CN111521817B - Method for identifying protein phosphorylation sites - Google Patents
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Abstract
本发明公开了一种用于识别蛋白质磷酸化位点的方法，涉及分子生物技术领域，具体地，该用于识别蛋白质磷酸化位点的方法包括将表达载体1表达的蛋白通过免疫沉淀实验分离，使用磷酸化抗体进行免疫印迹实验以检测待测磷酸化位点的修饰情况，表达载体1含具有待测位点的氨基酸序列片段，其在没有针对单个蛋白位点的磷酸化抗体的条件下，也能够有效准确地检测蛋白单个位点的磷酸化修饰情况，节省了针对特定位点的磷酸化抗体的制备成本，加快蛋白质磷酸化位点的研究进程。
The invention discloses a method for identifying protein phosphorylation sites, which relates to the field of molecular biology technology. Specifically, the method for identifying protein phosphorylation sites comprises separating the protein expressed by expression vector 1 by immunoprecipitation experiment. , using a phosphorylated antibody to carry out an immunoblotting experiment to detect the modification of the phosphorylation site to be tested, the expression vector 1 contains an amino acid sequence fragment with the site to be tested, which does not have a phosphorylated antibody against a single protein site. It can also effectively and accurately detect the phosphorylation modification of a single site of the protein, save the cost of preparing phosphorylated antibodies for specific sites, and speed up the research process of protein phosphorylation sites.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for identifying protein phosphorylation sites.
Background
The phosphorylation and dephosphorylation processes of proteins regulate important vital processes such as cell signal transduction, cell metabolism, cell differentiation and cell growth, and are molecular switches of cell physiological activities. Different protein kinases can recognize and modify different sites of different proteins, which increases the complexity of the study of phosphorylated proteins, making phosphorylated proteins a hotspot for post-translational modification studies of proteins.
By using32P-selective marker phosphorylated protein is a classical technique, which consists in purifying the kinase and32p for in vitro labelling, or by use of32P-ATP or32PO4 3-(orthophosphate) and then the proteins are separated by SDS-PAGE, 2D-GE or thin layer chromatography, and the labeled phosphorylated proteins are detected by autoradiography or optical imaging.
Whether a phosphorylated protein detected in vitro is of biological interest must be verified for its phosphorylation in vivo. Because in vitro kinases may interact with many proteins which are not at all accessible under physiological conditions due to their different cellular or subcellular structure, false positive knots may fall.
The in vivo phosphorylation marker study also depends on the efficiency of the body's own phosphorylation and the equilibrium relationship between the phosphorylation and non-phosphorylation states of the target protein. If a protein is already saturated with phosphate groups, there will be no insertion of radiolabeled phosphate groups regardless of kinase activity, and therefore phosphorylated proteins will not be detected.
By the 90 s of the 20 th century. The standard approach for phosphorylation site analysis is to use32P marks the purified phosphorylated protein, separates the obtained peptide segment by one-dimensional thin-layer electrophoresis and two-dimensional thin-layer chromatography (called two-dimensional peptide spectrum), detects the phosphorylated peptide segment by autoradiography, and carries out Edman degradation sequencing; the identification is then carried out by means of a specific retention time of the phosphorylated residues, the release of fluorescently labeled or radiolabeled amino acids ultimately localizes the amino acids released at the phosphorylation sites, and this method is clearly too labor intensive for large-scale applications.
With the continuous development of proteomics technology, proteomics research has entered into the quantitative analysis and functional proteomics stage, and phosphorylation modification becomes the focus of attention of many scholars.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for identifying protein phosphorylation sites.
The invention is realized by the following steps:
embodiments provide a method for identifying a phosphorylation site of a protein, comprising the steps of: separating the protein expressed by the expression vector 1 through an immunoprecipitation experiment, and carrying out an immunoblotting experiment by using a phosphorylated antibody to detect the modification condition of a phosphorylation site to be detected, wherein the expression vector 1 contains an amino acid sequence fragment 1 with the site to be detected;
the amino acid sequence fragment 1 is a fragment of the protein to be detected, wherein permanent non-phosphorylation site mutation is carried out on most possible reversible phosphorylation sites except the site to be detected.
