CN1305525A - Phagocytic assay method - Google Patents
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- CN1305525A CN1305525A CN99807222A CN99807222A CN1305525A CN 1305525 A CN1305525 A CN 1305525A CN 99807222 A CN99807222 A CN 99807222A CN 99807222 A CN99807222 A CN 99807222A CN 1305525 A CN1305525 A CN 1305525A
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Abstract
The invention provides assay methods for determining whether a compound is an enhancer or an inhibitor of a signal transduction pathway which promotes phagocytosis of apoptotic cells. The methods involve exposing a phagocytic cell to apoptotic cells, optionally transfected with a reporter gene, and measuring the extent of phagocytosis in the presence or absence of the test compound. Expression vectors are provided to transfect mammalian cells with DNA sequences which when expressed influence the rate of phagocytosis of apoptotic cells such as the human homologue of the C. elegans ced-6 gene.
Description
The present invention relates to apoptosis or apoptosis field, relate in particular to the phenomenon that apoptotic cell is engulfed rapidly by other cell.The invention provides and be used to measure analytical procedure and the material that apoptotic cell is engulfed.These analyses can be used for determining to influence the chemical substance that potential pharmacological activity is engulfed and had to apoptotic cell.Analysis of the present invention is applicable to substratum that carries out with porous plate and high flux screening.
In tissue development and maintenance process, a large amount of cells are gone through programmed death or apoptosis.In vertebrates and invertebrates, all can be observed.For example, in nematode C.elegans, 131 cell experience programmed deaths (Lui and Hengartner (1997) 1997 international worm meetings, summary 371).The cracking of apoptotic cell is potential deleterious, causes that content may cause the toxic damages to surrounding tissue.Also degraded by other cytophagies because of apoptotic cell thereupon, therefore can avoid this deleterious effect.In Mammals, phagocytic cell is professional or half professional phagocytic cell, and as neutrophil(e) cell or phagocytic cell, or phagocytic cell is the adjacent cells of dead cell.
One of key characteristic of apoptosis or apoptosis, be the dead cell speed being discerned by phagocytic cell and engulf (Sayill, J. etc., today immunology, 14:131-136,1993).Apoptosis triggers the incident of a series of uniquenesses, expresses as the cell surface phosphatidylserine, and dna fragmentationization or ladderization and membrane-bound cell fragment claim release (Cohen, immunologys such as J.J. summary annual, 10:267-293,1992. of apoptosis vesicle and corpusculum again; Kerr J.F.R. etc., Britain's cancer magazine, 26:239,1972.).Apoptotic cell and corpusculum by identification phosphatidylserine and other engulfed at the not isoacceptor of the undetermined part of the uniqueness on the surface of apoptosis material (Savill, J.S. etc., Journal of Clinical Investigation, 83:865-875,1989; Fadok, V.A. etc., Journal of Immunology, 148:2207-2216,1992.Savill, J. etc., nature, 343:170-173,1990.).By this kind mode, the apoptotic cell that contains potential inflammatory factor is removed and the reaction that do not cause inflammation (Fadok, V.A. etc., Journal of Clinical Investigation, 101:890-898,1998) rapidly by the adjacent cells of half professional phagocytic cell or macrophage system.
The process of apoptosis and multiple human disease-related comprise cancer, autoimmune disease, multiple neurodegenerative disease, as amyotrophic lateral sclerosis, huntington chorea, and senile dementia, apoplexy, myocardial infarction and acquired immune deficiency syndrome (AIDS) (Thompson, CB, science 267, pp1456-1462).Therefore, more and more many concerns are won in the research of apoptosis mechanism and this machine-processed gene of control, people try hard to develop the new methods of treatment to these diseases.
Some disease is with to engulf the damage that apoptotic body causes relevant.Such disease comprises autoimmune disease such as systemic lupus erythematous (Herrmann, M. etc., rheumatic arthritis, 41:1241-1250,1998.), AIDS (Zocchi, M.R. etc., AIDS, 11:1227-1235,1997.), acute lung infects (Cox, G. etc., U.S.'s breathing magazine, cellular elements biology, 12:232-237,1995) and anaphylaxis (Ying, S. etc., American physician federation proceedings, 109:42-50,1997).Clearly, the adjusting of apoptotic cell being engulfed by medicine is the direction of following treatment.
The phagolysis of apoptotic cell is very complicated process in the vertebrates, it be not immediately clear how signal that dead cell produces is received by phagocytic cell and conduct.
Observe engulfing rapidly of apoptotic cell in hermaphroditic nematode, this worm provides the effective means of research phagocytosis.For example, 6 genes that the influence of differentiating in nematode is engulfed, ced-1, ced-2, ced-5, ced-6, ced-7 and ced-10.The ced-6 that chooses separately of the present invention studies especially.Known ced-6 is at daf-4 (Lui and the Hengartner (1997) of the close nematode of collection of illustrative plates of karyomit(e) III; Lui and Hengartner (1996) East Coast worm conference summary 128).This work shows two clays from this district, and F56D2 and F43F12 can save nematode ced-6 (n1813) and engulf disappearance.It is active to have a rescue from the subclone of the 10kbXho I of F56D2, carries the ced-6 gene simultaneously.
The present inventor has differentiated 2 human homology's things of nematode ced-6, and (hlced-6 and h2ced-6), h2ced-6 are the shearing variants of h1ced-6, therefore as linear negative model.Two kinds of homologues all are present among the human cell line THP-1.Between hCED-6 and the nematode CED-6 surprising sequence homology is arranged.HCED-6 has phosphatidyl tyrosine land, from amino acid/11 1-amino acid/11 90, as shown in Figure 4, illustrates relevant with the tyrosine kinase signal conduction path.
H1CED-6 and h2CED-6 albumen and their coding nucleic acids can be used for carrying out the analysis of this paper, identify as the inhibitor that promotes the phagocytotic signal transduction pathway of apoptotic cell or the compound of toughener.Especially, they are at inhibitor or the toughener of differentiating h1CED-6 and h2CED-6, or in its inhibitor of transcribing or the toughener process effectively.These inhibitor or toughener can be used as useful therapeutical agent and are used for aforesaid treatment of diseases.
A first aspect of the present invention provides a kind of can express h1CED-6 or h2CED-6 expression vector, and this carrier comprises the aminoacid sequence of code pattern 4 or Fig. 5 or compares the deoxynucleoside acid sequence of the aminoacid sequence of the variation that the biological function conservative property only takes place with the amino acid of Fig. 4 or Fig. 5.
The term " biological function " of this paper definition refers to regulate or influence the phagocytotic ability of apoptotic cell." conservative " though the amino acid variation is that those are more high or low than the proteic biological function level of wild-type people CED-6, the variation that can keep biological function.These conservative variations comprise that one or more aminoacid insertion or disappearance or one or more amino acid are replaced by other amino acid or acid with similar chemical property.The method that amino acid generation conservative property is changed is well known to those skilled in the art.
In a preferred embodiment, expression vector is the carrier that comprises Fig. 2 or the deoxynucleoside acid sequence from the transcription initiation codon to the Transcription Termination codon shown in Figure 3 and comprise the deoxynucleoside acid sequence shown in Fig. 2 or Fig. 3 arbitrarily.
In an especially preferred embodiment, expression vector of the present invention comprises the nucleotide sequence of the reporter gene of encoding, and the expression of h1CED-6 or h2CED-6 can cause this report expression of gene.This reporter gene can place 3 ' or the 5 ' end of h1ced-6 or h2ced-6, can be used as the expressing fusion protein with h1CED-6 or h2CED-6.Suitable reporter gene is to express fluorescence-causing substance such as green fluorescent protein (GFP) gene.Other suitable reporter gene has enzyme, and as beta-galactosidase enzymes or luciferase, these enzymes can be crossed and act on substrate and produce detectable product, for example fluorescence-causing substance or luminous product.The example of expression product has pGA3103 and the pGA3104 that shows respectively in Figure 29 and 10 according to the present invention.
In another preferred embodiment, expression vector of the present invention is at h1CED-6 and proteic amino of h2CED-6 and/or C-terminal expression epi-position label.Plasmid pBAD/HisA-h1ced-6 for example, its sequence as shown in Figure 17.
Above-mentioned expression vector not only comprises the sequence of coding h1CED-6 and h2CED-6 or its functional variant, also comprises the exercisable adjusting sequence that links to each other with nucleic acid, if can influence dna fragmentation expression promoter district.Term " exercisable connection " refers to a kind of relation arranged side by side, and wherein the component of describing allows sequence to bring into play function by way of expectations.
Express required regulatory element and comprise with the RNA polymerase and combining, instruct transcription initiation proper level and promoter sequence and with rrna bonded translation initiation sequence.For example, bacterial expression vector can comprise the ribosome binding sequence and the initiator codon AUG of promotor such as lac promotor and translation initiation.Similar, carrier for expression of eukaryon can comprise the allos or the homologous promoter of RNA polymerase II, downstream polyadenylation signal, the terminator codon that initiator codon AUG and rrna break away from.These carriers can obtain or use method assembling well known in the art to describe from the commercial channel sequence.The promoter sequence that is used for expression vector comprises Δ HSP, CMV, SV40, EF-1 α, UbC, SG, RSV, TRE/minCMV, HSV TK, 5 ', LTR and QBISP136 enhanced CMV.
The embodiment of expression vector described herein is a plasmid, but also can be virus or phage vector.These carriers have the gene of a replication orgin and one or more selectable marker such as antibiotics resistance usually.Especially preferred expression vector of the present invention is suitable for the mammalian cell transfection, thereby provides corresponding selective marker for carrier.
A second aspect of the present invention provides with above-mentioned expression vector mammalian cells transfected system.The method of transfection mammalian cell is well-known.This clone can be grown in monolayer culture or suspension culture.Suitable cell is fibroblast or epithelial cell line such as COS1, BHK21, L929, pc12, CV1, SWISS3T3, HT144, IMR32, HEPG2, MDCK, MCF7,293, Hela, A549, SW48 or G361.Also available primary cell line such as human skin FIBs, skin keratin forms cell, white corpuscle, monocyte, lymphocyte, dendritic cell or scavenger cell.Engulf in the analysis of the present invention, especially professional the or half professional phagocytic cell of the Mammals of preferred expression h1CED-6 and h2CED-6 is J774 or human monocyte cell line THP1 (seeing embodiment 3 and Fig. 6) as mouse macrophage.More than two kinds of clones can be used as monocytic series, analyze used scavenger cell because of monocytic series can be divided into the present invention.
Third aspect present invention provides to be measured compound and is the toughener that promotes the signal transduction pathway that apoptotic cell is engulfed or the method for inhibitor, this method comprises that the mammalian cell that makes transfection that above-mentioned h1ced-6 or h2ced-6 be arranged is exposed to the apoptosis particle, measures particulate and engulf speed when compound exists or lack.Testing compound preferably adds before the apoptosis particle.
Suitable apoptosis particle is the cell that apoptosis takes place and depend on the needs and nurse one's health in serum, as neutrophilic granulocyte, and lymphocyte, red corpuscle, or dendritic cell.Be suitable for forming apoptosis particulate cell and comprise clone L929 and PC12.A kind of apoptosis particulate clone that is preferably used as especially is the mouse cell line Ba/F3 that somatomedin relies on.They can be grown in standard medium as described in the embodiment 5, can growths when growth factors I L-3 disappearance appropriate time (for example about 20 hours), and apoptosis-induced.The apoptosis state can be used, for example, and the kit measurement of annexin/propidium iodide mark of BoeringherMannheim (Brussels, Belgium).If about 20% annexin positive, and be no less than 5% propidium iodide feminine gender thinks that then cell is an early apoptosis.
PC12 clone can be in the standard medium of nerve growth factor disappearance the growth inducing apoptosis.
Remove above-mentioned extracellular, the apoptosis particle can be non-active material, as the latex particle of dye marker.The 0.1 μ M that has amino or carboxyl, 1 μ M, 4 μ M and 10 μ M particles can be available from Sigma-Aldrich, Bornem, Belgium, or Molecular Probes, Eugene, the U.S..
For making described analysis be suitable for high-throughout screening compound, preferred apoptosis particle carries certain detectable mark, and is then when the transfected mammalian cell of this particle absorbs, very obvious, can quantize.The inventor finds and can comprise that the expression vector stable transfection of reporter gene comprises apoptosis particulate cell by using, thereby realizes above-mentioned purpose.A specially suitable reporter gene coding beta-galactosidase, its can cracking fluorogenic substrate fluorescein two-fluorescent chemicals that b-D-galactopyranose glycosides detects for the available standards fluorescence detection device.Other fluorogenic substrate also can be used for beta-galactosidase enzymes.Plasmid pcDNA3.1/HIS/lacZ expresses beta-galactosidase enzymes and is suitable for transfection as apoptosis particulate cell, Ba/F3 for example, as shown in figure 11.Other suitable reporter gene is the proteic gene that coding fluorescence albumen such as green fluorescent protein maybe can produce luminous signal such as luciferase.Plasmid pEGFP-N3 or pEGFP-C2 (Clontech) are suitable for GFP-transfected to being used as apoptosis particulate cell, as Fig. 7, shown in 8,26 and 27.The plasmid of Promega " PGL contrast " is suitable for the gene of the plain enzyme of transfection coding fluorescence to being used as apoptosis particulate cell such as Ba/F3 cell.The dna sequence dna of " PGL contrast " as shown in figure 19.
Preferably the selection of apoptosis particulate reporter gene is controlled by the existence of mammalian cells transfected reporter gene.For example, the existence of same report gene in the mammal nuclear apoptotic cell of transfection h1 or h2ced-6, because of signal overlap not by preferably, although this situation is few.
More preferably can detect multiple compound, survey it for promoting the toughener or the inhibitor of the signal transduction pathway that apoptotic cell is engulfed.Compound can be any chemical formula, polymer or monomer.For example the compound of Jian Ceing can be genomic dna, cDNA, RNA, PNA, albumen or polypeptide, amino acid, nucleosides or Nucleotide.Compound can be known organism or pharmacologically active, unknown active known compound or the recruit who exists in the compound combinatorial libraries.
The amount that causes the transfected mammalian cell of apoptosis particle to be engulfed when the existence of any compound reduces or when not engulfing, should detect the viability of cell.The existence of viable cell confirm the shortage of engulfing because of the survey compound to the influence of activate the phagocytic capacity but not its non-specific toxicity.
Further, whether any is the inhibitor engulfed of apoptotic cell or the compound of toughener through above-mentioned Analysis and Identification, all will further detect this influence and mediate by CED-6.After identifying that a kind of compound is the apoptotic cell inhibitor or toughener engulfed, can be by carrying out the above-mentioned analysis of engulfing, but mammalian cell untransfected h1ced-6 or h2ced-6.If this compound illustrates that inducing the phenotype identical with the cell of transfection h1ced-6 in the cells transfected this compound not necessarily plays a role by CED-6 or CED-6 signal transduction pathway.
Similar, if the compound of the inhibitor that a kind of above-mentioned Analysis and Identification is an apoptotic cell engulfs, the phenotype that is tested and appraised the mammalian cell of the untransfected that is exposed to this compound confirms that whether its effect is by CED-6 or CED-6 signal transduction pathway.Recovery to the wild-type phenotype means that reaction is by CED-6 or CED-6 signal transduction pathway.
Fourth aspect present invention provide be identified for detecting a kind of compound whether be promote to engulf apoptotic cell signal transduction pathway the promotion thing or the method for inhibitor, this method comprises the following steps:
(1) to the mammalian cell microinjection comprise Fig. 4 or aminoacid sequence shown in Figure 5 or with Fig. 4 or aminoacid sequence shown in Figure 5 only have function conservative property amino acid change sequence people CED-6 albumen and
(2) mammalian cell with step 1 preparation is exposed to the apoptosis particle, measures particle by the speed of engulfing of cell when compound exists and lack.
