AF008445) has 70.3% identity (Fig. 3) on nucleic acid level, 48.6% identity is arranged on protein level and 13.6% amino acid similarity (Fig. 4) is arranged in addition.This shows that new albumen of the present invention is a member of phosphatide transferase family, and can infer the function of people PLS2 with the function of phosphatide transferase.
Studies show that the phospholipid molecule on the cytoplasmic membrane is asymmetrically distributed (Schrait A.J.et al Biochim.Biophys.Acta 1071:313-329,1991).In resting cell, the phospholipid molecule spontaneously upset speed between the cytolemma bilayer is very slow, and by Ca in the cell of initiations such as cell activation, cell injury, apoptosis
2+Level rises can cause quick, the two-way upset of phospholipid molecule on the lipid bilayer cytolemma, make originally outside the phosphatidylserine of tenuigenin side is positioned at, and the phosphoric acid acyl second ammonia alcohol in the outside is indexed to tenuigenin side (Williamson P.et al Biochemistry 31:6355-6360,1992).Phosphatide transferase is the protein molecular of wide expression on the cytolemma of various cells, and it can mediate Ca
2+The phospholipid molecule of dependent form is two-way move (Basse F.et al J.Biol.Chem.271 (29): 17205-17210,1996) on cytolemma.
Studies show that, phosphatide transferase is that (albumen of the present invention contains 15 proline(Pro) for the epicyte protein of a proline rich Type II, account for more than 6% of protein molecular total length), and a transmembrane domains (Zhou Q.et al J.Biol.Chem.272 (29): 18240-18244 is being arranged near C end place, 1997), the corresponding sequence of the present invention be among the SEQ ID NO.4 the 204th to the 224th amino acids, i.e. LDVKMKAMIFGACFLIDFMYF.This has confirmed that newer albumen of the present invention is new phosphatide transferase.
Experiment shows, amino phosphide fat is exposed to gathering and the activation that the outside can promote key enzyme in blood coagulation system, the complement system, and the acceleration reticuloendothelial system is to the removing (Bevers E.M.et alBlood Rev.5:146-154,1991) of damaging cells, apoptotic cell.The Ca of phosphatide transferase mediation
2+The dependent form phospholipid molecule is in the distribution again of the double-deck molecule of cell membrane lipid, to blood vessel hemostatic mechanism and cell purge mechanism play an important role (Zhou Q.et al J.Biol.Chem.272 (29): 18240-18244,1997).Quantitatively the immuning hybridization experiment shows that the molecule number of phosphatide transferase in the thrombocyte is higher about 10 times than erythrocyte, with the phosphatide transferase vigor consistent (Zhou Q.et al J.Biol.Chem.272 (29): 18240-18244,1997) that significantly rises in the platelet cell film.The expression level of phosphatide transferase usually and phosphatidylserine with Ca
2+Level rises and turn to degree corresponding (Zhou J.et al J.Biol.Chem.273 (12): 6603-6606,1998) outside the cytolemma.People and mouse and people's homologue relatively is presented at this proteic kytoplasm and partly contains conservative and the very similar Ca of known EF chiral structure sequence
2+In conjunction with the territory:
D(A/S)DNFGIQFPL?D
[annotating: " (A/S) " expression optional amino acid from these 2 amino acid in this sequence] (Zhou Q.et alBiochemistry 37:2356-2360,1998), the corresponding sequence of the present invention be among the SEQ ID NO.4 the 192nd to the 203rd amino acids, be DADHFDIHFPLD (wherein the 197th different with the 199th amino acids with the corresponding amino acid of the phosphatide transferase of having reported, the 195th amino acids and the corresponding amino acid similarity of the phosphatide transferase of having reported), this structure and Ca
2+The activity of regulating this enzyme is relevant.Experiment showed, that phosphatide transferase is away from Ca
2+Can be on the cysteine residues of the N end of binding site by palmitoylation.This posttranslational modification may with the dependence Ca of this enzyme
2+Vigor relevant (Zhou J.et al Biochemistry 37:6361-6366,1998).The recombinant protein of expression in escherichia coli shows enzyme activity and Ca
2+Bonding force all significantly descends.After the thioether bond hydrolysis, about 80% (Zhou J.et al Biochemistry 37:6361-6366,1998) of the afunction of this enzyme.
A kind of hereditary defect hemorrhagic disease---Scott syndromes (Scott Syndrome) is relevant with the afunction of phosphatide transferase (Zhou Q.et al J.Biol.Chem.272 (29): 18240-18244,1997) just.Leukemia by the people of people's new clones such as Kasukabe generates genes involved MmTRA1b, with phosphatide transferase fabulous coincideing arranged on aminoacid sequence, so these albumen may generate with leukemia and the differentiation relevant (Kasukabe T.et al Biochem Biophys.Res.Commun.249 (2): 449-455,1998) of monocytic leukemia cell.New phosphatide transferase of the present invention and MmTRA1b also have very high homology (Fig. 5).This hint, new albumen of the present invention and leukemia may also have necessarily gets in touch.