The invention has the following beneficial effects:
the embodiment of the invention provides a method for identifying protein phosphorylation sites, which can effectively and accurately detect phosphorylation modification conditions of single protein sites under the condition that no phosphorylation antibody aiming at the single protein sites exists, saves the preparation cost of the phosphorylation antibody aiming at specific sites, and accelerates the research process of the protein phosphorylation sites.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a map of the ATGL vector in example 2;
FIG. 2 is the expression diagram of ATGL-Ser47 locus expression vectors 1, 2 and 3 in example 2 of the present invention;
FIG. 3 is a graph showing the silver staining results and the immunoblotting experiment results of expression vector 1 corresponding to 10 phosphorylation sites of ATGL protein in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Noun definitions
As used herein, "protein phosphorylation" refers to the process of transferring the phosphate group of ATP to the amino acid residues (serine, threonine and tyrosine) of the substrate protein, or the binding of GTP by a signal, catalyzed by protein kinases. The reason that the analysis of protein phosphorylation sites is difficult in the prior art is that protein phosphorylation is an unstable dynamic process in vivo, phosphorylated proteins are low in intracellular abundance, and phosphate groups of phosphorylated proteins are easily lost in a separation process and are difficult to protonate due to electronegativity.
As used herein, the term "phosphorylated antibody" refers to an antibody produced against a phosphorylation site of a substrate in a phosphorylated state, and an immunogen is phosphorylated, and an antibody produced using the phosphorylated antibody is referred to as a phosphorylated antibody. However, the prior art has the disadvantages that the preparation of specific phosphorylated antibodies directed against a single site of a specific protein is difficult, the preparation cost is high, the preparation time is long, and the preparation may not be successful.
As used herein, the term "reversible phosphorylation site" refers to a site on a protein at which phosphorylation is possible, wherein the phosphorylation is catalyzed by a protein kinase, and the transfer of a phosphorylated gene of ATP or GTP to an amino acid residue of the protein is reversed, wherein the dephosphorylation and the non-phosphorylation of the protein are reversed, and wherein the phosphorylation is catalyzed by a protein phospho (ester) enzyme.
Reference herein to "the majority of possible reversible phosphorylation sites" is to: after all of the "most likely reversible phosphorylation sites" are subjected to permanent non-phosphorylation site mutation, the vector containing a fragment of the most likely reversible phosphorylation sites can be used as a negative control to the extent that the vector does not react with a phosphorylated antibody.
Reference herein to "permanent non-phosphorylation site mutation" and "permanent phosphorylation site mutation" is a reference to the mutation of an amino acid that is reversibly phosphorylated to a site to a permanent non-phosphorylated and permanently phosphorylated amino acid.
The embodiment of the invention provides a method for identifying protein phosphorylation sites, which can detect phosphorylation site modification conditions of protein single sites under the condition of no specific phosphorylation antibody aiming at the protein single sites.
Specifically, the method specifically comprises the following steps: and (3) separating the protein expressed by the expression vector 1 through an immunoprecipitation experiment, and carrying out an immunoblotting experiment by using a phosphorylation antibody to detect the modification condition of the phosphorylation site to be detected, wherein the expression vector 1 contains an amino acid sequence fragment 1 with the site to be detected.
The amino acid sequence fragment 1 is a fragment of the protein to be detected, wherein permanent non-phosphorylation site mutation is carried out on most possible reversible phosphorylation sites except the site to be detected.
Specifically, in the expression vector 1, the purpose of carrying out permanent non-phosphorylation site mutation on most possible reversible phosphorylation sites except for the site to be detected is as follows: the possibility of phosphorylation of other reversible phosphorylation sites is eliminated, so that the object detected by the phosphorylation antibody which is not specifically directed to a single site is directed only to the site to be detected, namely under the condition, the phosphorylation condition on the site to be detected can be detected without the phosphorylation antibody which is specifically directed to a single site of a certain protein.
In an alternative embodiment, the method further comprises separating the protein expressed by the expression vector 2 through an immunoprecipitation experiment, and performing a Western Blot experiment using a phosphorylated antibody to detect the modification of the phosphorylation site to be detected, wherein the expression vector 2 contains an amino acid sequence fragment 2 having the phosphorylation site to be detected.
The amino acid sequence segment 2 is a segment in which most possible reversible phosphorylation sites on the segment of the protein to be detected are subjected to permanent non-phosphorylation site mutation. As a negative control for the method.