Preferably, comprise the fusion rotein of h1Ced-6 or h2CED-6 and reporter gene to the mammalian cell microinjection, reporter gene can be above-mentioned any reporter gene.Preferred fusion protein obtains by the GFP and the h1ced-6 encoding sequence of expressing the plasmid shown in Fig. 9 or 28.
The feature and the embodiment of the mammal cell line analysis of above-mentioned all transfections can be used for the above-mentioned cell with h1Ced-6 or h2CED-6 or its fusion rotein microinjection.
Fifth aspect present invention be provided for detecting a kind of compound whether be promote to engulf apoptotic cell signal transduction pathway the promotion thing or the method for inhibitor, this method comprises the following steps:
(1) to the carrier of the RNA of all or part antisense of mammalian cell microinjection or transfection expression Fig. 2 or nucleotide sequence shown in Figure 3;
(2) mammalian cell with step 1 preparation is exposed to the apoptosis particle, measures particle by the speed of engulfing of cell when compound exists and lack.
Preferably, this sense-rna comprise can with the nucleotide sequence of Fig. 2 or nucleotide sequence hybridization shown in Figure 3, the stringency of hybridization is higher than 2 * SSC; 0.1%SDS; 25 ℃ to 50 ℃.
All preferred characteristics of the analysis of above-mentioned useful mammalian cells transfected system and the clone that embodiment may be used on the sense-rna injection.
Be preferred for the mammalian cells transfected transfection h1ced-6 or the h2ced-6 of above-mentioned analysis.Because of h2ced-6 is regarded as the recessive form of the dominance of h1ced-6, have opposite biological action, cell depends on the selection of transfection wishes to differentiate the inhibitor engulfed as apoptotic cell or the compound of toughener.For example the cell of transfection h1ced-6 is particularly suitable for the inhibitor of differentiating that apoptotic cell is engulfed, and the cell of transfection h2ced-6 is particularly suitable for differentiating toughener.
Therefore illustrate and the present invention also relates to, be accredited as and promote apoptotic cell to engulf the inhibitor of signal transduction pathway or any compound of toughener according to any analytical procedure described herein.
The nucleotide sequence of h1ced-6 and h2ced-6 is respectively shown in Fig. 2 and 3.In addition, the cDNAs of coding alternative splicing h2CED-6 and be preserved in Belgian microorganism according to budapest treaty on June 8th, 1998 from the insertion fragment of h2ced-6 reorganization h1ced-6 and coordinate preservation center (BCCM) Molecular Biology Lab-plasmid preservation (LMBP) B9000, Gent, Belgium, preserving number is respectively 3868 and 3869.
From using T2 or T7 primer to obtain sequence (available or use 2 primers simultaneously) from depositing species in different actual insertion sites.Primer can be available from Clontech (～1227 and～1228), and sequence is as follows:
T7 primer: 5 ' (TAATACGACTCACTATAGGGAGA) 3 '
T3 primer: 5 ' (ATTAACCCTCACTAAAGGGA) 3 '
Outside developing based on the analysis of crossing expression or the proteic mammalian cell of low expressing human CED-6, the present inventor identifies the epi-position of h1CED-6 and has prepared its useful antibody.
Sixth aspect present invention comprises the people Ced-6 protein fragments with aminoacid sequence shown in Figure 4, and wherein fragment comprises aminoacid sequence HRAFSRKKDKTC, FLGSTEVEQPKGTE or TRNGTQPPPVPSRST.Preparation comprises the antibody of above-mentioned one or more epi-positions.Press the method acquisition antibody preparations that embodiment 6 describes, their specificity such as embodiment 7 use WesternBlots to confirm (Figure 20 to 25).
Above-mentioned antibody be used in phagocytic cell cross express or patient that low expressing human Ced-6 is relevant in the method that diagnoses the illness.Especially, this paper provides a kind of method to be used for diagnosing the relevant disease of crossing expression or low expressing human Ced-6 at individuality and phagocytic cell, comprising:
(a) obtain cytophagous sample from individuality;
(b) expose phagocytic cell and give aforesaid antibody preparations;
(c) existence of any immunocomplex that between antibody and Ced-6 albumen, forms of quantitative measurment; And
(d) with the amount of using the immunocomplex that relatively forms from the immunocomplex that contrasts individual phagocytic cell formation.
It is the analysis that promotes that apoptotic cell is engulfed the inhibitor or the toughener of signal transduction pathway that above-mentioned antibody can be further used for measuring a kind of compound.Especially, this paper provides a kind of method, and whether be used to measure a kind of compound is to promote apoptotic cell to engulf inhibitor or the toughener that signal forwards path to, and this method comprises:
(a) expose the aforesaid expression vector mammalian cell of transfection and give testing compound;
(b) expose mammalian cell and give above-mentioned a kind of antibody preparations;
(c) existence of any immunocomplex of formation is seen in quantitative measurment at the albumen of antibody and cell expressing; And
(d) with the level of the immunocomplex of the amount comparison detection that is not exposed to the immunocomplex of surveying in mammalian cells transfected compound, that describe as step (a).
In aforesaid method, mammalian cell can be selected from COS1, BHK21, and L929, CU1, SWISS 3T3, HT144, IMR32, HEPG2, MDCK, MCF7,293, Hela, A549, SW48 or G361, the COS1 cell is especially preferred.Mammalian cell also can be human skin FIB, people's keratinocyte, white corpuscle, monocyte, lymphocyte, dendritic cell or scavenger cell.Preferred professional phagocytic cell such as mouse macrophage clone J774 or person monocytic cell's clone THP-1.
Other application of antibody of the present invention comprise h1CED-6 purifying and with the interactional proteic discriminating of CED-6, but then signal transduction pathway characterization, also comprise survey that crossing of hCED-6 expressed or lowly express, location or posttranslational modification in the cell, epitope mapping and activation site and in suitably carrier or thinner, comprise the evaluation of the pharmacology composition of described antibody.
Seventh aspect present invention comprise a kind of be used for diagnosing crossing with phagocytic cell people CED-6 at individuality express or the low method of expressing relevant disease, this method comprises:
(a) obtain the phagocytic cell sample from individuality,
(b) from the phagocytic cell isolation of RNA,
(c) prepare cDNA from RNA,
(d) in cDNA, carry out one-level PCR reaction,
(e) on the reaction product of one-level PCR reaction, carry out secondary (nested type) PCR,
(f) existence of and quantitative measurment people ced-6 RNA qualitative by the product of analyzing firsts and seconds PCR, and
(g) reaction product of the phagocytic cell formation of reaction product that will form in firsts and seconds PCR and use contrast individuality compares the amount and the type of product.
Preferably, use the sequence deutero-primer of the h1ced-6 of this paper definition or h2ced-6 or carry out PCR from the deutero-primer that cDNA produces the carrier that uses.Especially, described one-level PCR can be with the primer with following nucleotide sequence:
1)cgcaaggatccccatgaaccgtgcttttagcaggaag
2)gatctactaggtactggag
Secondary PCR can be with the primer with following nucleotide sequence:
1)cgcaaggatccccatgaaccgtgcttttagcaggaag
2)gcggatggtaccgtcgactgctgatacttgagttattctcag
Analytical procedure described herein, improve by the present inventor and whether to be used to measure toughener or the inhibitor of compound behaviour CED-6, can be common to evaluation influences the compound that apoptotic cell is engulfed under any mechanism, and not only relates to people CED-6 or serve as the signal transduction pathway of component by it.
Liu etc.(Liu, Y. etc., AAi, 1:1999) having described a kind of the evaluation influences the compound that apoptotic cell is engulfed, and wherein the compound of different concns joins phagocytic cell, inserts apoptotic cell subsequently in phagocytic cell.Be engulfing of quantification apoptotic cell, the micro-quantification of author to engulfing, wherein the absorption to apoptotic cell is shown by electron microscope, and the minimum cell (Sayill of the every slide that calculates with the optics microscopic counting, J.S.Wyllie, A.H.Henson, J.E.Walport, M.J.Henson, P.M.Haslett, C., J Clin Inyest, 83:865-875,1989).
Becoming known for quantizing the technology that apoptotic cell engulfs is not easy compound is carried out high material throughput screening potential pharmacologic activity.Main because known analytical technology depends on when being exposed to detection compound, to the cytophagous ratio microscopic count of picked-up apoptotic cell.But the present invention overcomes this shortcoming, because of they can carry out in the porous analysis plates, and provides the probe microscope system that do not need.
Therefore, the present invention further provides a kind of method, identify whether a kind of compound is toughener or the inhibitor that apoptotic cell is engulfed, and it comprises:
A) exist or during disappearance at testing compound, can produce the professional or professional phagocytic cell partly of the mammalian cell exposure Mammals of reporter gene apoptosis of the signal that non-microscope surveys to stable transfection,
B) remove not any apoptotic cell of being engulfed by phagocytic cell also
C) signal of any next Autophagocytic reporter gene of detection;
Wherein when compound exists and any signal difference explanation compound in compound when disappearance be toughener or the inhibitor that apoptotic cell is engulfed.
Usually, preferred earlier with the phagocytic cell and the compound incubation of surveying in aforesaid method, add apoptotic cell then.The suitable incubation time is 15-30 minute.
The suitable phagocytic cell that uses method of the present invention is as mouse J774 cell or people THP-1 cell, and is as described herein.These cells are monocytic series, but make it to be divided into scavenger cell through cultivation before adding apoptotic cell.
The apoptotic cell that is used for aforesaid method can be the neutrophil(e) cell of apoptosis, the lymphocyte of apoptosis or the red corpuscle of apoptosis.Apoptotic cell can be chosen wantonly by being exposed to serum and nurse one's health.Preferred apoptotic cell be attached cell be L929 or PC12 and, the mouse cell line Ba/F3 that somatomedin described herein relies on.
Utilization aforesaid method stable transfection reporter gene is to apoptotic cell.The special problem that the utilization reporter gene can run into is an expression of gene in effectively dead apoptotic cell, than weak a lot of in the complete viable cell.If or cell is in and joins in the cytophagous apoptosis particle, insufficient particle of not engulfing of washing can be covered any signal of the cell of engulfing from apoptosis from the signal of viable cell.Though can be with after scouring, the present inventor has improved a kind of special embodiment and has avoided this problem.
Especially, it comprises that use is expressed in transit cell and changes low albumen, the reporter gene of preferred enzyme, and then viable cell has identical approximately protein concentration or enzymic activity with the apoptosis particle.Thereby overcome above-mentioned shortcoming.Several possible reporter proteins and substrate be at fluorescent probe and research pharmaceutical chemicals handbook, and P.Haugland compiles that (OR has description in USA) for Molecular Probes, Eugene.The present inventor finds that also beta-galactosidase enzymes (lacZ) is specially suitable reporter gene.This enzyme is low relatively to be upgraded, and cell has the activity of relative same amount with the apoptosis particle.Have several substrates (seeing Molecular Probes, Eugene, or USA) that are suitable for beta-galactosidase enzymes in addition, the present inventor uses above-mentioned FDG.The compound that it engulfs a kind of high material throughput screening of development change apoptosis particle becomes possibility.
The phagocytic cell that method of the present invention is used can be wild-type cell or transgenosis or mutant cell.The cell of sudden change is compared with wild-type, can reduce or increase activate the phagocytic capacity.But the transgenic cell stable transfection influences the gene of the speed of apoptotic cell activate the phagocytic capacity when expressing.For example, h1ced-6 that the mammalian cell transfection is above-mentioned or h2ced-6 preferably use any carrier of mentioning in description or accompanying drawing.
In another embodiment of the present invention, but the DNA of phagocytic cell transfection Codocyte surface antigen CD36.The human macrophage of utilization phosphatidylserine receptor or glass connection protein receptor is engulfed the expression that apoptotic cell needs CD36.(Fadok V.A. etc., Journal of Immunology 1998 Dec1.161 (11): 6250-7).Transfection can adopt any method described herein to carry out, the preferred carrier of the dna sequence dna that comprises coding CD36 as shown in figure 31 or the complete vector of Figure 31 of using.
Method of the present invention is all carried out in porous plate, in therefore being particularly suitable for-and high flux screening.In a preferred embodiment, use 96 orifice plates, but the present invention also can use the orifice plate of other number, includes but not limited to 6,12,24,384,864,1536 orifice plates.
All methods of the present invention need be surveyed when testing compound exists the signal that the quantification apoptotic cell is engulfed.The key features of method of the present invention is that this signal (also claiming indicating value) uses non-visual detection method to detect.Term used herein ' non-visual detection method ' refers to a kind of method that does not need human eye to comprise the signal of microscope visual inspection of any detection.The utilization of non-visual detection system is main advantage, and surmounting knownly needs pair cell to carry out visual inspection by eyes to detect apoptotic cell by the screening method of cytophagous absorption.
For carrying out non-visual detection to apoptotic cell in engulfing the height of analysis-middle through-put screening, reporter gene must be able to produce can be by automatic plate count device, as the signal of victor2 (Wallac, Turku, Finland) detection.Most preferably detect the automatic plate count device of fluorescent signal.
By producing the signal of automatic porous plate meter reading, measures of quantization can be carried out, thereby the effect of multiple compound can be once evaluated, and the effect of comparative compound.
Need point out further that testing compound can be the compound of above-mentioned any kind, the signal when specific compound causes phagocytic cell to reduce or disappear will detect cytophagous vigor, thereby get rid of the non-specific toxicity reason of compound.
In following non-restrictive example, with reference to figure below
Fig. 1 is from EST, the structure (seeing embodiment 1) of the consensus sequence of the 2416bp that RACE and colony hybridization obtain.The following assembling of sequence is a template with aa159394, and primer is shown in multiple sequence.The complement that the rcc representative reverses.Visible ced-6 and hced-6 in multiple sequence; PGA101 is selected by colony hybridization.
Fig. 2 shows the total dna sequence dna (2416bp) of h1ced-6.Enlightenment and terminator codon are black matrix and underscore.The alternative splicing district is underscore also.
Fig. 3 shows the dna sequence dna of the alternative splicing of h2ced-6.Enlightenment and terminator codon are black matrix and underscore.
Fig. 4 shows the aminoacid sequence of h1CED-6.The residue number: 304, molecular weight 34.4kDa.The alternative splicing district is underscore also.
Fig. 5 shows the aminoacid sequence of h2CED-6, the alternative splicing district.