People PLS2 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 PLS2 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor PLS2 and the N end of mouse PLS or people PLS are exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor PLS2, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family, such as mouse PLS).
For example, inventor PLS2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people PLS2 or the overexpression that suppresses people PLS2.People PLS2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people PLS2 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 PLS2 in intestinal bacteria
The cDNA sequence of coding people PLS2 is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, is that template increases with the fragment that obtains among the embodiment 1, with synthetic insertion fragment.
5 ' Oligonucleolide primers sequence is
5′-TTGCGGATCCATGCCAGGGCCAACTCCTAT-3′(SEQ?ID?NO.5)，
This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 20 Nucleotide of the people PLS2 encoding sequence that begun by initiator codon;
3 ' Oligonucleolide primers sequence is
5’-GTTCGTCGACTTAACCATAGGTGATGGCT-3’(SEQ?ID?NO.6)，
This primer contains the restriction enzyme site of SalI restriction enzyme, the partial sequence of the coding of translation termination and people PLS2.
The restriction enzyme site of restriction enzyme is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp
r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.
With BamHI and SalI digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan
r).Screen transformant containing on the LB culture dish of Amp and Kan, 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).The extracting plasmid, sequence verification result shows that the cDNA of people PLS2 inserts the fragment carrier of correctly packing into.
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 PLS2 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people PLS2 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, 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 28KDa.
In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
The expression of people PLS2 in eukaryotic cell (Chinese hamster ovary celI strain)
In this embodiment, the cDNA sequence of coding people PLS2 being used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, is that template increases with the fragment that obtains among the embodiment 1, with synthetic insertion fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TTGCAAGCTTATGCCAGGGCCAACTCCTAT-3′(SEQ?ID?NO.7)，
This primer contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 20 Nucleotide of the people PLS2 encoding sequence that begun by initiator codon;
3 ' end primer sequence is:
5’-GTTCGGATCCTTAACCATAGGTGATGGCT-3’(SEQ?ID?NO.8)
This primer contains the partial sequence of the coding of the restriction enzyme site of BamHI restriction enzyme, translation termination and people PLS2.
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 HindIII, BamHI 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, sequence verification result shows that the cDNA of people PLS2 inserts the fragment carrier of correctly packing into.
Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 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 28KDa.
In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 5
Preparation antibody
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): new human phosphatide transferase, its encoding sequence and method for making and purposes be the sequence number (iii): 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:TACAGTCCAC AGCAACCCAG TAC 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:AGTGCTGACT GTAAGCCCAA TCC 23 (2) SEQ ID NO.3: (i) sequence signature:
(A) length: 892bp
(B) type: nucleic acid
(C) chain: strand
( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3TACAGTCCAC AGCAACCCAG TACCTTCCCT TTGTACCAGC CAGTTGGTGG TATCCATCCT 60GTCCGGTATC AGCCTGGAAA ATATCCTATG CCAAATCAGT CTGTTCCAAT AACATGGATG 120CCAGGGCCAA CTCCTATGGC AAACTGCCCT CCTGGTCTGG AATACTTAGT TCAGTTGGAC 180AACATACATG TTCTTCAGCA TTTTGAGCCT CTGGAAATGA TGACATGTTT TGAAACTAAT 240AATAGATATG ATATTAAAAA CAACTTAGAC CAGATTGGTT TTACATTTGT AACCGAAGAC 300ACAGATGACG GTTACCCGGG AGGAACTGCC TATCGGACAC TAAGGCCCTT CGGTCTCCGG 360GTCACTGATT GTATGGGCCG AGAAATCATG ACAATGCAGA GACCCTTCAG ATGCACCTGC 420TGTTGCTTCT GTTGCCCCTC TGCCAGACAA GAGCTGGAGG TGCAGTGTCC TCCTGGTGTC 480ACCATTGGCT TTGTTGCGGA ACATTGGAAC CTGTGCAGGG CGGTGTACAG CATCCAAAAT 540GAGAAGAAAG AAAATGTGAT GAGAGTTCGT GGGCCATGCT CAACCTATGG CTGTGGTTCA 600GATTCTGTTT TTGAGGTCAA ATCCCTTGAT GGCATATCCA ACATCGGCAG TATTATCCGG 660AAGTGGAATG GTTTGTTATC AGCAATGGCA GATGCTGACC ATTTTGACAT TCACTTCCCA 720CTAGACCTGG ATGTGAAGAT GAAAGCCATG ATTTTTGGAG CTTGCTTCCT CATTGACTTC 780ATGTATTTTG AAAGATCTCC ACACAACGTT CAAGATAGAG AGACACAGCA AGCCATCACC 840TATGGTTAAT TTTGAAAAAT GGAAAAGTTG GATTGGGCTT ACAGTCAGCA CT 892 ( 2 ) SEQ ID NO.4： ( i ) ：