In an alternative embodiment, the method further comprises separating the protein expressed by the expression vector 3 through an immunoprecipitation experiment, and performing a Western Blot experiment using a phosphorylated antibody to detect the modification of the phosphorylation site to be detected, wherein the expression vector 3 contains the amino acid sequence fragment 3 having the phosphorylation site to be detected.
And the amino acid sequence fragment 3 is a fragment in which permanent phosphorylation site mutation is carried out on a site to be detected on a fragment of a protein to be detected, and permanent non-phosphorylation site mutation is carried out on most of possible reversible phosphorylation sites except the site to be detected. As a positive control for the method.
In the case of expression vectors 1-3, the method comprises separating proteins expressed by the expression vectors 1-3, and performing Western Blot experiments using phosphorylated antibodies to detect the modification of the phosphorylated sites to be detected. In addition, "amino acid sequence fragment 1, amino acid sequence fragment 2, and amino acid sequence fragment 3" in the specification of the present application are all amino acid sequence fragments of a protein to be detected, and the differences are only in the treatment modes of phosphorylation sites on the sequence fragments.
The protein expressed by the expression vector 1 is used as a wild type sample for detecting the locus to be detected, the protein expressed by the expression vector 2 is used as a negative control for detecting the locus to be detected, and the protein expressed by the expression vector 3 is used as a positive control for detecting the locus to be detected. In the embodiment with the expression vectors 1-3, comparison can be carried out according to the detection result, and the difference of the phosphorylation site modification degree can be compared.
In an alternative embodiment, the phosphorylated antibody is a phosphorylated antibody that is non-specific for a single site.
Preferably, the phosphorylated antibody comprises: an antibody that is resistant to phosphorylation by any one or more of the following amino acids: serine, threonine and tyrosine.
Further, the above-mentioned "antibody resistant to phosphorylation of any one amino acid" includes: serine phosphorylated antibodies, threonine phosphorylated antibodies, tyrosine phosphorylated antibodies; further examples are antibodies that are resistant to phosphorylation of various amino acids: serine/threonine phosphorylated antibodies, serine/tyrosine phosphorylated antibodies, tyrosine/threonine phosphorylated antibodies, and tyrosine/serine/threonine phosphorylated antibodies.
In an alternative embodiment, the means of screening for reversible phosphorylation sites on the test protein sequence is a phosphoproteomic analysis. It should be noted that the potential reversible phosphorylation sites can also be identified by screening in other ways, such as by searching the available literature.
In alternative embodiments, the permanent non-phosphorylation site mutation is a mutation of a serine, threonine, and/or tyrosine to an alanine or phenylalanine.
Specifically, "mutation of serine, threonine and/or tyrosine to alanine or phenylalanine" can be understood as 2 cases, and when a permanent non-phosphorylation site mutation is made for a single reversible phosphorylation site, any one of serine, threonine and tyrosine is mutated to alanine or phenylalanine; when permanent non-phosphorylation site mutation is made for multiple phosphorylation sites on a sequence, serine, threonine and tyrosine of most possible reversible phosphorylation sites on the sequence are mutated to alanine or phenylalanine.
The permanent phosphorylation site is mutated to serine, threonine and/or tyrosine to aspartic acid or glutamic acid. It should be noted that the understanding of the permanent phosphorylation site mutation is analogized to the understanding of the permanent non-phosphorylation site mutation.
In an alternative embodiment, the method is used to identify the phosphorylation site of triglyceride lipase.
Enzymes responsible for triglyceride metabolism include triglyceride lipase (ATGL), Hormone Sensitive Lipase (HSL) and monoglyceride lipase (MGL). Among them, research on HSL and MGL has been over 40 years old, and ATGL has been discovered only in recent years, and its research is progressing.
ATGL was originally found in adipose tissue, and has been found to be widely present in many tissues (including liver, cardiac and skeletal). ATGL is the rate-limiting enzyme involved in the first step of triglyceride hydrolysis. Under the action of ATGL, triglycerides are hydrolyzed to Diglycerides (DAG), then diglycerides are hydrolyzed by HSL to monoglycerides, and then completely hydrolyzed to glycerol and fatty acids by monoglyceride lipase MGL. Although phosphorylation of ATGL plays an important role in the synthesis and breakdown of triglycerides, the lack of antibodies directed to single-site phosphorylation of ATGL has limited the study of ATGL phosphorylation modifications in relation to their function. The phosphorylation sites of ATGL can be rapidly and effectively analyzed by adopting the method provided by the embodiment of the application.