Fig. 6 shows the gel analysis to the nested PCR product of describing among the embodiment 3; Swimming lane is respectively (1) 100bp marker, (2) elementary neutrophil(e) cell alive, (3) elementary apoptosis neutrophil(e) cell, (4) elementary scavenger cell, (5) and the elementary scavenger cell that withers and act on toward the neutrophil(e) cell, (6) J774, (7) COS-1, (8) THP-1
Fig. 7 shows the dna sequence dna of the Clonetech carrier pEGFP-N3 that comprises reporter gene GFP that uses in embodiment 4 and 8;
Fig. 8 shows the plasmid map of pEGFP-N3;
The dna sequence dna of the plasmid pGA3104 that uses in Fig. 9 diagrammatic sketch 4 and 8 comprises h1ced-6 in the multiple clone site of pEGFP-N3;
Figure 10 shows the plasmid map of plasmid pGA3104;
Figure 11 shows the dna sequence dna (seeing embodiment 4) of the plasmid pcDNA3.1/His/LacZ that can obtain from the commercial channel that is used for stable transfection Ba/F3 cell;
Figure 12 shows fluorescence intensity, and when beta-galactosidase enzymes and fluorogenic substrate fluorescein-b-D-galactopyranose glycosides (FDG) reaction, fluorescence intensity is as cells transfected per-cent;
Figure 13 shows the influence of FDG concentration to the reading of the analysis of embodiment 5;
Figure 14 temperature indicating is educated the influence of time to the reading of the analysis of embodiment 5;
Figure 15 shows that serum-concentration is to the influence of the analysis of embodiment 5 in the Ba/F3 cell cultures;
Figure 16 shows the location in the epi-position of h1CED-6 that is used for producing polyclonal antibody;
Figure 17 shows the dna sequence dna (pBAD/HisA/-hced-6) of the plasmid pGA1028 that embodiment 7 is used;
Figure 18 shows the plasmid map of pGA1028;
Figure 19 shows the dna sequence dna of the commercial plasmid pGL2 of the reporter gene luciferase that is suitable for changing over to the Ba/F3 cell;
Figure 20 to 25 shows the result that mark is drawn in immunity among the embodiment 7.In all figure, swimming lane is as follows:
Swimming lane 1: prestained SDS-PAGE lower molecular weight standard (Bio Rad-Hercules, CA, USA),
Swimming lane 2:pBAD/His A (Invitrogen, Leek, The Netherlands)
Swimming lane 3:pGA1028 (pBAD/HisA/-h1CED-6)
Figure 20 shows antibody and the control antibodies Anti-Xpress antibody (Invitrogen that uses at the epi-position EP 990044 that identifies among the embodiment 7, Leek, The Netherlands) and mouse Ig, complete antibody (sheep) stained gel (the ECL Western trace detection reagent and the analytical system of horseradish peroxidase connection, Amersham Pharmacia Biotech, UK, England):
Figure 21 shows antibody and the immune serum stained gel of describing with as embodiment 7 to epi-position 990044;
Figure 22 shows that usefulness is as the antibody of the epi-position 990045 of evaluation among the embodiment 7 and the control antibodies stained gel of describing as Figure 20;
Figure 23 shows as epi-position of describing among the embodiment 7 990045 and immune serum dyeing stained gel;
Figure 24 shows epi-position 990046 and the control antibodies stained gel of describing as among the embodiment 7;
Figure 25 shows epi-position 990046 and the immune serum stained gel of describing as among the embodiment 7
Figure 26 shows the dna sequence dna as the used commercial Clonetech carrier pEGFP-C2 that comprises reporter gene GFP of embodiment 8;
Figure 27 shows the plasmid map of pEGFP-C2;
Figure 28 shows that wherein the multiple clone site at pEGFP-C2 comprises h1ced-6 as the dna sequence dna of the used plasmid pGA3103 of embodiment 8;
Figure 29 shows the plasmid map of pGA3103;
Figure 30 shows from transfection MOCK (negative control of transfection), pEGFP-N3, the cell lysate of the COS-1 cell of pGA3103 and pGA3104; Contrast lysate with effective co-immunoprecipitation of a Ba/F3 cell anti-or that do not educate with a temperature resistance; The Western trace of the positive control lysate of the A431 cell that stimulates from EGF; , use anti--phosphatidyl tyrosine antibody.Trace A detects the albumen of tyrosine-phosphatide acidylate in the cell lysate with mouse monoclonal antibody IgG 2; Trace B detects with the polyclonal antibody of anti-green fluorescent protein; Trace C detects with the rabbit anti-serum of h1Ced-6.The molecular weight of H1CED-6 is 34435.39; The molecular weight of GFP is 26886.32; The molecular weight of fusion rotein GFP-CED-6 or CED-6-GFP is 62385.95;
Figure 31 shows the dna sequence dna of plasmid pGA1058, leads the multiple clone site of pEGFP-N3 comprising the dna sequence dna insertion of Codocyte surface receptor CD36;
Figure 32 shows the plasmid map of pGA1059;
Figure 33 is shown in IL3 and withdraws per-cent in back annexin and the cell colony of propidium iodide positive cell in the Ba/F3 cell;
Figure 34 temperature indicating degree is to the influence of FDF incubation, among the Ba/F3 cell adding scavenger cell clone J774 that live and apoptosis.After engulfing analysis, FDG (10 μ M) is at 4 ℃, 20 ℃ of incubations 1 hour, 37 ℃ of temperature in 10 and 20 minutes.
With ced-6 sequence (the consensus dna sequence dna of Fig. 2 hced-6) to public domain database (EST, Genbank, EMBL, Swissport and PIR) broad research shown the significant homologue (AA443368 of statistics at some ESTS in proteic C-terminal district, AA431995, R33389, R53881).A kind of Est (T48513) shows and the carboxyl terminal in PTB district and the initiator homology of live zone.Marathonready 5 ' RACE analyzes, and cDNA adenocarcinoma of colon storehouse is from Clontech.The position of the primer of utilization as shown in Figure 1 in RACE and the order-checking.By clone and sequential analysis subsequently, obtain extra sequence information.Use this extra sequence information, in database, search for, disclose extra EST, make us be made up the consensus sequence of about 2400bp.This sequence can further enlarge and change by colony hybridization and extra RACE product order-checking.
The RNA trace:
The people is multiple organize Northern (MTN-1, Clontech), every swimming lane comprises respectively from 8 kinds of different people tissues (heart, brains, placenta, lung, liver, skeletal muscle, kidney and pancreas) 2mg poly+RNA and the multiple Northern that organizes of MTN-II people, every swimming lane comprises respectively from spleen, thymus gland, prostate gland, testis, ovary, small intestine, colon and peripheral leukocytes the 2mg poly+RNA, hybridize according to manufacturer's explanation, at 0.1 * SSC, 0.2%SDS washes at 55 ℃.From Clontech, detect human cancer clone (melanoma G361, lung cancer A549 equally, colorectal carcinoma SW480, burkitt's lymphoma Raji, lymphoblast leukemia Molt-4, chronic granulocytic leukemia 62, Hela S3 and former myelocytic leukemia HL60) poly+RNA.
The expression pattern of HCED-6 in health adult tissue and cancer cells is as shown in table 1 through the Northern trace:
A) multiple Northern (MTN) the trace II C that organizes of the people multiple Northern of organizing of people (MTN) trace B)) human carcinoma cell line organizes Northern (MTN more
TM) trace.
Table I A)
B)
C)
The detection of embodiment 3 CED-6 (h1CED-6) and splice variant (h2CED-6) thereof in phagocytic cell system
Clone THP-1 (American type culture collection ATCC number: TIB-202), a kind of person monocytic cell's clone can be used PMA (Sigma-Aldrich, St-Louis, MO, USA) make it to be divided into scavenger cell, cell under standard conditions, contain 2mM L-glutaminate, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10mM HEPES, the 1mM Sodium.alpha.-ketopropionate is cultivated in the RPMI1640 substratum of 0.05mM beta-mercaptoethanol.(medicine is all available from gibcoBRL, Life Technologies, and Merelbeke, Belgium).
According to manufacturer's explanation, or change slightly, use Rneasy mini test kit (Westburg, Leusden, the Netherlands) clone isolation of RNA from then on.
According to manufacturer's explanation, or change slightly, (Pharmacia Biotech Piscataway, NJ USA) produce the first chain cDNA to use the Ready-To-Go T-primed first chain test kit.
The cDNA that produces is used to produce dna fragmentation in the PCR step, use primer: oGA131:5 '-CGCAAGGATCCCCATGAACCGTGCTTTTAGCAGGAAG-3 ' 445-10934-13R:5 '-GATCTACTAGGTACTGGAG-3 ', PCR uses TaKaRa ex Taq test kit (Takara Shuzo Co., LTD, Shiga, Japan) carry out, according to manufacturer's explanation, or change plasmid pGA1025 slightly, comprise the h1ced-6 gene, as positive control.
Brief summary:
PCR produces the first chain cDNA, use the Ready-To-Go T-primed first chain test kit, the first chain cDNA that obtains comprises complete cDNA, other material is 0.4 μ loGA131 (100pmol/ μ l), 0.4 μ l 445-10934-13R (100pmol/ μ l), 0.5 μ l exTaq 5U/ μ l, 65.7 μ l water.
Positive control is carried out PCR, comprise 10 μ l damping fluid exTaq 10 *, 10 μ l dNTP mixing exTaq 10 *, 0.4 μ l oGA131 (100pmol/ μ l), 0.4 μ l 445-10934-13R (100pmol/ μ l), 2 μ l pGA1025,76.2 μ l water.
The PCR program:
The product of each PCR reaction product of 2 μ l and 1 μ l positive control PCR reaction is used to carry out nested PCR, adopts following primer:
OGA131: the same
oGA141：
5’-GCGGATGGTACCGTCGACTGCTGATACTTGAGTTATTCTCAG-3’
PCR uses TaKaRa ex Taq test kit (Takara Shuzo Co., LTD, Shiga, Japan) to carry out, and according to manufacturer's explanation, or changes slightly.
Main mixture: 5 μ l damping fluid exTaq 10 *, 5 μ l dNTP mixing exTaq 10 *, 0.2 μ l oGA131 (100pmol/ μ l), 0.2 μ l oGA141 (100pmol/ μ l), 0.5 μ l exTaq 5U/ μ l, 37.1 μ l water.
Program:
According to standard method 10 μ l nested PCR products are carried out gel analysis (molecular cloning experiment guide, Sambrook etc., 1989, CSHL press).
Elementary neutrophil(e) cell alive, elementary apoptosis neutrophil(e) cell, elementary scavenger cell with the interactional elementary scavenger cell of apoptosis neutrophil(e) cell, repeats above-mentioned steps in mouse monocyte clone J774 and the COS-1 cell.The result as shown in Figure 6.Significantly, only in clone THP-1, can record h1ced-6 and its splice variant h2ced-6 (seeing the swimming lane 8 of Fig. 6).
The stable cell lines of people CED-6
J774 mouse monocyte tumor cell line (Morland and Kaplan, 1978, experimental cytology research .115:53-61; Morland and Kaplan, 1978, experimental cytology research .115:63-72 ATCC registration number TIB67, be called J774.1 again) in DMEM, cultivate, add glutamaxI, 10%myclone serum is (all available from GIBCoBRL, LifeTechnologies, Merelbeke, Belgium), electroporation transfection plasmid pEGFP-N3 (Clonetech, Palo Alto, CA, USA), (Fig. 7 and 8), simulation transfection, pGA3104, h1CED-6/GFP fusions (Fig. 9 and 10), salmon sperm dna; Negative control.
Electroporation Easyject Plus electric perforating system (Equibio Ltd, Immunosource, Halle-Zoersel, Belgium), according to the following steps:
3 * 10
6Cell places 800 μ l cell culture mediums, adds 30 μ g DNA.EasyjectPlus electroporation apparatuss and is made as: two pulse:
Voltage I=750V, capacitor I=25 μ F, resistance I=99R, interpulse delay=0
Voltage I=150V, capacitor I=1500 μ F, resistance I=99R, Optipulse is optional
The cell inoculation of per 3500 μ l electroporations is to 175cm
2In the culturing bottle, the G418 resistance is selected (400 μ g/ml) (Duchefa, Haarlem, The Netherlands) 72 hours.Obtain and detect the clone's of GFP expression subclone.
Phagolysis is analyzed
The preparation phagocytic cell
The monocyte clone J774 of stable transfection pEGFPn3 or pGA3104 or pGA1058 is with 1 * 10
5Density is seeded in black 96 orifice plates 37 ℃, and 5%CO2 engulfs and cultivated 36 hours before analyzing.
The preparation of apoptotic cell
The stable transfection beta-galactosidase enzymes is as the clone Ba/F3 of the somatomedin dependence of reporter gene, as the source of apoptotic cell.The suitable plasmid of transfection Ba/F3 is pcDNA3.1/His/LacZ, as shown in figure 11.The Ba/F3 cell is mouse clone (Palacios and Steinmetz, 1985, the cell 41:727-734 that IL-3 relies on; Palacios etc., 1984, natural 309:126-131), adding glutamazI, 10%FCS, 1% microbiotic (all available from GIBCOBRL, ibid.), is cultivated in the supernatant of 10%WEHI-3 culture.
WEHI-3 (ATCC number: TIB-68) at substratum: RPMI 1640, glutamaxI, 1O%FCS, 3.6 μ l beta-mercaptoethanols/liter in growth, produce IL-3.
The Ba/F3 cell is at two days forward pass of transactional analysis generation (exponential phase of growth), Ba/F3 cell (5 * 10
6Cell/ml) cultivated 20 hours under no growth factors I L-3 condition is then analyzed.The Ba/F3 cell of apoptosis uses annexin/propidium iodide labelling kit (Boeringher Mannheim, Brussels, Belgium) to detect.Be less than 5% propidium iodide feminine gender as the 20% annexin positive, the Ba/F3 cell is an early apoptosis.The Ba/F3 cell that is added with growth factors I L-3 is as negative control.The result that annexin/propidium iodide detects as shown in figure 33.
Add apoptotic cell to phagocytic cell
200 μ l Ba/F3 cells (1 * 10
7Cell/ml) joins in the hole of the J774 that contains stable transfection and negative control, 37 ℃ of incubations, and the time is from 20 minutes to 5 hours, afterwards removal apoptotic cell from institute is porose.(GIBCOBRL washes 3 times in ibid) phagocytic cell, notes not removing cell at the PBS damping fluid.
Reading
The cytophagic apoptotic body of J774 detects by detecting the beta-galactosidase enzymes of expressing in the Ba/F3 cell and realizes.Detect adopting fluorogenic substrate, fluorescein is two-b-D-galactopyranose glycosides (FDG) (Molecular Probes, Eugene, OR, the U.S.).Xiang Kongzhong adds 10 μ M FDG, and the room temperature incubation is 1 hour in the dark.FDG is hydrolyzed to FMG and fluorescein because of betagalactosidase activity, at Standard Plate Count device counting, and the 480mm exciting light, 520mm launches light, and suitably susceptibility detects green fluorescence.
Be the purpose of calibration, the fluorescence reading depends on different B a/F3 concentration.As the fluorescence reading of cell concn as shown in figure 12.
Further different FDG concentration are used in experiment, the different incubation times of analyzing, the concentration of serum in different temperature of analyzing and the Ba/F3 substratum.The result is respectively as Figure 13, and 14,15 and 34.
Screening compound
The above-mentioned analysis of engulfing is used for SCREENED COMPOUND, judges that it influences the level that apoptotic cell is engulfed by professional phagocytic cell.The compound of surveying can be added to and detect in the hole, and about 30 minutes, the back added the Ba/F3 cell.Because the porous plate form of analyzing, and automatic fluorescence reading uses standard equipment, the typical screening compound that is fit to the high material throughput of this analysis.
When the existence of any compound causes not having fluorescence or compares with the phagocytic cell that is not exposed to compound, fluorescent weakening, the cell vigor that carries out of then tackling in this hole detects, practical commercial reagents such as Live/Dead Viability/Cytotoxicity test kit (MolecularProbes, Eugene, USA).This test kit provides the activation analysis of two kinds of color fluorocytes, and alive and dead cell are determined fluorexon AM and bromination second pyridine homodimer simultaneously with two kinds of probes.Thereby measure the two-parameter of cell viability, cell lactonase activity and membrane integrity.This test kit is suitable for fluorescence porous plate scanner.The existence of viable cell will be confirmed the disappearance of fluorescence because of the influence of compound to activate the phagocytic capacity, but not only nonspecific toxicity.
Further, anyly be accredited as the inhibitor that apoptotic cell engulfs or the compound of toughener, will further detect the adjusting of this effect by CED-6 through this analysis.When compound identification be apoptotic cell engulf toughener the time, be that the J774 cell of transfection h1ced-6 or h2ced-6 carries out the phagocytic cell analysis by carrying out as above-mentioned usefulness.If compound can be induced identical phenotype in the cell with transfection hced-6 in the J774 cell, illustrate that compound is not necessarily by CED-6 or the effect of CED-6 signal transduction pathway.