(A) length: 243 amino acid
(B) type: amino acid
( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4：Met Pro Gly Pro Thr Pro Met Ala Asn Cys Pro Pro Gly Leu Glu 15Tyr Leu Val Gln Leu Asp Asn Ile His Val Leu Gln His Phe Glu 30Pro Leu Glu Met Met Thr Cys Phe Glu Thr Asn Asn Arg Tyr Asp 45Ile Lys Asn Asn Leu Asp Gln Ile Gly Phe Thr Phe Val Thr Glu 60Asp Thr Asp Asp Gly Tyr Pro Gly Gly Thr Ala Tyr Arg Thr Leu 75Arg Pro Phe Gly Leu Arg Val Thr Asp Cys Met Gly Arg Glu Ile 90Met Thr Met Gln Arg Pro Phe Arg Cys Thr Cys Cys Cys Phe Cys 105Cys Pro Ser Ala Arg Gln Glu Leu Glu Val Gln Cys Pro Pro Gly 120Val Thr Ile Gly Phe Val Ala Glu His Trp Asn Leu Cys Arg Ala 135Val Tyr Ser Ile Gln Asn Glu Lys Lys Glu Asn Val Met Arg Val 150Arg Gly Pro Cys Ser Thr Tyr Gly Cys Gly Ser Asp Ser Val Phe 165Glu Val Lys Ser Leu Asp Gly Ile Ser Asn Ile Gly Ser Ile Ile 180Arg Lys Trp Asn Gly Leu Leu Ser Ala Met Ala Asp Ala Asp His 195Phe Asp Ile His Phe Pro Leu Asp Leu Asp Val Lys Met Lys Ala 210Met Ile Phe Gly Ala Cys Phe Leu Ile Asp Phe Met Tyr Phe Glu 225Arg Ser Pro His Asn Val Gln Asp Arg Glu Thr Gln Gln Ala Ile 240Thr Tyr Gly 243 ( 2 ) SEQ ID NO.5 ( i )
(A) length: 30 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:TTGCGGATCC ATGCCAGGGC CAACTCCTAT 30 (2) SEQ ID NO.6
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.6:GTTCGTCGAC TTAACCATAG GTGATGGCT 29 (2) SEQ ID NO.7
(i) sequence signature
(A) length: 30 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.7:TTGCAAGCTT ATGCCAGGGC CAACTCCTAT 30 (2) SEQ ID NO.8
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO.8:GTTCGGATCC TTAACCATAG GTGATGGCT 29
Claims (14)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people PLS2 protein-active,
Show at least 70% homology from the nucleotides sequence of Nucleotide 118-849 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 118-849 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 118-849 position.
4. isolating people PLS2 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. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.
9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.
10. a generation has the method for the polypeptide of people PLS2 protein-active, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people PLS2 protein-active operationally is connected in expression regulation sequence, form people PLS2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 118-849 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 PLS2;
(c) under the condition that is fit to expressing human PLS2 protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people PLS2 protein-active.
11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 118-849 position among the SEQ ID NO.3.
12. energy and the described people PLS2 of claim 4 protein polypeptide specificity bonded antibody.
13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.
14. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the described dna molecular of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98123826 CN1259574A (en) | 1998-10-29 | 1998-10-29 | New human phosphatide transferase, its code sequence, prepn. and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98123826 CN1259574A (en) | 1998-10-29 | 1998-10-29 | New human phosphatide transferase, its code sequence, prepn. and use thereof |
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ID=5228370
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|---|---|---|---|
| CN 98123826 Pending CN1259574A (en) | 1998-10-29 | 1998-10-29 | New human phosphatide transferase, its code sequence, prepn. and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001087942A1 (en) * | 2000-04-29 | 2001-11-22 | Shanghai Biowindow Gene Development Inc. | A novel polypeptide -human phosphatidase 13 and the polynucleotide encoding said polypeptide |
| WO2001096362A1 (en) * | 2000-05-16 | 2001-12-20 | Shanghai Biowindow Gene Development Inc. | A novel peptide --- human phosphatidase 16 and the polynucleotide coding this novel peptide |
-
1998
- 1998-10-29 CN CN 98123826 patent/CN1259574A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001087942A1 (en) * | 2000-04-29 | 2001-11-22 | Shanghai Biowindow Gene Development Inc. | A novel polypeptide -human phosphatidase 13 and the polynucleotide encoding said polypeptide |
| WO2001096362A1 (en) * | 2000-05-16 | 2001-12-20 | Shanghai Biowindow Gene Development Inc. | A novel peptide --- human phosphatidase 16 and the polynucleotide coding this novel peptide |
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