In an alternative embodiment, the phosphorylation site of the triglyceride lipase is selected from the group consisting of: any one of Ser47, Ser87, Thr101, Thr210, Thr372, Tyr378, Ser393, Ser396, Ser406, and Ser 430.
In an alternative embodiment, the phosphorylation site of the triglyceride lipase is Ser 47.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present example provides a method for identifying a phosphorylation site of a protein, which includes the following steps.
(1) Determination of phosphorylation sites of a test sample
Most of the possible reversible phosphorylation sites are screened for their possible existence by literature search means or phosphorylation proteomics analysis.
(2) Construction of expression vectors 1-3
The expression vectors 1-3 are plasmids respectively containing amino acid sequence fragments 1-3, the amino acid sequence fragments 1-3 are sequence fragments containing to-be-detected sites of to-be-detected proteins, and the sequence lengths are the same.
Wherein, the amino acid sequence segment 1 is a segment of which most possible reversible phosphorylation sites except the site to be detected on the segment of the protein to be detected are subjected to permanent non-phosphorylation site mutation;
the amino acid sequence segment 2 is a segment in which permanent non-phosphorylation site mutation is carried out on most possible reversible phosphorylation sites on a segment of the protein to be detected;
and the amino acid sequence fragment 3 is a fragment in which permanent phosphorylation site mutation is carried out on a site to be detected on a fragment of a protein to be detected, and permanent non-phosphorylation site mutation is carried out on most of possible reversible phosphorylation sites except the site to be detected.
(3) Detection of phosphorylation sites
And (3) respectively carrying out transfection experiments on the expression vectors 1-3 in the step (2), culturing host cells, and obtaining proteins expressed by the host cells. And (3) separating the proteins expressed by the expression vectors 1-3 through an immunoprecipitation experiment, and performing an immunoblotting experiment by using a phosphorylation antibody to detect the modification condition of the phosphorylation sites to be detected.
Example 2
This example provides a method for detecting ATGL single-site phosphorylation, and the phosphorylation site of Ser47 is used as a research object in this example.
(1) Screening for possible phosphorylation sites of ATGL
The mouse ATGL is reported to be modified by 10 phosphorylation sites in the literature, including Ser47, Ser87, Thr101, Thr210, Thr372, Tyr378, Ser393, Ser396, Ser406 and Ser 430.
(2) Construction of expression vectors
In this example, the phosphorylation site of Ser47 was used as a study object, and a 10-site permanent non-phosphorylation mutant plasmid controlled by CMV promoter (FLAG-FLAG ditag, p9-ATGL-FLAG) was designed as a negative control, i.e., expression vector 2, and the ATGL vector map is shown in FIG. 1. The base sequence of the amino acid sequence fragment is shown as SEQ ID No. 2.
The plasmid contains 10 permanent non-phosphorylation mutation sites of amino acid sequence fragment: S47A, S87A, T101A, T210A, T372A, Y378F, S393A, S396A, S406A and S430A (a is an abbreviation for alanine, F is an abbreviation for phenylalanine, it should be noted that in other embodiments, Y378 may be mutated to alanine for the negative control, and the other 9 phosphorylation sites may be mutated to phenylalanine).
Then, a Ser47 permanently phosphorylated plasmid (p10-ATGL-S47D-FLAG) and a Ser47 wild-type plasmid (p10-ATGL-S47-FLAG) were constructed on the basis of negative controls.
Specifically, the Ser47 permanently phosphorylated plasmid is an expression vector 3, and the base sequence of the amino acid sequence fragment 3 is shown as SEQ ID No. 3; the Ser47 wild type plasmid is an expression vector 1, and the base sequence of the amino acid sequence segment 3 is shown as SEQ ID No. 1.
It should be noted that, in the examples, when Ser87 is the research site, the operation process of the method is the same as Ser47, except that in the plasmid construction, the positive control is p10-ATGL-S87D-FLAG, the wild type plasmid is p10-ATGL-S87-FLAG, and the rest sites are analogized in turn.