Similar, if during the inhibitor that compound identification is an apoptotic cell engulfs, be exposed to the phenotype of J774 of the transfection of compound by detection, confirm that this effect is by CED-6 or the effect of CED-6 signal transduction pathway.The CED-6 signal transduction pathway is passed through in reverse explanation reaction to the wild-type phenotype.
Anti-people CED-6 gets polyclonal antibody
Polyclonal antibody is cultivated in rabbit, anti-following ced-6 epi-position:
EP990044?H2N-NRA?FSR?KKD?KTC?CONH2
EP990045?H2N-CFL?GST?EVE?QPK?GTE?CONH2
EP990046?H2N-CTR?NGT?QPP?PVP?SRS?T?CONH2
The location of these epi-positions in ced-6 albumen as shown in figure 16.
Polyclonal antibody is by Eurogentec Bel (Herstal, Belgium) preparation, according to the following step:
The 0th day: remove preimmune serum, for the first time immunization
The 14th day: immunization for the second time
The 28th day: immunization for the third time
The 38th day: blood-sample withdrawal (getting blood 2ml)
The 56th day: the 4th immunity
The 66th day: blood-sample withdrawal (getting blood 2+20ml)
The 80th day: finish and get blood
Embodiment 7
The Western trace detects antibody
In the TOP10 competent cell, transform plasmid pGA1028 (pBAD/His A-hCED-6) (seeing Figure 17 and 18).Transform identical Bacillus coli cells as negative control with pBAD/His.PBAD/His A and intestinal bacteria TOP10 available from Invitrogen (Leek, TheNetherlands).
Use the pBAD-vector expression system, it is a kind of known efficient expression system.When pectinose existed, the expression of pbAD began, and when lacking pectinose, the pBAD transcriptional level is extremely low.Carry out guide's expression according to manufacturer explanation, the amount difference of pectinose is wherein determined the amount of the pectinose that proteic high expression level is required.Method is according to manufacturer (Invitrogen, Lekk, The Netherlands).With carrying out great expression with quadrat method.
Proteic purifying carries out in the Bacillus coli cells that transforms pGA1028: 5ml lysis buffer (10ml TE 1 * pH9,0.5mg./ml N,O-Diacetylmuramidase, 0.1mg/ml DNAse, 100 μ l 1M CaCl
2400 μ l proteinase inhibitor 25 *) join the culture throw out of 50ml abduction delivering, be deposited in the lysis buffer resuspended.Resuspended thing was placed on ice 30 minutes, and ultrasonic 3 times, each 5 seconds (high-density), resuspended subsequently thing was placed 30 minutes at 37 ℃ through 3 freeze-thaw cycle (liquid nitrogen-42 ℃).Resuspended thing centrifugal 5 minutes with maximum speed of revolution.Precipitation comprises soluble part and hCED-6 fusion rotein, and is resuspended in 1ml 2M urea, 1200rpm jolting 5 minutes.Resuspended thing centrifugal 5 minutes with maximum speed of revolution, supernatant is used for gel electrophoresis and Western trace.Cleer and peaceful 25 μ l premix SDS-polyacrylamide gel electrophoresis sample buffers (Bio Rad-Hercules, CA, the U.S.) mix on the 25 μ l.
From the albumen of negative control purifying not.The intestinal bacteria that pBAD/His transforms are prepared as follows, and precipitation 1ml inductive culture of Escherichia coli is deposited in the 1ml premix SDS-polyacrylamide gel electrophoresis sample buffer (Bio Rad-Hercules, CA, the U.S.) resuspended.Resuspended thing is used for the PAGE gel electrophoresis.
The sample of preparation joins gel: sample boiled 5 minutes, and application of sample is prepended on ice.To the gel TrisHCl of preparation, 4-15% (Bio Rad-Hercules, CA, the U.S.) 50 μ l wells add 25 μ l samples, and electrophoresis carries out according to manufacturer's explanation.Use MiniTransBlot electrophoresis cell (Bio Rad-Hercules, CA, the U.S.) albumen on the glue is forwarded to nitrocellulose filter (Trans-blot Transfermedim, Bio Rad-Hercules, CA, the U.S.), carry out (BioRad-Hercules according to manufacturer's explanation, CA, the U.S.).
Supplier and detection kit according to antibody are carried out the Western trace.One is anti-, immune serum of rabbit or preimmune serum PBST (1.44g/L KH2P04,90g/LNaCl, 7.75g/L Na2HPO4.7H20,0.1%Tween) in by 1/2000 dilution.Two is anti-: anti-rabbit, the complete antibody of horseradish peroxidase (donkey) in PBST (0.1%) by 1/4000 dilution (ECL Western trace detection reagent and analytical system, AmershamPharmacia Biotech, UK, England).
Incubation is undertaken by giving birth to manufacturer's explanation, does change slightly.First antibody is incubated overnight at PBST rather than in TBST.Detection of antibodies according to manufacturer's instruction carry out (ECL Western trace detection reagent and analytical system, Amersham PharmaciaBiotech, UK, England).Detect at the ECL test kit, scrape off and label film again according to manufacturer's instruction.
Carry out the Western trace of anti-HisA antibody (Invitrogen, Leek, Holland) according to manufacturer's instruction, control antibodies is arranged simultaneously.Be diluted among the PBST (0.1%) (Invitrogen, Leek, Holland) anti--Xpress antibody as first antibody with 1/5000.Two is anti-: mouse Ig, the full length antibody of horseradish peroxidase (deriving from sheep), with 1/4000 be diluted in (0.1%) among the PBST (ECL Western trace detection reagent and analytical system, Amersham Pharmacia Biotech, UK, England).
In the experiment antibody staining according to carry out according to manufacturer explanation (ECL Western trace detection reagent and analytical system, Amersham Pharmacia Biotech, UK, England).
The result is shown in Figure 20 to 25.
Embodiment 8
H-CED-6 and tyrosine phosphorylated proteins
Co-immunoprecipitation
The antibody of 3 epi-positions of anti-h1CED-6 is used for the Western trace, detects the interaction of CED-6 and tyrosine phosphorylated proteins, identifies the CED-6 interaction protein.
Transfection
Plasmid pGA3103 (seeing Figure 26-29) and pGA3104 (seeing Fig. 7-10) transfection 175cm
2COS-1 cell (1 * 10 in the square vase
7Cell).Negative control: MOCK and pEGFP-N3.Investigation total length people ced-6 (be in GFP and read in the frame, N end and C end merge internal control).Lipofection amine Plus reagent (GIBCO-BRL) transfection of COS-1 cell.The method of following attached LifeTechnologies, volume is as shown in table 2.
The albumen of MOCK transfection is the shade contrast of transfection.In cell, only add the solvent of DNA and do not add DNA.The solvent of DNA is the TE damping fluid, and pH=8:1M Tris (ICN) ph=8 and 0.5 M EDTA (Merck-Belgolabo) pH=8 are soluble in water.
Table 2 175cm
2Lipofection amine transfection (Life Technologies) COS-1 cell in the square vase
| Culturing bottle | Works | DNA concentration | DNA | ?DNA | ?Optimem | Add reagent | Optimem | ?Lipo fecta ?mine | ?Optimem | ?Optimem |
| μg/μl | ?μg | ?μl | ?μl | ?μl | ?μl | ?μl | ?μl | ?ml | ||
| ?175cm 2 | MOCK | ?- | ?- | ?12(TE) | ?1125 | ?60 | ?1125 | ?90 | ?2250 | ?15 |
| ?175cm 2 | pEGFP-N3 | ?1 | ?12 | ?12 | ?1125 | ?60 | ?1125 | ?90 | ?2250 | ?15 |
| ?175cm 2 | PGA3103 | ?1 | ?12 | ?12 | ?1125 | ?60 | ?1125 | ?90 | ?2250 | ?15 |
| ?175cm 2 | PGA3104 | ?1 | ?12 | ?12 | ?1125 | ?60 | ?1125 | ?90 | ?2250 | ?15 |
The beta chain of the IL-3 acceptor of the Ba/F3 cell of phosphorylation is as the positive control of phosphorylation.
Use digitoxin to prepare COS-1 cell lysate (soft as far as possible, as not destroy interacting), in lysis buffer, add inhibitors of phosphatases (proteinase inhibitor, preferred mixture or pefablock).
Damping fluid based on low stringent type digitoxin:
The damping fluid of digitoxin among the 10ml bidi
1% digitoxin (Serva 19551, molecular weight 1229.3) SERVA
2% mother liquor=250mg is dissolved in 12.5ml
10mM Triethanolamine 99(TEA 99) pH7.8 (Sigma-Aldrich; Bornem, Belgium 10 * mother liquor 100mM=185.7mg are dissolved in 10ml pH7.8 (the 5ml dilution is 50ml)
O.15M?NaCl(MW?58.44)?87.66?mg/ml
2mM EDTA (Titriplex III) molecular weight 372.24)
(Darmstadt, Germany)
7.444mg every 10ml
200U/ml press down enzyme peptide-Trazilol (Sigma-Aldrich, Bornem, Belgium)
1mg=11TIU=9900KU
200x mother liquor: 10mg is dissolved among the 2ml PBS=49500KU (containing 50 μ l among the 10ml)
The every 10ml of 1mM Pefabloc (Merck, Darmstadt, Germany) 2.4mg
The cracking of cell
Transfectional cell DulbecccoShi PBS (GIBCOBRL) in falcon washes 2 times
Cell scraping is deposited in the 300 μ l lysis buffers resuspended
All operations carries out at 4 ℃
Prepared product 4000rpm is centrifugal, and supernatant is transferred in the new pipe
Preclearing
(Amersham Pharmacia, Roosendaal theNetherlands) provide with freeze dried additive form Protein G agarose CL-4B pearl.These additives can substitute lysis buffer by ethanol at the neutral pH flush away.
50%v/v Protein G agarose suspension:
Draw 1ml 50/50/v/v Protein G agarose and with maximum speed centrifugal 5 seconds, resuspended in the equal-volume lysis buffer subsequently.Wash 3 times.
(AmershamPharmacia ibid.), acts on 1 hour to add 50 μ l Protein G agarose CL-4B pearls in 300 μ l lysates.
Centrifugal 10 seconds of 4 ℃ of 14000rpm, supernatant changes new pipe over to.
Co-immunoprecipitation
COS-1 lysate: add 5 μ l and resist-green fluorescent protein (GFP) multi-clone rabbit anti-(Immunosource, Halle-Zoersel, Belgium)
Lyophilized form is dissolved in the 100 μ l distilled water, and-20 ℃ frozen.
No. 5, Ba/F3 lysate: anti-(the Vander heyden J. of rabbit that adds the beta chain of the anti-IL-3 acceptor of 5 μ l, Devos R., Plaetinck G., Fache I., Fiers W., J.1991., Tavernier uses the evaluation of serial monoclonal antibody to the mIL5 receptor complex, the relation of this mixture and mouse IL-3 acceptor.Journal of Immunology.147:3412-3418)
No. 6, Ba/F3 lysate: do not add antibody
Sample rotates 4 ℃ of incubations 4 hours and 24 hours (spending the night)
Add 50 μ l albumin A pearls, 4 ℃ of incubations 1 hour
Centrifugal 3 minutes of 4 ℃ of 3000rpm of sample
Pearl is resuspended in the 800 μ l lysis buffers, reversing several times or the rotation several minutes, centrifugal 3 minutes of 4 ℃ of 3000rpm.Repeat 3 this, remove cleaning buffer solution with kapillary at last
Pearl is resuspended in 20 μ l SDS sample loading buffers (containing sulfydryl).
The A431 cell pyrolysis liquid (anti-Tyrosine O-phosphate) that No. 7=EGF-of lysate stimulates
(Upstate?Biotechnology,Cat.No.12-302)
2.5 μ l beta-mercaptoethanol joins in the 100 μ l lysates and sample that boil.
The centrifugal 3000rpm of sample, 3 minutes (4 ℃), SN is added among the SDS PAGE, uses the SDS-PAGE lower molecular weight standard (BioRad cat.no161-0305) of dying in advance
The Western trace
Cell lysate is transferred on the nitrocellulose filter
Transfering buffering liquid=4BmM Tris, 39 mM glycine, 20% methyl alcohol, pH9.2 (200ml methyl alcohol is dissolved in 1 premium on currency for 5.82g Tris, 2.93g glycine)
Sealing damping fluid: 1 * PBS, 0.1%Tween, 5% milk powder is incubated overnight
Anti-Tyrosine O-phosphate mark Gel (cat.05-321, Upstate Biotechnology): 1 μ g/ml 3 hours, PBS 0.1%Tween wash 2 times, each 5 minutes
The albumen colour developing uses 1:4000 mountain sheep anti mouse horseradish peroxidase (cat.No RPN2108, Amersham Pharmacia biotech) two anti-, room temperature 1 hour.
PBS 0.1%Tween washes film 2 times, and each 5 minutes, confining liquid was washed film 2 times, and each 5 minutes, washing film 2 times, each 5 minutes.
ECL Western engram analysis system
ECL Western trace detection reagent is available from Amersham PharmaciaBiotech (cat.no RPN 2108).
The ECL detection kit detects the back flush away binding antibody label film of laying equal stress on
The ECL detection kit detects the back flush away binding antibody label film of laying equal stress on.After each immunodetection, it is moistening that film keeps in 4 ℃ of preservative films.
Film immerses in the strip buffer (100mM beta-mercaptoethanol, 2%SDS, 62.5mM Tris-HCl pH6.7), and 50 ℃ of incubations 30 minutes stir once in a while.
Film was washed among the 0.1%Tween 2 * 10 minutes at PBS, used large volume to wash the film damping fluid under the room temperature.
Film room temperature in the sealing damping fluid was sealed 1 hour.
Use anti-green fluorescence GFP immunodetection, repeat with anti-people CED-6
The result
The anti-Tyrosine O-phosphate of Western trace of all cells lysate, the rabbit anteserum mark of anti--green fluorescent protein (GFP) or anti-CED-6 is as Figure 30 trace (A), (B) and (C).Anti-Tyrosine O-phosphate is painted, and the band between 49 and 74KD appears in the COS-1 cell lysate of the fusion rotein that specially dyes GFP and CED-6, and does not occur in contrast COS-1 cell lysate.The Western trace of anti--GFP and the anti--CED-6 band between identical 49 and 74KD that also dyes.
Conclusion
Fusion rotein CED-6-GFP and GFP-CED-6 are tyrosine phosphorylation.Their molecular weight is 62385.95, show 49 and 74KD between band be anti-Tyrosine O-phosphate, the positive staining of anti-green fluorescent protein (GFP) and anti--CED-6.
Embodiment 9
Stable cell lines personnel selection cell surface receptor CD36 transfection.
J774 mouse monocyte tumor cell line is by electroporation plasmid pGA1058 transfection, shown in Figure 31 and 32.Method is described as embodiment 4 transfection people ced-6.
Cells transfected system is as the positive control of engulfing analysis that uses embodiment 5 methods.
Produce the apoptosis particle from PC12.
(ATCC number: CRL-1712) (Mesner P.W., Winters T.R., Green S.H., (1992) cytobiology magazine .119:1669-1680) is tending towards agglomerating growth to PC-12 clone.(the 50ng/ml final concentration, Sigma), the PC-12 cytodifferentiation is a neuronal cell to add nervegrowthfactor-.After nerve growth factor is removed 5 days, induce neurone P of Rats C12 programmed cell cell death.Cell is containing the 2mM L-glutaminate, the 1.5g/L sodium bicarbonate, and 4.5g/L glucose, 10mM HEPES and 1mM Sodium.alpha.-ketopropionate are cultivated in 10% horse serum, the RPMI1640 of 5% foetal calf serum (Life Technologies).
These cells can use above-mentioned annexin/PI test kit to detect its apoptosis characteristic.
Sequence table
This paper Nucleotide and corresponding following SEQ ID NOs of aminoacid sequence as shown in the figure.
SEQ ID NO:1 Fig. 1
SEQ ID NO:2 Fig. 2
SEQ ID NO:3 Fig. 3
SEQ ID NO:4 Fig. 4
SEQ ID NO:5 Fig. 5
SEQ ID NO:6 Fig. 7
SEQ ID NO:7 Fig. 9
SEQ ID NO:8 Figure 11
SEQ ID NO:9 Figure 17
SEQ ID N0:10 Figure 19
SEQ ID N0:11 Figure 26
SEQ ID N0:12 Figure 28
SEQ ID N0:13 Figure 31
sequence table＜110〉DEVGEN, NV＜120〉engulf analytical method＜130〉SCB/50784/023＜140〉＜141＜160〉15＜170〉PatentIn, Ver.2.0＜210〉1＜211〉2416＜212〉DNA＜213〉mankind＜220〉＜221〉CDS＜222, (430) .., (1344)＜400〉1ggtgatgagc, ccttgggttc, tcgctccgac, tgctaaattc, gcttggccgg, gtccaccttc, 60tcgtggcctc, actcgccaca, cggatcagaa, tccggagcag, gcagttctct, ctattctgag, 120gctcctgcgg, ctgccgcgct, gacttccctg, tgtggnggag, ggaactctgg, gcaggctggt, 180tttcttggaa, tgtgtttacg, atgttgaatg, ggacttgaac, aggaagctgg, acgctgcagc, 240tggaactagc, gtgccaagtt, atttatgatt, ccatctgata, tacataggag, agaaactgat, 300agaagaattc, tgatggcaac, tgtatgatag, aagctatata, aagtcaagtg, tccattttct, 360ttcaactata, tttgagcata, cccaggattt, aagtcgtgga, actgaacatt, tatttggctg, 420atcctcatc, atg, aac, cgt, gct, ttt, agc, agg, aag, aaa, gac, aaa, aca, tgg, atg, 471
Met?Asn?Arg?Ala?Phe?Ser?Arg?Lys?Lys?Asp?Lys?Thr?Trp?Met
1???????????????5??????????????????10cat?aca?cct?gaa?gct?tta?tca?aaa?cat?ttc?att?ccc?tat?aat?gca?aag???519His?Thr?Pro?Glu?Ala?Leu?Ser?Lys?His?Phe?Ile?Pro?Tyr?Asn?Ala?Lys?15??????????????????20??????????????????25??????????????????30ttt?ctt?ggc?agt?aca?gaa?gtg?gaa?cag?cca?aaa?gga?aca?gaa?gtt?gtg???567Phe?Leu?Gly?Ser?Thr?Glu?Val?Glu?Gln?Pro?Lys?Gly?Thr?Glu?Val?Val
35??????????????????40??????????????????45aga?gat?gct?gta?agg?aaa?cta?aag?ttt?gca?aga?cat?atc?aag?aaa?tct???615Arg?Asp?Ala?Val?Arg?Lys?Leu?Lys?Phe?Ala?Arg?His?Ile?Lys?Lys?Ser
50??????????????????55??????????????????60gaa?ggc?cag?aaa?att?cct?aaa?gtg?gag?ttg?caa?ata?tca?att?tat?gga???663Glu?Gly?Gln?Lys?Ile?Pro?Lys?Val?Glu?Leu?Gln?Ile?Ser?Ile?Tyr?Gly
65??????????????????70??????????????????75gta?aaa?att?cta?gaa?ccc?aaa?aca?aag?gaa?gtt?caa?cac?aat?tgc?cag???711Val?LVs?Ile?Leu?Glu?Pro?Lys?Thr?Lys?Glu?Val?Gln?His?Asn?Cys?Gln
80??????????????????85??????????????????90ctt?cat?aga?ata?tct?ttt?tgt?gca?gat?gat?aaa?act?gac?aag?agg?ata????759Leu?His?Arg?Ile?Ser?Phe?Cys?Ala?Asp?Asp?Lys?Thr?Asp?Lys?Arg?Ile?95?????????????????100?????????????????105?????????????????110ttc?act?ttc?ata?tgc?aaa?gat?tct?gag?tca?aat?aaa?cat?ttg?tgc?tat????807Phe?Thr?Phe?Ile?Cys?Lys?Asp?Ser?Glu?Ser?Ash?Lys?His?Leu?Cys?Tyr
115?????????????????120?????????????????125gta?ttt?gac?agc?gaa?aag?tgt?gct?gaa?gag?atc?act?tta?aca?att?ggc????855Val?Phe?Asp?Ser?Glu?Lys?Cys?Ala?Glu?Glu?Ile?Thr?Leu?Thr?Ile?Gly
130??????????????????135?????????????????140caa?gca?ttt?gac?ctg?gca?tac?agg?aaa?ttt?cta?gaa?tca?gga?gga?aaa????903Gln?Ala?Phe?Asp?Leu?Ala?Tyr?Arg?Lys?Phe?Leu?Glu?Ser?Gly?Gly?Lys
145?????????????????150?????????????????155gat?gtt?gaa?aca?aga?aaa?cag?atc?gca?ggg?tta?caa?aaa?aga?atc?caa????951Asp?Val?Glu?Thr?Arg?Lys?Gln?Ile?Ala?Gly?Leu?Gln?Lys?Arg?Ile?Gln
160?????????????????165?????????????????170gac?tta?gaa?aca?gaa?aat?atg?gaa?ctt?aaa?aat?aaa?gta?caa?gat?ttg????999Asp?Leu?Glu?Thr?Glu?Asn?Met?Glu?Leu?Lys?Asn?Lys?Val?Gln?Asp?Leu175?????????????????180?????????????????185?????????????????190gaa?aac?caa?ctg?aga?ata?act?caa?gta?tca?gca?cct?cca?gca?ggc?agt????1047Glu?Asn?Gln?Leu?Arg?Ile?Thr?Gln?Val?Ser?Ala?Pro?Pro?Ala?Gly?Ser
195?????????????????200?????????????????205atg?aca?cct?aag?tcg?ccc?tcc?act?gac?atc?ttt?gat?atg?att?cca?ttt????1095Met?Thr?Pro?Lys?Ser?Pro?Ser?Thr?Asp?Ile?Phe?Asp?Met?Ile?Pro?Phe
210?????????????????215?????????????????220tct?cca?ata?tca?cac?cag?tct?tcg?atg?cct?act?cgc?aat?ggc?aca?cag????1143Ser?Pro?Ile?Ser?His?Gln?Ser?Ser?Met?Pro?Thr?Arg?Asn?Gly?Thr?Gln
225?????????????????230?????????????????235cca?cct?cca?gta?cct?agt?aga?tct?act?gag?att?aaa?cgg?gac?ctg?ttt????1191Pro?Pro?Pro?Val?Pro?Ser?Arg?Ser?Thr?Glu?Ile?Lys?Arg?Asp?Lcu?Phe
240?????????????????245?????????????????250gga?gca?gaa?cct?ttt?gac?cca?ttt?aac?tgt?gga?gca?gca?gat?ttc?cct????1239Gly?Ala?Glu?Pro?Phe?Asp?Pro?Phe?Asn?Cys?Gly?Ala?Ala?Asp?Phe?Pro255?????????????????260?????????????????265?????????????????270cca?gat?att?caa?tca?aaa?tta?gat?gag?atg?cag?gag?ggg?ttc?aaa?atg????1287Pro?Asp?Ile?Gln?Ser?Lys?Leu?Asp?Glu?Met?Gln?Glu?Gly?Phe?Lys?Met
275?????????????????280?????????????????????285gga?cta?act?ctt?gaa?ggc?aca?gta?ttt?tgt?ctc?gac?ccg?tta?gac?agt????1335Gly?Leu?Thr?Leu?Glu?Gly?Thr?Val?Phe?Cys?Leu?Asp?Pro?Leu?Asp?Ser
290?????????????????295?????????????????300agg?tgc?tga?catcaagaac?aagaaatcct?gattcatgtt?aaatgtgttt????????????1384Arg?Cys
305gtatacacat, gtcatttatt, attattactt, taagataggt, attattcatg, tgtcaatgtg, 1444tttgaatatt, ttaatatttt, gaaaattttc, tcagttaaat, ttcctcacct, tcactattga, 1504tctgtaattt, ttattttaaa, aacagcttac, tgtaaagtag, atcatacttt, tatgttcctt, 1564tctgtttcta, ctgtagatga, atttgtaatt, gaaagacata, ttatacaaat, acctgccttg, 1624tgtctgagtt, ctatttagtt, agcatcttga, aatttgtatt, cattttccag, atggctagtt, 1684tattaatgat, ttcccaaaag, ccatacctta, aagataactt, tttaaattct, gaagagacat, 1744gccaatgtca, aactaaacat, gttctgtttt, taaaccaaca, aacatgttac, tattcattgg, 1804acagatstca, ttttatgtat, aaatactgtt, cacatcactg, ggaaaatgta, aactttaaac, 1864staatgccac, aaggtcacta, atttctagca, ggtaasatta, taaggatata, aattccaata, 1924staaaccaaa, tgtatttags, gtatttatta, gtaaatgcaa, ggtgatgtta, gttatgatca, 1984gttatactct, aaatatttaa, tttgttttat, aaaggtagtg, aaaaaatgaa, aatttgctat, 2044ttattaaaaa, acattaaatt, tcattccaaa, tgagataagt, gatattacta, taacatctaa, 2104gcatcatctg, atttgatatt, ccctaaaaaa, catttggaat, atatgctatc, tatagattca, 2164gtatctacta, cccatattta, ctttaccaaa, tatatttctc, ctcactgcat, aaggactsct, 2224cttctcatat, tttcttcttt, gatgaagata, tttttcacca, aagtttattt, tgtgatgccc, 2284tcttggtttt, gatactttaa, aatctgtggc, acccgttcta, catgaattat, caatatttgg, 2344taaattcaat, ctgtatttgt, tttgttaaag, tcaaaaatct, cattttccaa, aaaaaaaaaa, 2404aaaaaactcg, ag, 2416＜210〉2＜21l〉304＜212〉protein＜213〉mankind＜400〉2Met, Asn, Arg, Ala, Phe, Ser, Arg, Lys, Lys, Asp, Lys, Thr, Trp, Met, His, Thr, 1, 5, 10, 15Pro, Glu, Ala, Leu, Ser, Lys, His, Phe, Ile, Pro, Tyr, Asn, Ala, Lys, Phe, Leu
20??????????????????25??????????????????30Gly?Ser?Thr?Glu?Val?Glu?Gln?Pro?Lys?Gly?Thr?Glu?Val?Val?Arg?Asp
35??????????????????40?????????????????45Ala?Val?Arg?Lys?Leu?Lys?Phe?Ala?Arg?His?Ile?Lys?Lys?Ser?Glu?Gly
50?????????????????55??????????????????60Gln?Lys?Ile?Pro?Lys?Val?Glu?Leu?Gln?Ile?Ser?Ile?Tyr?Gly?Val?Lys?65??????????????????70?????????????????75??????????????????80Ile?Leu?Glu?Pro?Lys?Thr?Lys?Glu?Val?Gln?His?Asn?Cys?Gln?Leu?His
85??????????????????90??????????????????95Arg?Ile?Ser?Phe?Cys?Ala?Asp?Asp?Lys?Thr?Asp?Lys?Arg?Ile?Phe?Thr
100????????????????105?????????????????110Phe?Ile?Cys?Lys?Asp?Ser?Glu?Ser?Asn?Lys?His?Leu?Cys?Tyr?Val?Phe
115?????????????????120?????????????????125Asp?Ser?Glu?Lys?Cys?Ala?Glu?Glu?Ile?Thr?Leu?Thr?Ile?Gly?Gln?Ala
130?????????????????135?????????????????140Phe?Asp?Leu?Ala?Tyr?Arg?Lys?Phe?Leu?Glu?Ser?Gly?Gly?Lys?Asp?Val145?????????????????150?????????????????155?????????????????160Glu?Thr?Arg?Lys?Gln?Ile?Ala?Gly?Leu?Gln?Lys?Arg?Ile?Gln?Asp?Leu
165?????????????????170?????????????????175Glu?Thr?Glu?Ash?Met?Glu?Leu?Lys?Ash?Lys?Val?Gln?Asp?Leu?Glu?Ash
180?????????????????185?????????????????190Gln?Leu?Arg?Ile?Thr?Gln?Val?Ser?Ala?Pro?Pro?Ala?Gly?Ser?Met?Thr
195?????????????????200?????????????????205Pro?Lys?Ser?Pro?Ser?Thr?Asp?Ile?Phe?Asp?Met?Ile?Pro?Phe?Ser?Pro
210?????????????????215?????????????????220Ile?Ser?His?Gln?Ser?Ser?Met?Pro?Thr?Arg?Asn?Gly?Thr?Gln?Pro?Pro225?????????????????230?????????????????235?????????????????240Pro?Val?Pro?Ser?Arg?Ser?Thr?Glu?Ile?Lys?Arg?Asp?Leu?Phe?Gly?Ala
245?????????????????250?????????????????255Glu?Pro?Phe?Asp?Pro?Phe?Asn?Cys?Gly?Ala?Ala?Asp?Phe?Pro?Pro?Asp
260?????????????????265?????????????????270Ile?Gln?Ser?Lys?Leu?Asp?Glu?Met?Gln?Glu?Gly?Phe?Lys?Met?Gly?Leu
275?????????????????280?????????????????285Thr?Leu?Glu?Gly?Thr?Val?Phe?Cys?Leu?Asp?Pro?Leu?Asp?Ser?Arg?Cys
290, 295, 300＜210〉3＜211〉2278＜212〉DNA＜213〉mankind＜220〉＜221〉CDS＜222 〉, (430) .., (1206)＜400〉3ggtgatgagc, ccttgggttc, tcgctccgac, tgctaaattc, gcttggccgg, gtccaccttc, 60tcgtggcctc, actcgccaca, cggatcagaa, tccggagcag, gcagttctct, ctattctgag, 120gctcctgcgg, ctgccgcgct, gacttccctg, tgtggnggag, ggaactctgg, gcaggctggt, 180tttcttggaa, tgtgtttacg, atgttgaatg, ggacttgaac, aggaagctgg, acgctgcagc, 240tggaactagc, gtgccaagtt, atttatgatt, ccatctgata, tacataggag, agaaactgat, 300agaagaattc, tgatggcaac, tgtatgatag, aagctatata, aagtcaagtg, tccattttct, 360ttcaactata, tttgagcata, cccaggattt, aagtcgtgga, actgaacatt, tatttggctg, 420atcctcatc, atg, aac, cgt, gct, ttt, agc, agg, aag, aaa, gac, aaa, aca, tgg, atg, 471
Met?Asn?Arg?Ala?Phe?Ser?Arg?Lys?Lys?Asp?Lys?Thr?Trp?Met
1???????????????5??????????????????10cat?aca?cct?gaa?gct?tta?tca?aaa?cat?ttc?att?ccc?tat?aat?gca?aag???519His?Thr?Pro?Glu?Ala?Leu?Ser?Lys?His?Phe?Ile?Prc?Tyr?Asn?Ala?Lys?15??????????????????20??????????????????25??????????????????30ttt?ctt?ggc?agt?aca?gaa?gtg?gaa?cag?cca?aaa?gga?aca?gaa?gtt?gtg???567Phe?Leu?Gly?Ser?Thr?Glu?Val?Glu?Gln?Pro?Lys?Gly?Thr?Glu?Val?Val
35??????????????????40??????????????????45aga?gat?gct?gta?agg?aaa?cta?aag?ttt?gca?aga?cat?atc?aag?aaa?tct???615Arg?Asp?Ala?Val?Arg?Lys?Leu?Lys?Phe?Ala?Arg?His?Ile?Lys?Lys?Ser
50??????????????????55??????????????????60gaa?ggc?cag?aaa?att?cct?aaa?gtg?gag?ttg?caa?ata?tca?att?tat?gga???663Glu?Gly?Gln?Lys?Ile?Pro?Lys?Val?Glu?Leu?Gln?Ile?Ser?Ile?Tyr?Gly
65??????????????????70??????????????????75gta?aaa?att?cta?gaa?ccc?aaa?aca?aag?gct?gaa?gag?atc?act?tta?aca???711Val?Lys?Ile?Leu?Glu?Pro?Lys?Thr?Lys?Ala?Glu?Glu?Ile?Thr?Leu?Thr
80??????????????????85??????????????????90att?ggc?caa?gca?ttt?gac?ctg?gca?tac?agg?aaa?ttt?cta?gaa?tca?gga???759Ile?Gly?Gln?Ala?Phe?Asp?Leu?Ala?Tyr?Arg?Lys?Phe?Leu?Glu?Ser?Gly?95?????????????????100?????????????????105?????????????????110gga?aaa?gat?gtt?gaa?aca?aga?aaa?cag?atc?gca?ggg?tta?caa?aaa?aga???807Gly?Lys?Asp?Val?Glu?Thr?Arg?Lys?Gln?Ile?Ala?Gly?Leu?Gln?Lys?Arg
115?????????????????120?????????????????125atc?caa?gac?tta?gaa?aca?gaa?aat?atg?gaa?ctt?aaa?aat?aaa?gta?caa???855Ile?Gln?Asp?Leu?Glu?Thr?Glu?Asn?Met?Glu?Leu?Lys?Asn?Lys?Val?Gln
130?????????????????135?????????????????140gat?ttg?gaa?aac?caa?ctg?aga?ata?act?caa?gta?tca?gca?cct?cca?gca???903Asp?Leu?Glu?Asn?Gln?Leu?Arg?Ile?Thr?Gln?Val?Ser?Ala?Pro?Pro?Ala
145?????????????????150?????????????????155ggc?agt?atg?aca?cct?aag?tcg?ccc?tcc?act?gac?atc?ttt?gat?atg?att???951Gly?Ser?Met?Thr?Pro?Lys?Ser?Pro?Ser?Thr?Asp?Ile?Phe?Asp?Met?Ile
160?????????????????165?????????????????170cca?ttt?tct?cca?ata?tca?cac?cag?tct?tcg?atg?cct?act?cgc?aat?ggc???999Pro?Phe?Ser?Pro?Ile?Ser?His?Gln?Ser?Ser?Met?Pro?Thr?Arg?Asn?Gly175?????????????????180?????????????????185?????????????????190aca?cag?cca?cct?cca?gta?cct?agt?aga?tct?act?gag?att?aaa?cgg?gac??1047Thr?Gln?Pro?Pro?Pro?Val?Pro?Ser?Arg?Ser?Thr?Glu?Ile?Lys?Arg?Asp
195?????????????????200?????????????????205ctg?ttt?gga?gca?gaa?cct?ttt?gac?cca?ttt?aac?tgt?gga?gca?gca?gat??1095Leu?Phe?Gly?Ala?Glu?Pro?Phe?Asp?Pro?Phe?Asn?Cys?Gly?Ala?Ala?Asp
210?????????????????215?????????????????220ttc?cct?cca?gat?att?caa?tca?aaa?tta?gat?gag?atg?cag?gag?ggg?ttc??1143Phe?Pro?Pro?Asp?Ile?Gln?Ser?Lys?Leu?Asp?Glu?Met?Gln?Glu?Gly?Phe
225?????????????????230?????????????????235aaa?atg?gga?cta?act?ctt?gaa?ggc?aca?gta?ttt?tgt?ctc?gac?ccg?tta??1191Lys?Met?Gly?Leu?Thr?Leu?Glu?Gly?Thr?Val?Phe?Cys?Leu?Asp?Pro?Leu
240, 245, 250gac, agt, agg, tgc, tga, catcaagaac, aagaaatcct, gattcatgtt, aaatgtgttt, 1246Asp, Ser, Arg, Cys255gtatacacat, gtcatttatt, attattactt, taagataggt, attattcatg, tgtcaatgtt, 1306tttgaatatt, ttaatatttt, gaaaattttc, tcagttaaat, ttcctcacct, tcactattga, 1366tctgtaattt, ttattttaaa, aacagcttac, tgtaaagtag, atcatacttt, tatgttcctt, 1426tctgtttcta, ctgtagatga, atttgtaatt, gaaagacata, ttatacaaat, acctgccttg, 1486tgtctgagtt, ctatttagtt, agcatcttga, aatttgtatt, cattttccag, atggctagtt, 1546tattaatgat, ttcccaaaag, ccatacctta, aagataactt, tttaaattct, gaagagacat, 1606gccaatgtca, aactaaacat, gttctgtttt, taaaccaaca, aacatgttac, tattcattgg, 1666acagatatca, ttttatgtat, aaatactgtt, cacatcactg, ggaaaatgta, aactttaaac, 1726ataatgccac, aaggtcacta, atttctagca, ggtaaaatta, taaggatata, aattccaata, 1786ataaaccaaa, tgtatttaga, gtatttatta, gtaaatgcaa, ggtgatgtta, gttatgatca, 1846gttatactct, aaatstttaa, tttgttttat, aaaggtagtg, aaaaaatgaa, aatttgctat, 1906ttattaaaaa, acattaaatt, tcattccaaa, tgagataagt, gatattacta, taacatctaa, 1966gcatcatctg, atttgatatt, ccctaaaaaa, catttggaat, atatgctatc, tatagattca, 2026gtatctacta, cccatattta, ctttaccaaa, tatatttctc, ctcactgcat, aaggactact, 2086cttctcatat, tttcttcttt, gatgaagata, tttttcacca, aagtttattt, tgtgatgccc, 2146tcttggtttt, gatactttaa, aatctgtggc, acccgttcta, catgaattat, caatatttgg, 2206taaattcaat, ctgtatttgt, tttgttaaag, tcaaaaatct, cattttccaa, aaaaaaaaaa, 2266aaaaaactcg, ag, 2278＜210〉4＜211〉258＜212〉protein＜213〉mankind＜400〉4Met, Asn, Arg, Ala, Phe, Ser, Arg, Lys, Lys, Asp, Lys, Thr, Trp, Met, His, Thr, 1, 5, 10, 15Pro, Glu, Ala, Leu, Ser, Lys, His, Phe, Ile, Pro, Tyr, Asn, Ala, Lys, Phe, Leu