(3) Detection of phosphorylation sites
3.1 cell culture and plasmid transfection
Transfecting a mouse-derived cardiomyocyte cell line HL-1 cell with the negative plasmid (expression vector 2), the positive plasmid (expression vector 3) and the wild-type plasmid (expression vector 1) obtained in the step (2), respectively, growing the cell in Claycomb medium (Sigma) supplemented with 10% FBS, 1% penicillin and streptomycin, and culturing the cell at 37 ℃ and 5% CO2Culturing under the conditions of (1).
3.2 harvesting of cells
After plasmid transfection for 24-48 h protein expression, the culture medium is discarded, the culture dish is washed with 4 ℃ precooled PBS for 1-2 times, and the liquid is sucked dry. At 4 ℃ or on ice, add the appropriate amount of 4 ℃ pre-cooled cell IP lysis buffer (10 mM NaF, 10mM Na added)3VO4And protease inhibitor), the cells were harvested with a cell scraper, lysed at 4 ℃ or on ice for 30 minutes, centrifuged at 12000g for 30 minutes in a centrifuge, and the supernatant was collected.
A small amount of lysate was taken for Western blot analysis and the remaining lysate was added to 50. mu.L of FLAG magnetic beads (Sigma-Aldrich M8823) and incubated at 4 ℃ for 4h with slow shaking. After incubation, the beads were separated with a magnetic rack and the supernatant was aspirated. The beads were washed 1 to 2 times with 1mL lysis buffer. Finally, 15. mu.L of 2 XSDS loading buffer was added and the mixture was heat denatured by boiling for 10 min. Total protein was separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked at 4 ℃ for 8h in TBS buffer with 0.1% Tween 20 and 5% bovine albumin, and incubated at 4 ℃ for 8h as primary antibody. Incubation with horseradish-peroxidase conjugated secondary antibody for 8h and detection with ECL system.
The ATGL protein was separated by FLAG magnetic bead immunoprecipitation, silver staining and immunoblotting as shown in FIG. 2. In FIG. 2, Input is a transfected cell lysate; output is supernatant after immunoprecipitation of transfected cell lysate; wash is protein washed by FLAG magnetic beads after immunoprecipitation; beads are FLAG magnetic bead binding protein. The Heavy chain refers to the Heavy chain of the FLAG antibody on the magnetic beads, and the Light chain refers to the Light chain of the FLAG antibody on the magnetic beads; ATGL-FLAG refers to FLAG-tagged ATGL protein expressed by an expression vector.
Silver staining results show that the FLAG magnetic bead binding protein is mainly the ATGL protein with the FLAG label expressed by an expression vector. The immunoblotting result shows that the phosphorylation modification of the carrier 3 is positive, the phosphorylation modification of the carrier 2 is negative, and the phosphorylation modification of the carrier 1 is weak.
Example 3
The present embodiment provides a method for detecting ATGL single-site phosphorylation, which uses the phosphorylation sites of Ser47, Ser87, Thr101, Thr210, Thr372, Tyr378, Ser393, Ser396, Ser406, and Ser430 as the research targets, respectively, as in embodiment 2.
TABLE 1 sequence information
Transfecting HL-1 cells with ATGL10 phosphorylation site plasmid vector 1 for 48 hours to collect protein, and separating ATGL protein by FLAG magnetic bead immunoprecipitation reaction. Silver staining results and immunoblotting results please refer to fig. 3.
Silver staining results show that the FLAG magnetic bead binding protein is mainly an ATGL protein with a FLAG label expressed by an expression vector. Immunoblot results showed phosphorylation modification of ATGL ten phosphorylation site vector 1.