20??????????????????25??????????????????30Gly?Ser?Thr?Glu?Val?Glu?Gln?Pro?Lys?Gly?Thr?Glu?Val?Val?Arg?Asp
35??????????????????40??????????????????45Ala?Val?Arg?Lys?Leu?Lys?Phe?Ala?Arg?His?Ile?Lys?Lys?Ser?Glu?Gly
50??????????????????55?????????????????60Gln?Lys?Ile?Pro?Lys?Val?Glu?Leu?Gln?Ile?Ser?Ile?Tyr?Gly?Val?Lys?65??????????????????70??????????????????75??????????????????80Ile?Leu?Glu?Pro?Lys?Thr?Lys?Ala?Glu?Glu?Ile?Thr?Leu?Thr?Ile?Gly
85??????????????????90??????????????????95Gln?Ala?Phe?Asp?Leu?Ala?Tyr?Arg?Lys?Phe?Leu?Glu?Ser?Gly?Gly?Lys
100?????????????????105?????????????????110Asp?Val?Glu?Thr?Arg?Lys?Gln?Ile?Ala?Gly?Leu?Gln?Lys?Arg?Ile?Gln
115?????????????????120?????????????????125Asp?Leu?Glu?Thr?Glu?Asn?Met?Glu?Leu?Lys?Asn?Lys?Val?Gln?Asp?Leu
130?????????????????135?????????????????140Glu?Asn?Gln?Leu?Arg?Ile?Thr?Gln?Val?Ser?Ala?Pro?Pro?Ala?Gly?Ser145?????????????????150?????????????????155?????????????????160Met?Thr?Pro?Lys?Ser?Pro?Ser?Thr?Asp?Ile?Phe?Asp?Met?Ile?Pro?Phe
165??????????????????170????????????????175Ser?Pro?Ile?Ser?His?Gln?Ser?Ser?Met?Pro?Thr?Arg?Asn?Gly?Thr?Gln
180?????????????????185?????????????????190Pro?Pro?Pro?Val?Pro?Ser?Arg?Ser?Thr?Glu?Ile?Lys?Arg?Asp?Leu?Phe
195?????????????????200?????????????????205Gly?Ala?Glu?Pro?Phe?Asp?Pro?Phe?Asn?Cys?Gly?Ala?Ala?Asp?Phe?Pro
210?????????????????215?????????????????220Prc?Asp?Ile?Gln?Ser?Lys?Leu?Asp?Glu?Met?Gln?Glu?Gly?Phe?Lys?Met225?????????????????230?????????????????235?????????????????240Gly?Leu?Thr?Leu?Glu?Gly?Thr?Val?Phe?Cys?Leu?Asp?Pro?Leu?Asp?Ser
245250255
Arg Cys
<210> 5
<211> 5619
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of artificial sequence
pGA3104-a construct including direct cloned into the cloning vector pEGFP-N3 points
The hlced-6cDNA
<400> 5
gatccccatg aaccgtgctt ttagcaggaa gaaagacaaa acatggatgc atacacctga 60
agctttatca aaacatttca ttccctataa tgcaaagttt cttggcagta cagaagtgga 120
acagccaaaa ggaacagaag ttgtgagaga tgctgtaagg aaactaaagt ttgcaagaca 180
tatcaagaaa tctgaaggcc agaaaattcc taaagtggag ttgcaaatat caatttatgg 240
agtaaaaatt ctagaaccca aaacaaagga agttcaacac aattgccagc ttcatagaat 300
atctttttgt gcagatgata aaactgacaa gaggatattc actttcatat gcaaagattc 360
tgagtcaaat aaacatttgt gctatgtatt tgacagcgaa aagtgtgctg aagagatcac 420
tttaacaatt ggccaagcat ttgacctggc atacacgaaa tttctagaat caggaggaaa 480
agatgttgaa acaagaaaac agatcgcagg gttacaaaaa agaatccaag acttagaaac 540
agaaaatatg gaacttaaaa ataaagtaca agatttggaa aaccaactga gaataactca 600
agtatcagca cctccagcag gcagtatgac acctaagtcg ccctccactg acatctttga 660
tatgattcca ttttctccaa tatcacacca gtcttcgatg cctactcgca atggcacaca 720
gccacctcca gtacctagta gatctactga gattaaacgg gacctgtttg gagcagaacc 780
ttttgaccca tttaactgtg gagcagcaga tttccctcca gatattcaat caaaattaga 840
tgagatgcag gaggggttca aaatgggact aactcttgaa ggcacagtat tttgtctcga 900
cccgttagac agtaggtgcg tcgacggtac cgcgggcccg ggatccatcg ccaccatggt 960
gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 1020
cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa 1080
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 1140
gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 1200
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 1260
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 1320
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 1380
ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 1440
caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 1500
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 1560
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 1620
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg 1680
ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa 1740
aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac 1800
ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 1860
aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa 1920
ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 1980
ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 2040
cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 2100
ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 2160
accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 2220
gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 2280
gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 2340
caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg 2400
aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 2460
catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc 2520
ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 2580
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 2640
ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 2700
gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 2760
ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 2820
ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca 2880
agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 2940
ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 3000
tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 3060
cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac 3120
gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 3180
gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 3240
agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 3300
attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 3360
tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 3420
caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 3480
cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 3540
gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 3600
tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 3660
gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 3720
atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 3780
tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 3840
ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag 3900
gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac 3960
ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat 4020
accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac 4080
cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc 4140
atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 4200
aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 4260
tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 4320
tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4380
ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4440
ataccaaata Ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4500
gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4560
aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4620
ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4680
agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4740
aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4800
aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4860
ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4920
cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 4980
tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc 5040
attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 5100
tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 5160
aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 5220
cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 5280
taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 5340
gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 5400
tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 5460
tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 5520
cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg 5580
tttagtgaac cgtcagatcc gctagcgcta ccggactca 5619
<210> 6
<211> 5021
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial sequence pGA1028-a construct comprising the plasmid cloning site of vector pBAD His A person
...
hlced-6 cDNA <400> 6gatccccatg aaccgtgctt ttagcaggaa gaaagacaaa acatggatgc atacacctga 60agctttatca aaacatttca ttccctataa tgcaaagttt cttggcagta cagaagtgga 120acagccaaaa ggaacagaag ttgtgagaga tgctgtaagg aaactaaagt ttgcaagaca 180tatcaagaaa tctgaaggcc agaaaattcc taaagtggag ttgcaaatat caatttatgg 240agtaaaaatt ctagaaccca aaacaaagga agttcaacac aattgccagc ttcatagaat 300atctttttgt gcagatgata aaactgacaa gaggatattc actttcatat gcaaagattc 360tgagtcaaat aaacatttgt gctatgtatt tgacagcgaa aagtgtgctg aagagatcac 420tttaacaatt ggccaagcat ttgacctggc atacacgaaa tttctagaat caggaggaaa 480agatgttgaa acaagaaaac agatcgcagg gttacaaaaa agaatccaag acttagaaac 540agaaaatatg gaacttaaaa ataaagtaca agatttggaa aaccaactga gaataactca 600agtatcagca cctccagcag gcagtatgac acctaagtcg ccctccactg acatctttga 660tatgattcca ttttctccaa tatcacacca gtcttcgatg cctactcgca atggcacaca 720gccacctcca gtacctagta gatctactga gattaaacgg gacctgtttg gagcagaacc 780ttttgaccca tttaactgtg gagcagcaga tttccctcca gatattcaat caaaattaga 840tgagatgcag gaggggttca aaatgggact aactcttgaa ggcacagtat tttgtctcga 900cccgttagac agtaggtgct gagtcgacgg taccatatgg gaattcgaag cttggctgtt 960ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc 1020tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga 1080actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag 1140ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 1200atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg 1260aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 1320catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 1380tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 1440aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1500ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1560aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 1620acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 1680ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg 1740gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 1800atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 1860acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 1920tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 1980ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 2040aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 2100aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 2160ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 2220atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2280aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 2340accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 2400tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 2460tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2520tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2580cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2640caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2700cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2760cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2820gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 2880acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 2940atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3000cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3060gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3120tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 3180tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 3240agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta 3300cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 3360ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 3420ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 3480ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 3540accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcataatg 3600tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg tcaagccgtc 3660aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca ctttttcttc 3720acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa 3780atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt 3840gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat 3900ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc 3960gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc 4020gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag 4080taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc 4140ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg 4200aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg 4260cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg 4320atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc 4380ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc 4440cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc 4500caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat 4560acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg 4620ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa 4680aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta 4740taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat 4800agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac 4860tgtttctcca tacccgtttt tttgggctaa caggaggaat taaccatggg gggttctcat 4920catcatcatc atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg 4980tacgacgatg acgataagga tcgatgggga tccgagctcg a 5021 <210> 7 <211> 5628 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: pGA3103-a...