As can be seen from FIG. 3, the phosphorylation modifications of the protein expressed by the expression vector 1 corresponding to each of the 10 phosphorylation sites of ATGL protein were detected by the reaction system. In conclusion, the method provided in example 1 can effectively detect the phosphorylation state of the target site. ATGL is a key enzyme in triglyceride metabolism, and phosphorylation is a major pathway for regulating ATGL activity. The method can effectively replace ATGL single-site phosphorylation antibody, and is convenient for various functional researches on ATGL. The same principle can be applied to other proteins, and provides a powerful tool for researching protein phosphorylation modification.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Beijing thoracic Hospital affiliated to capital medical university
BEIJING TUBERCULOSIS AND THORACIC TUMOR Research Institute
<120> a method for identifying protein phosphorylation sites
<160> 21
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<400> 3
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccga cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 4
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 4
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatccctc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 5
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 5
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccga cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 6
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 6
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
accctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 7
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 7
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gacctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 8
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 8
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacacc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 9
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 9
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgac agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 10
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 10
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag cagacgggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 11
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 11
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggacggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 12
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 12
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gtatctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 13
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 13
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca ggagctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 14
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 14
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgccttcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 15
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 15
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgaca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 16
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 16
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactgtctga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 17
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 17
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggacga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 18
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 18
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccagtctct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 19
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 19
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggacct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgcc ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 20
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 20
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctctca ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
<210> 21
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 21
atgttcccga gggagaccaa gtggaacatc tcattcgctg gctgcggctt cctcggggtc 60
taccacattg gcgtggcctc ctgcctccgt gagcacgcgc ccttcctggt ggccaacgcc 120
actcacatct acggagccgc cgcaggggcg ctcaccgcca cagcgctggt cactggggcc 180
tgcctgggtg aagcaggtgc caacattatt gaggtgtcca aggaggcccg gaagcggttc 240
ctgggtcctc tgcatcccgc cttcaacctg gtgaagacca tccgtggctg tctactaaag 300
gccctgcctg ctgattgcca tgagcgcgcc aatggacgcc tgggcatctc cctgactcgt 360
gtttcagacg gagagaacgt catcatatcc cactttagct ccaaggatga gctcatccag 420
gccaatgtct gcagcacatt tatcccggtg tactgtggcc tcattcctcc taccctccaa 480
ggggtgcgct atgtggatgg cggcatttca gacaacttgc cactttatga gctgaagaat 540
accatcacag tgtccccatt ctcaggcgag agtgacatct gccctcagga cagctccacc 600
aacatccacg agcttcgcgt caccaacgcc agcatccagt tcaaccttcg caatctctac 660
cgcctctcga aggctctctt cccgccagag cccatggtcc tccgagagat gtgcaaacag 720
ggctacagag atggacttcg attccttagg aggaatggcc tactgaacca acccaaccct 780
ttgctggcac tgcccccagt tgtcccccag gaagaggatg cagaggaagc tgctgtggtg 840
gaggagaggg ctggagagga ggatcaattg cagccttata gaaaagatcg aattctagag 900
cacctgcctg ccagactcaa tgaggccctg ctggaggcct gtgtggaacc aaaggacctg 960
atgaccaccc tttccaacat gctaccagtg cgcctggcaa cggccatgat ggtgccctat 1020
actctgccgc tggagagtgc agtgtccttc accatccgct tgttggagtg gctgcctgat 1080
gtccctgaag atatccggtg gatgaaagag caggccggta gcatctgcca gttcctggtg 1140
atgagggcca agaggaaatt gggtgaccat ctgcctgcca gactggccga gcaggtggaa 1200
ctgcgacgtg cccaggccct gccctctgtg ccactgtctt gcgccaccta cagtgaggcc 1260
ctacccaact gggtacgaaa caacctcgac ctgggggacg cgctggccaa gtgggaagaa 1320
tgccagcgtc agctactgct gggtctcttc tgcaccaatg tggccttccc gccggatgcc 1380
ttgcgcatgc gcgcacctgc cagccccact gccgcagatc ctgccacccc acaggatcca 1440
cctggcctcc cgccttgctg a 1461
Claims (8)
1. A method for identifying phosphorylation sites of a protein, comprising the steps of: separating proteins expressed by the expression vector 1, the expression vector 2 and the expression vector 3 through an immunoprecipitation experiment, and carrying out a Western Blot experiment by using a phosphorylated antibody to detect the modification condition of a phosphorylation site to be detected, wherein the expression vector 1, the expression vector 2 and the expression vector 3 sequentially comprise an amino acid sequence fragment 1, an amino acid sequence fragment 2 and an amino acid sequence fragment 3 with the site to be detected;
the amino acid sequence fragment 1 is a fragment of which most of possible reversible phosphorylation sites except the site to be detected on the fragment of the protein to be detected are subjected to permanent non-phosphorylation site mutation;
the amino acid sequence segment 2 is a segment in which permanent non-phosphorylation site mutation is carried out on most possible reversible phosphorylation sites on a segment of the protein to be detected;
an amino acid sequence fragment 3, which is a fragment in which permanent phosphorylation site mutation is performed on a site to be detected on a fragment of a protein to be detected, and permanent non-phosphorylation site mutation is performed on most of possible reversible phosphorylation sites except the site to be detected;
the phosphorylated antibody is a phosphorylated antibody that is non-specific for a single site.
2. The method for identifying phosphorylation sites of a protein according to claim 1, wherein the phosphorylated antibody comprises: an antibody that is resistant to phosphorylation of any one or more of the following amino acids: serine, threonine and tyrosine.
3. The method for identifying protein phosphorylation sites according to claim 1, wherein the method further comprises screening reversible phosphorylation sites on a test protein sequence before separating proteins expressed by expression vectors 1-3 through an immunoprecipitation experiment and performing a Western Blot experiment using a phosphorylation antibody to detect modifications of the phosphorylation sites to be tested, respectively.
4. The method according to claim 3, wherein the reversible phosphorylation sites on the test protein sequence are selected by: phosphoproteomics analysis.
5. The method for identifying phosphorylation sites of a protein according to claim 1, wherein the permanent non-phosphorylation site is mutated to a serine, threonine and/or tyrosine to an alanine or phenylalanine;
the permanent phosphorylation site is mutated to serine, threonine and/or tyrosine to aspartic acid or glutamic acid.
6. The method for identifying the phosphorylation site of a protein according to claim 1, wherein the method is used for identifying the phosphorylation site of triglyceride lipase.
7. The method for identifying the phosphorylation site of a protein according to claim 6, wherein the phosphorylation site of the triglyceride lipase is selected from the group consisting of: any one of Ser47, Ser87, Thr101, Thr210, Thr372, Tyr378, Ser393, Ser396, Ser406, and Ser 430.
8. The method for identifying the phosphorylation site of a protein according to claim 7, wherein the phosphorylation site of the triglyceride lipase is Ser 47.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101233228A (en) * | 2005-06-10 | 2008-07-30 | 马德里自治大学 | Novel phosphorylation sites, modified proteins and uses of mitogen-activated protein kinases |
| CN101506226A (en) * | 2005-11-14 | 2009-08-12 | 维兹曼科学研究所耶达研究与发展有限公司 | Improved variants of pigment epithelium derived factor and uses thereof |
| WO2011053565A2 (en) * | 2009-10-29 | 2011-05-05 | Biomedical Sciences Research Centre "Alexander Fleming" | Compositions and methods for detecting a tauopathy |
| CN103163010A (en) * | 2011-12-14 | 2013-06-19 | 天津市国际生物医药联合研究院 | Method for relative quantitative analysis of protein phosphorylation modification level |
| CN109342592A (en) * | 2018-10-31 | 2019-02-15 | 华中科技大学 | A method for identifying specific phosphorylated amino acid sites of kinases in vitro |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030215899A1 (en) * | 2002-02-13 | 2003-11-20 | Ceptyr, Inc. | Reversible oxidation of protein tyrosine phosphatases |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101233228A (en) * | 2005-06-10 | 2008-07-30 | 马德里自治大学 | Novel phosphorylation sites, modified proteins and uses of mitogen-activated protein kinases |
| CN101506226A (en) * | 2005-11-14 | 2009-08-12 | 维兹曼科学研究所耶达研究与发展有限公司 | Improved variants of pigment epithelium derived factor and uses thereof |
| WO2011053565A2 (en) * | 2009-10-29 | 2011-05-05 | Biomedical Sciences Research Centre "Alexander Fleming" | Compositions and methods for detecting a tauopathy |
| CN103163010A (en) * | 2011-12-14 | 2013-06-19 | 天津市国际生物医药联合研究院 | Method for relative quantitative analysis of protein phosphorylation modification level |
| CN109342592A (en) * | 2018-10-31 | 2019-02-15 | 华中科技大学 | A method for identifying specific phosphorylated amino acid sites of kinases in vitro |
Non-Patent Citations (1)
| Title |
|---|
| 大麦条纹花叶病毒γb蛋白的磷酸化及其与寄主激酶STY46互作的功能分析;张璇;《中国优秀博士学位论文全文数据库 农业科技辑》;20190115;第2018年卷(第12期);D046-20 * |
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