Cloned into the vector construct including the pEGFP-C2-cloning site of human hlced-6 cDNA
<400> 7
tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata atcagccata 60
ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 120
aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 180
aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 240
gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg 300
ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 360
ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 420
tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 480
tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 540
tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 600
aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 660
ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 720
ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 780
tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 840
caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc 900
agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 960
tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 1020
aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 1080
ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 1140
atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 1200
ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg 1260
attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 1320
tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 1380
caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 1440
gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 1500
gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 1560
ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 1620
cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 1680
gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 1740
catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 1800
gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 1860
cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1920
gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1980
gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 2040
gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 2100
catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 2160
tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 2220
accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta 2280
tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg 2340
gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat 2400
acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc 2460
tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt 2520
agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 2580
atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 2640
aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 2700
caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 2760
ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 2820
cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 2880
tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 2940
gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 3000
ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 3060
gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 3120
caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 3180
ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 3240
tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 3300
ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc 3360
attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 3420
cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 3480
ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 3540
caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 3600
ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 3660
tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 3720
accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 3780
ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 3840
cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 3900
gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc 3960
taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 4020
tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 4080
gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 4140
tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 4200
ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 4260
agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 4320
agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 4380
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 4440
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 4500
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 4560
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 4620
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 4680
agctgtacaa gtccggccgg actcagatcc ccatgaaccg tgcttttagc aggaagaaag 4740
acaaaacatg gatgcataca cctgaagctt tatcaaaaca tttcattccc tataatgcaa 4800
agtttcttgg cagtacagaa gtggaacagc caaaaggaac agaagttgtg agagatgctg 4860
taaggaaact aaagtttgca agacatatca agaaatctga aggccagaaa attcctaaag 4920
tggagttgca aatatcaatt tatggagtaa aaattctaga acccaaaaca aaggaagttc 4980
aacacaattg ccagcttcat agaatatctt tttgtgcaga tgataaaact gacaagagga 5040
tattcacttt catatgcaaa gattctgagt caaataaaca tttgtgctat gtatttgaca 5100
gcgaaaagtg tgctgaagag atcactttaa caattggcca agcatttgac ctggcataca 5160
cgaaatttct agaatcagga ggaaaagatg ttgaaacaag aaaacagatc gcagggttac 5220
aaaaaagaat ccaagactta gaaacagaaa atatggaact taaaaataaa gtacaagatt 5280
tggaaaacca actgagaata actcaagtat cagcacctcc agcaggcagt atgacaccta 5340
agtcgccctc cactgacatc tttgatatga ttccattttc tccaatatca caccagtctt 5400
cgatgcctac tcgcaatggc acacagocac ctccagtacc tagtagatct actgagatta 5460
aacgggacct gtttggagca gaaccttttg acccatttaa ctgtggagca gcagatttcc 5520
ctccagatat tcaatcaaaa ttagatgaga tgcaggaggg gttcaaaatg ggactaactc 5580
ttgaacgcac agtattttgt ctcgacccgt tagacagtag gtgctgag 5628
<210> 8
<211> 6121
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: a construct comprising cloned into the vector pEGFP-N3
...
Cloning site of human CD36 cDNA
<400>
gatctatggg ctgtgaccgg aactgtgggc tcatcgctgg ggctgtcatt ggtgctgtcc 60
tggctgtgtt tggaggtatt ctaatgccag ttggagacct gcttatccag aagacaatta 120
aaaagcaagt tgtcctcgaa gaaggtacaa ttgcttttaa aaattgggtt aaaacaggca 180
cagaagttta cagacagttt tggatctttg atgtgcaaaa tccacaggaa gtgatgatga 240
acagcagcaa cattcaagtt aagcaaagag gtccttatac gtacagagtt cgttttctag 300
ccaaggaaaa tgtaacccag gacgctgagg acaacacagt ctctttcctg cagcccaatg 360
gtgccatctt cgaaccttca ctatcagttg gaacagaggc tgacaacttc acagttctca 420
atctggctgt ggcagctgca tcccatatct atcaaaatca atttgttcaa atgatcctca 480
attcacttat taacaagtca aaatcttcta tgttccaagt cagaactttg agagaactgt 540
tatggggcta tagggatcca tttttgagtt tggttccgta ccctgttact actacagttg 600
gtctgtttta tccttacaac aatactgcag atggagttta taaagttttc aatggaaaag 660
ataacataag taaagttgcc ataatcgaca catataaagg taaaaggaat ctgtcctatt 720
gggaaagtca ctgcgacatg attaatggta cagatgcagc ctcatttcca ccttttgttg 780
agaaaagcca ggtattgcag ttcttttctt ctgatatttg caggtcaatc tatgctgtat 840
ttgaatccga cgttaatctg aaaggaatcc ctgtgtatag attcgttctt ccatccaagg 900
cctttgcctc tccagttgaa aacccagaca actattgttt ctgcacagaa aaaattatct 960
caaaaaattg tacatcatat ggtgtgctag acatcagcaa atgcaaagaa gggagacctg 1020
tgtacatttc acttcctcat tttctgtatg caagtcctga tgtttcagaa cctattgatg 1080
gattaaaccc aaatgaagaa gaacatagga catacttgga tattcaacct ataactggat 1140
tcactttaca atttgcaaaa cggctgcagg tcaacctatt ggtcaagcca tcagaaaaaa 1200
ttcaagtatt aaagaatctg aagaggaact atattgtgcc tattctttgg cttaatgaga 1260
ctgggaccat tggtgatgag aaggcaaaca tgttcagaag tcaagtaact ggaaaaataa 1320
acctccttgg cctgatagaa atgatcttac tcagtgttgg tgtggtgatg tttgttgctt 1380
ttatgatttc atattgtgca tgcagatcga aaacaataaa agtcgacggt accgcgggcc 1440
cgggatccat cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 1500
tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 1560
agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 1620
ccgtgccctg gcccaccctc gtgaccaccc tgadctacgg cgtgcagtgc ttcagccgct 1680
accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 1740
aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 1800
tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 1860
gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 1920
ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 1980
gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 2040
tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 2100
agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 2160
acgagctgta caagtaaagc ggccgcgact ctagatcata atcagccata ccacatttgt 2220
agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 2280
gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa 2340
tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 2400
caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg 2460
cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 2520
cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga 2580
gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 2640
atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag 2700
cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 2760
acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 2820
tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 2880
cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2940
tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 3000
gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg 3060
tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 3120
agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 3180
tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 3240
gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 3300
cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 3360
aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag 3420
atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 3480
cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 3540
cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag 3600
cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 3660
ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 3720
ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 3780
cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 3840
gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 3900
tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg 3960
tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 4020
gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 4080
cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 4140
gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 4200
gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga 4260
tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc 4320
cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg 4380
gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat 4440
aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc 4500
ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt 4560
ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa 4620
cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt 4680
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 4740
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 4800
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 4860
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 4920
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 4980
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 5040
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 5100
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 5160
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 5220
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 5280
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 5340
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 5400
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 5460
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt 5520
aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 5580
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 5640
taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 5700
agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 5760
cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 5820
tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 5880
tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 5940
gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 6000
caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 6060
aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggactc 6120
a 6121
<210> 9
<211> 12
<212> Protein
<213> human
<400> 9
His Arg Ala Phe Ser Arg Lys Lys Asp Lys Thr Cys
1510
<210> 10
<211> 14
<212> Protein
<213> human
<400> 10
Phe Leu Gly Ser Thr Glu Val Glu Gln Pro Lys Gly Thr Glu
1510
<210> 11
<211> 15
<212> Protein
<213> human
<400> 11
Thr Arg Asn Gly Thr Gln Pro Pro Pro Val Pro Ser Arg Ser Thr
151015
<210> 12
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 12
cgcaaggatc cccatgaacc gtgcttttag caggaag 37
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 13
gatctactag gtactggag 19
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: T7 primer
<400> 14
taatacgact cactataggg aga 23
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: T3 primer
<400> 15
attaaccctc actaaaggga 20
...
Claims (114)
1. expression vector, comprise coding people CED-6 proteic deoxynucleoside acid sequence, wherein people CED-6 albumen comprises the aminoacid sequence of Fig. 4 or Fig. 5 or the aminoacid sequence of the amino acid variation that its difference with the aminoacid sequence of Fig. 4 or Fig. 5 only has the function conservative property.
2. the expression vector of claim 1 comprises Fig. 2 or the deoxynucleoside acid sequence from the transcription initiation codon to the Transcription Termination codon shown in Figure 3.
3. claim 1 or 2 expression vector, it comprises that coding is arranged in the deoxynucleoside acid sequence of the reporter gene of described carrier, the result is that the expression of the conservative variant of people CED-6 albumen or its function causes this report genetic expression reporter protein.
4. the expression vector of claim 3, wherein reporter gene is positioned at 5 ' end of the deoxynucleoside acid sequence of the conservative variant of coding people's CED-6 albumen or its function.
5. the expression vector of claim 3, wherein reporter gene is positioned at 3 ' end of the deoxynucleoside acid sequence of the conservative variant of coding people's CED-6 albumen or its function.
6. any one expression vector in the claim 3 to 5, wherein reporter gene encoding green fluorescent protein (GFP).
7. the expression vector of claim 4, it is for having inserted the pEGFP-C2 of the proteic deoxynucleoside acid sequence of coding people CED-6 in multiple clone site, and wherein people CED-6 albumen comprises as Fig. 4 or aminoacid sequence or the conservative variant of its function shown in Figure 5.
8. the expression vector of claim 5, it is for having inserted the pEGFP-N3 of the proteic deoxynucleoside acid sequence of coding people CED-6 in multiple clone site, and wherein people CED-6 albumen comprises as Fig. 4 or aminoacid sequence or the conservative variant of its function shown in Figure 5.
9. claim 4 or 7 expression vector, wherein said carrier comprises the nucleotide sequence (pGA3103) of Figure 28.
10. claim 5 or 8 expression vector, wherein said carrier comprises the nucleotide sequence (pGA3104) of Fig. 9.
11. the expression vector of claim 1 or 2, the variant that wherein said vector expression people CED-6 albumen or its function are conservative, and comprise the epi-position mark at the amino and/or the carboxyl terminal of carrier.
12. the expression vector of claim 11, wherein epi-position is labeled as HisA.
13. the expression vector of claim 11, it is pBAD/HisA, is inserted with the proteic deoxynucleoside acid sequence of coding people CED-6, and people CED-6 albumen comprises as Fig. 4 or aminoacid sequence shown in Figure 5, or the conservative variant of its function.
14. the expression vector of claim 12, it has deoxynucleoside acid sequence (pGA1028) shown in Figure 17.
15. be with each expression vector mammalian cells transfected in the claim 1 to 13.
16. the mammal cell line of claim 15, wherein said cell are selected from inoblast clone or epithelial cells system.
17. the mammal cell line of claim 16, wherein said clone is selected from COS1, BHK21, L929, CV1, SWISS 3T3, HT144, IMR32, HEPG2, MDCK, MCF7,293, Hela, A549, SW48, or G361.
18. the mammal cell line of claim 15, wherein said cell are primary cell system.
19. the mammal cell line of claim 18, wherein said clone is selected from human skin FIBs, skin keratin cell, white corpuscle, monocyte, lymphocyte, dendritic cell or scavenger cell.
20. the mammal cell line of claim 19, it is J774 or person monocytic cell's clone THP-1 for mouse macrophage.
21. method, this method is used for determining that whether a kind of compound is the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed, this method comprises that the mammalian cell with conversion any in the claim 15 to 20 is exposed to the apoptosis particle, and be determined at that compound exists or when lacking, the transfected cytophagic speed of particle.
22. the method for claim 21 wherein is exposed to compound with cells transfected before adding the apoptosis particle.
23. the method for claim 21 or 22, wherein the apoptosis particle is selected from the apoptosis neutrophil(e) cell, the red corpuscle of the apoptosis lymphocyte or the process conditioning that depends on the needs.
24. any one method in the claim 21 to 23, wherein the apoptosis particle comprises that attached cell is PC12.
25. any one method in the claim 21 to 23, wherein the apoptosis particle comprises the mouse cell lines Ba/F3 of growth factor dependency.
26. the method for claim 25, wherein the Ba/F3 cell is by cultivating when lacking growth factors I L-3 so that apoptosis to take place.
27. any one method in the claim 23 to 26, comprising the transfection of apoptosis particulate cytotostatic reporter gene.
28. the method for claim 27, wherein reporter gene is selected from coding beta-galactosidase gene, and proteic gene of coding fluorescence or coding can produce the proteic gene of fluorescence.
29. the method for claim 28, wherein albumen that can be luminous is luciferase.
30. the method for claim 28, wherein said fluorescin are green fluorescent protein.
31. the method for claim 26, wherein the Ba/F3 cell of apoptosis particle beta-galactosidase enzymes that comprised stable transfection or luciferase.
32. the method for claim 26, wherein phagocytotic level a kind ofly can be converted into that the substrate of fluorescent chemicals detects by beta-galactosidase enzymes by adding.
33. if any one method in the claim 21 to 32 wherein when being exposed to testing compound, is not observed phagolysis or the amount of engulfing reduces, and should detect the vigor of Mammals transfectional cell.
34. the method for claim 33 if wherein cell has vigor, is compared the phenotype of Mammals cells transfected with the mammalian cell phenotype of the untransfected of same cell system.
35. any one method in the claim 21 to 32, if wherein when testing compound exists, observe the increase of the amount of engulfing, this method comprises the mammalian cell of the same cell system that further described compound is exposed to untransfected, and observes compound and whether induce the identical phenotype of phenotype that shows with transfection mammalian cell.
36. by any one the method compounds identified of claim 21 to 35, it is accredited as the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed.
37. a method, this method are used for determining that whether a kind of compound is the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed, this method may further comprise the steps:
(1) comprises that to the mammalian cell microinjection difference of the aminoacid sequence of the aminoacid sequence of Fig. 4 or Fig. 5 or itself and Fig. 4 or Fig. 5 only has the people CED-6 albumen of the aminoacid sequence that the amino acid of function conservative property changes;
(2) mammalian cell that step (1) is produced is exposed to the apoptosis particle and measures the speed that particle is engulfed when compound exists or lack by cell.
38. the method for claim 37, wherein the mammalian cell of microinjection is exposed to compound before adding the apoptosis particle.
39. the method for claim 38, wherein the apoptosis particle is selected from the apoptosis neutrophil(e) cell, the apoptosis lymphocyte or depend on the needs through the conditioning the apoptosis red corpuscle.
40. the method in claim 37 or 38, wherein the apoptosis particle comprises that attached cell is PC12.
41. any one method in the claim 37 to 39, wherein apoptotic cell comprises the mouse cell lines Ba/F3 that somatomedin relies on.
42. the method for claim 41, wherein the Ba/F3 cell produces apoptosis by cultivating when growth factors I L-3 lacks.
43. any one method in the claim 39 to 42, comprising the transfection of apoptosis particulate cytotostatic reporter gene.
44. the method for claim 27, wherein reporter gene is selected from coding beta-galactosidase gene, the proteic gene that proteic gene of coding fluorescence or coding can be luminous.
45. the method for claim 44, the fluorescence that wherein said albumen can produce is luciferase.
46. the method for claim 44, wherein fluorescin is a green fluorescent protein.
47. the method for claim 42, wherein the apoptosis particle Ba/F3 cell of beta-galactosidase enzymes that comprised stable transfection.
48. the method for claim 47, wherein phagocytotic level can be surveyed by the substrate of beta-galactosidase enzymes conversionization fluorescent chemicals by adding.
49. any one method in the claim 37 to 48, wherein mammalian cell is inoblast or epithelial cell.
50. the method for claim 49, wherein mammalian cell is selected from COS1, BHK21, L929, CV1, SWISS 3T3, HT144, IMR32, HEPG2, MDCK, MCF7,293, Hela, A549, SW48 or G361.
51. any one method in the claim 37 to 48, wherein mammalian cell is a kind of primary cell.
52. the method for claim 51, wherein said mammalian cell is selected from human skin FIBs, skin keratin cell, white corpuscle, monocyte, lymphocyte, dendritic cell or scavenger cell.
53. the method for claim 52, wherein mammalian cell is mouse macrophage J774 or person monocytic cell THP-1.
54. if any one method in the claim 37 to 53 when being exposed to testing compound, is not observed phagolysis or the amount of engulfing reduces, and should detect the vigor of Mammals transfectional cell.
55. the method for claim 54 if wherein cell has vigor, is compared the phenotype of Mammals cells transfected with the mammalian cell phenotype of the untransfected of same cell system.
56. any one method in the claim 21 to 32, if wherein when to be measuredization and thing exist, observe the increase of the amount of engulfing, this method comprises the mammalian cell that further exposure compound is for the same cell of untransfected, and observes compound and whether induce the identical phenotype of phenotype that shows with transfection mammalian cell.
57. by any one the method compounds identified of claim 37 to 56, it is accredited as the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed.
58. a method, this method are used for determining that whether a kind of compound is the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed, this method may further comprise the steps:
(1) to mammalian cell microinjection or a kind of carrier of transfection, this vector expression with as all of Fig. 2 or nucleotide sequence shown in Figure 3 or the RNA sequence of a part of antisense;
(2) to the mammalian cell of apoptosis particle exposing step 1 preparation and measure when compound exists or lack particle by cytophagic speed.
59. the method for claim 58, wherein sense-rna is included in and is higher than 2 * SSC; 0.1%SDS; 25 ℃ of-50 ℃ of stringent conditions down can with the nucleotide sequence of the nucleotide sequence hybridization shown in Fig. 2 or Fig. 3.
60. the method for claim 58 or 59 comprises the feature that claim 38 to 56 is any.
61. by the compound that any one method of claim 58 to 60 is determined, method wherein can determine that whether described compound is the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed.
62. a peptide, it comprises the fragment that has as the people CED-6 homologue of the aminoacid sequence of Fig. 4, and wherein said fragment comprises aminoacid sequence NRAFSRKKDKTC, FLGSTEVEQPKGTE or TRNGTQPPPVPSRST.
63. the peptide of claim 62, by aminoacid sequence NRAFSRKKDKTC, FLGSTEVEQPKGET or TRNGTQPPPVPSRST form.
64. an antibody preparations, it comprises the antibody of the one or more epi-positions of anti-following people CED-6 homologue as Fig. 4: NRAFSRKKDKTC, FLGSTEVEQPKGTE or TRNGTQPPPVPSRST.
65. an antibody preparations comprises the antibody of anti-people CED-6 homologue epi-position NRAFSRKKDKTC.
66. an antibody preparations comprises the antibody of anti-people CED-6 homologue epi-position FLGSTEVEQPKGTE.
67. an antibody preparations comprises the antibody of anti-people CED-6 homologue epi-position TRNGTQPPPVPSRST.
68. any one antibody preparations in the claim 63 to 66, wherein said antibody is polyclonal antibody.
69. a method is used for diagnosing with proteic mistake of individual phagocytic cell people CED-6 and expresses or the relevant disease of low expression, comprising:
(a) from the described individual phagocytic cell sample that obtains;
(b) any one antibody preparations exposes phagocytic cell in claim 64 to 68;
(c) existence of the immunocomplex arbitrarily that between antibody and described CED-6 albumen, forms of quantitative assay;
(d) with the amount that relatively forms immunocomplex from the individual phagocytic cell of contrast.
70. a method is used for determining that whether a kind of compound is the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed, comprising:
(a) to the compound that the detects mammalian cells transfected of any one expression vector in the claim 1 to 14 that exposed transfection;
(b) expose any one antibody preparations in the claim 64 to 68 to mammalian cell;
(c) existence of the immunocomplex that forms between the albumen of quantitative measurment antibody and described cell expressing;
(d) the immunocomplex amount that detects and the level as the amount of the immunocomplex that detects in step (a) mammalian cells transfected that is not exposed to compound are compared.
71. the method for claim 70, wherein lactation send the thing cell to be selected from COS1, BHK21, L929, CU1 SWISS 3T3, HT144, IMR32, HEPG2, MDCK, MCF7,293, Hela, A549, SW48 or G361.
72. the method for claim 71, wherein mammalian cell is the COS1 cell.
73. the method for claim 70, wherein mammalian cell is human skin FIB, skin keratin cell, white corpuscle, monocyte or scavenger cell.
74. the method for claim 73, wherein said cell are mouse monokaryon cell J774 or person monocytic cell THP-1.
75. a fusion rotein comprises:
(1) difference of the sequence of the aminoacid sequence of a kind of Fig. 4 or Fig. 5 or itself and Fig. 4 or Fig. 5 only has the aminoacid sequence that the amino acid of the conservative type of function changes; With
(2) a kind of is the albumen of the expression product of reporter gene.
76. the fusion rotein of claim 75, it obtains by expressing the h1ced-7 encoding sequence shown in GFP and Fig. 9 or 28.
77. a fusion rotein comprises:
(1) a kind of as Fig. 4 or Fig. 5 aminoacid sequence or the difference of the aminoacid sequence of itself and Fig. 4 or Fig. 5 only on the function conservative property, take place sequence that amino acid changes and
(2) a kind of epi-position mark.
78. the fusion rotein of claim 77, it obtains by expressing HisA and hlced-6 encoding sequence shown in Figure 17.
79. a method, this method are used for determining that whether a kind of compound is the inhibitor or the toughener of the signal transduction pathway that promotes that apoptotic cell is engulfed, this method comprises:
(a) when testing compound exists or lack, expose the professional or half professional phagocytic cell of Mammals to the mammalian cell of apoptosis, the mammalian cell stable transfection of this apoptosis can produce the reporter gene that need not the signal that microscope surveys,
(b) remove the apoptotic cell of not engulfed by described phagocytic cell
(c) survey any next Autophagocytic signal;
This compound of Discrepancy Description of comparing any signal when compound exists when wherein lacking with compound is phagocytotic inhibitor of apoptotic cell or toughener.
80. the method for claim 79, wherein phagocytic cell is mouse cell lines J774 or human monocyte cell line THP-1.
81. the method for claim 80, wherein monocytic series is divided into scavenger cell before cultivation makes it be exposed to the apoptosis particle.
82. any one method in the claim 79 to 81, wherein phagocytic cell is a transgenic cell.
83. the method for claim 82, wherein phagocytic cell any one expression vector transfection of claim 1 to 14.
84. the method for claim 82, the wherein phagocytic cell expression vector transfection of Codocyte surface receptor CD36.
85. the method for claim 84, the wherein phagocytic cell carrier transfection of Figure 31.
86. the method for claim 79 to 85, wherein apoptotic cell comprises attached cell PC12.
87. the method for claim 79 to 85, wherein apoptotic cell comprises the mouse cell lines Ba/F3 that somatomedin relies on.
88. apoptosis takes place in the method for claim 26 when wherein the Ba/F3 cell is cultivated under growth factors I L-3 shortage condition.
89. the method for claim 88, wherein positive and be less than 5% propidium iodide when negative at about 20% annexin, cell is considered to apoptosis.
90. the method for claim 79, wherein reporter gene is selected from coding beta-galactosidase gene, and proteic gene of coding fluorescence or coding can produce the proteic gene of fluorescence.
91. the method for claim 90, the albumen that wherein can produce fluorescence is luciferase.
92. the method for claim 91, wherein the apoptotic cell plasmid of expression characterization of PGL2 contrast of performance Figure 19 that has been stabilized transfection.
93. the method for claim 92, wherein apoptotic cell has been stabilized transfection and has comprised the plasmid of the deoxyribonucleotide sequence of Figure 19.
94. any one method among the claim 79-89, wherein fluorescin is green fluorescent protein (GFP).
95. the method for claim 94, wherein apoptotic cell be stabilized transfection the performance expression characterization plasmid or as the plasmid of Figure 10 or 29.
96. being stabilized transfection, the method for claim 95, wherein said apoptotic cell comprise the plasmid of the deoxyribonucleotide sequence of Fig. 9 or 28.
97. any one method among the claim 79-89, wherein apoptotic cell has been stabilized transfection and has expressed the plasmid of beta-galactosidase enzymes.
98. the method for claim 97, wherein said plasmid has the expression characterization of plasmid shown in Figure 11.
99. the method for claim 98, wherein said plasmid comprise deoxyribonucleotide sequence shown in Figure 11.
100. any one method among the claim 79-89, wherein the apoptosis particle clone Ba/F3 of beta-galactosidase enzymes that comprised stable transfection.
101. the method for claim 100, the level of wherein engulfing a kind ofly can be converted to the substrate of fluorescent chemicals by beta-galactosidase enzymes and detected by adding.
102. if any one method among the claim 79-101 wherein when being exposed to testing compound, is not observed phagolysis or the amount of engulfing reduces, and should detect the vigor of Mammals transfectional cell.
103. any one method among the claim 79-102, wherein phagocytic cell is cultivated in porous plate, adds apoptotic cell and testing compound to each hole wherein.
104. the method for aforementioned each claim, wherein the signal from reporter gene detects by automatic plate reader.
105. the method for claim 101, wherein the signal from reporter gene detects by the automatic plate reader that can detect fluorescent signal.
106. a compound is accredited as inhibitor or the toughener that apoptotic cell is engulfed through any one method among the claim 79-105.
107. claim 22-35,39-56, any one method among the 58-60 is wherein engulfed by non-microscope mode and is measured.
108. the method for claim 107, wherein non-microscope mode is the porous flat plate readout instrument.
109. the method for claim 108, wherein the porous flat plate readout instrument measure luminous, fluorescence or carry out Spectrophotometric Assays.
110. any one method among the claim 79-105 has the characteristics of claim 108 and 109.
111. a method is used for diagnosing with the cytophagous people CED-6 of individuality and crosses expression or the relevant disease of low expression, comprising:
(a) from described individuality, obtain the phagocytic cell sample,
(b) from the phagocytic cell isolation of RNA,
(c) prepare DNA from RNA
(d) on described cDNA, carry out the PCR reaction first time,
(e) on the reaction product of the described PCR reaction first time, carry out (nested type) PCR for the second time,
(f) by analyzing the reaction product of the 1st time and the 2nd time PCR, the existence of quantitative and observational measurement CED-6RNA,
(g) with the reaction product that forms from the individual phagocytic cell of contrast relatively in the amount and the type of the reaction product of the 1st time and the 2nd time PCR formation.
112. the method for claim 111, wherein PCR uses the sequence deutero-primer from people CED-6, or the carrier deutero-primer that uses when cDNA produces.
113. the method for claim 111 or 112, wherein the primer of the 1st PCR use has nucleotide sequence:
1)cgcaaggatccccatgaaccgtgcttttagcaggaag
2)gatctactaggtactggag
114. the method for claim 111,112 or 113, wherein the primer of the 2nd PCR use has nucleotide sequence:
1)cgcaaggatccccatgaaccgtgcttttagcaggaag
2)gcggatggtaccgtcgactgctgatacttgagttattctcag。
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9812660.0A GB9812660D0 (en) | 1998-06-11 | 1998-06-11 | Engulfment gene and uses thereof |
| GB9812660.0 | 1998-06-11 | ||
| GBGB9820816.8A GB9820816D0 (en) | 1998-09-24 | 1998-09-24 | Engulfment gene and uses thereof |
| GB9820816.8 | 1998-09-24 |
Publications (1)
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| CN1305525A true CN1305525A (en) | 2001-07-25 |
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|---|---|---|---|
| CN99807222A Pending CN1305525A (en) | 1998-06-11 | 1999-06-10 | Phagocytic assay method |
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| EP (1) | EP1084242A2 (en) |
| JP (1) | JP2002517238A (en) |
| KR (1) | KR20010071453A (en) |
| CN (1) | CN1305525A (en) |
| AU (1) | AU4608499A (en) |
| BR (1) | BR9911097A (en) |
| CA (1) | CA2332993A1 (en) |
| HU (1) | HUP0102349A2 (en) |
| IL (1) | IL140016A0 (en) |
| PL (1) | PL345951A1 (en) |
| WO (1) | WO1999064586A2 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020004041A1 (en) * | 1999-02-19 | 2002-01-10 | Albert Matthew L. | Methods for abrogating a cellular immune response |
| US6489535B1 (en) | 1999-03-18 | 2002-12-03 | The Board Of Trustees Of The Leland Stanford Junior University | Non-mammalian transgenic animal having an adult onset neurodegenerative phenotype |
| US6316690B1 (en) | 1999-08-04 | 2001-11-13 | Tosk, Inc. | Non-mammalian transgenic animal model for cellular proliferative diseases |
| US6365129B1 (en) | 1999-08-04 | 2002-04-02 | Tosk, Inc. | Invivo high throughput toxicology screening method |
| US7615676B2 (en) | 2001-11-30 | 2009-11-10 | U.S. Department Of Veterans Affairs | Transgenic screen and method for screening modulators of brain-derived neurotrophic factor (BDNF) production |
| US7491810B2 (en) * | 2001-11-30 | 2009-02-17 | U.S. Department Of Veterans Affairs | Transgenic screen and method for screening modulators of brain-derived neurotrophic factor (BDNF) production |
| WO2018212612A1 (en) * | 2017-05-17 | 2018-11-22 | 울산과학기술원 | Method and device for evaluating immune cells using magnetic particle |
| KR102213080B1 (en) * | 2017-05-17 | 2021-02-08 | 울산과학기술원 | Method for evaluating immune cells using magnetic particles |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6232061B1 (en) * | 1992-04-01 | 2001-05-15 | Cambridge Neuroscience, Inc. | Homology cloning |
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1999
- 1999-06-10 AU AU46084/99A patent/AU4608499A/en not_active Abandoned
- 1999-06-10 WO PCT/EP1999/004043 patent/WO1999064586A2/en not_active Application Discontinuation
- 1999-06-10 JP JP2000553576A patent/JP2002517238A/en active Pending
- 1999-06-10 CA CA002332993A patent/CA2332993A1/en not_active Abandoned
- 1999-06-10 HU HU0102349A patent/HUP0102349A2/en unknown
- 1999-06-10 IL IL14001699A patent/IL140016A0/en unknown
- 1999-06-10 PL PL99345951A patent/PL345951A1/en not_active Application Discontinuation
- 1999-06-10 KR KR1020007014063A patent/KR20010071453A/en not_active Application Discontinuation
- 1999-06-10 CN CN99807222A patent/CN1305525A/en active Pending
- 1999-06-10 EP EP99929185A patent/EP1084242A2/en not_active Withdrawn
- 1999-06-10 BR BR9911097-0A patent/BR9911097A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP1084242A2 (en) | 2001-03-21 |
| KR20010071453A (en) | 2001-07-28 |
| WO1999064586A3 (en) | 2000-04-27 |
| AU4608499A (en) | 1999-12-30 |
| JP2002517238A (en) | 2002-06-18 |
| BR9911097A (en) | 2001-02-13 |
| CA2332993A1 (en) | 1999-12-16 |
| HUP0102349A2 (en) | 2001-10-28 |
| PL345951A1 (en) | 2002-01-14 |
| IL140016A0 (en) | 2002-02-10 |
| WO1999064586A2 (en) | 1999-12-16 |
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