CN112630197B - Method for judging whether liquid-liquid phase separation of protein can occur - Google Patents
Method for judging whether liquid-liquid phase separation of protein can occur Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
A method for judging whether protein can generate liquid-liquid phase separation belongs to the technical field of genetic engineering. In order to solve the problems that the steps of the method for researching whether the protein can be subjected to liquid-liquid phase separation are complicated, time and labor are wasted, and the prokaryotic expression is easy to form an inclusion body, the invention provides a method for judging whether the protein can be subjected to liquid-liquid phase separation. The method specifically comprises the following steps: constructing a vector containing an in vitro transcription promoter, a protein to be detected and a marker protein gene, taking the vector as a template, carrying out in vitro transcription after PCR amplification to obtain mRNA of a fusion gene, injecting the mRNA into a fertilized egg, and observing whether liquid-liquid phase separation occurs or not through a fluorescence microscope after culturing for 15-20 hours. The method takes the fertilized eggs as the carrier, and the mRNA transcribed in vitro is expressed in the fertilized eggs in an injection mode, so the method has the advantages of very simple operation, clear result and capability of obtaining the result in a short time.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for judging whether liquid-liquid phase separation of protein can occur.
Background
In recent years, Liquid-Liquid Phase Separation (LLPS) of proteins has become one of the hot spots in biological research. It plays a very important role in both normal metabolism and disease processes of the living body. With the increasing reports of exploring the specific mechanisms of LLPS, it was found that the factors affecting protein LLPS are mainly derived from two aspects: one is the influence of protein sequence properties, such as mutations and post-translational modifications; secondly, the micro-environment of the system, such as temperature, protein concentration, pH value, salt concentration, pressure and the like is regulated. At present, the main research on whether a certain protein can be subjected to liquid-liquid phase separation is to express a purified protein in vitro and then observe whether the protein can be subjected to phase transition in a certain buffer solution. The method has complex steps, wastes time and labor, relates to the links of expression vector design and construction, (eukaryotic or prokaryotic system) induced expression of protein, protein purification, protein dialysis, protein concentration and the like, and if the prokaryotic expression system is adopted, the situation that the expressed protein is folded by mistake to form an inclusion body can occur, so that denaturation and renaturation treatment are needed, and the complexity of the experiment is increased.
Disclosure of Invention
In order to solve the problems that the steps of the method for researching whether the protein can be subjected to liquid-liquid phase separation are complicated, time and labor are wasted, and the prokaryotic expression is easy to form an inclusion body, the invention provides a method for judging whether the protein can be subjected to liquid-liquid phase separation. The method specifically comprises the following steps:
(1) constructing an in vitro transcription element of the protein to be detected, wherein the in vitro transcription element is a fusion gene formed by connecting an in vitro transcription promoter, a gene sequence of the protein to be detected and a gene encoding the fluorescent protein;
(2) in vitro transcription to obtain mRNA of the fusion gene;
(3) injecting the mRNA obtained in the step (2) into a fertilized egg;
(4) culturing the fertilized eggs after injection for 15-20 hours, and judging whether the protein to be determined is subjected to liquid-liquid phase separation or not through a fluorescence signal.
Preferably, the in vitro transcription promoter in the step (1) is T7 promoter, and the sequence is shown as SEQ ID NO. 2.
Preferably, the mRNA of step (3) is injected into the cytoplasm or nucleus of a fertilized egg.
Preferably, the concentration of mRNA injected in step (3) is 50-300 ng/. mu.l.
Preferably, the mRNA injection concentration in the step (3) is 150 ng/. mu.l.
Preferably, the mRNA injection parameters in step (3) are injection pressure of 80-200hpa, injection time of 0.1s, and compensation pressure of 100-300 hpa.
Preferably, the fertilized egg in step (3) is a mouse fertilized egg.
Preferably, the fertilized egg culture conditions in step (1) are 37 ℃ and CO2The concentration of (2) is 5%.
Preferably, the determination of whether the protein to be measured undergoes liquid-liquid phase separation in step (4) is performed by observing the protein through a fluorescence microscope, wherein if the formed fluorescence signal is a circle, liquid-liquid phase separation occurs, and if the protein exhibits a diffuse distribution, liquid-liquid phase separation does not occur.
Advantageous effects
The method takes the fertilized egg as a carrier, and expresses the mRNA transcribed in vitro in the fertilized egg by an injection mode, the operation is very simple, and the protein capable of generating liquid-liquid phase separation is in a liquid drop shape after being expressed in the fertilized egg, so the protein is a very regular circular signal observed under a microscope, the result is clear, and the result can be obtained in a short time.
Drawings
FIG. 1.pcDNA3.1-NONO-mCherry vector map;
FIG. 2 shows the result of phase transition of a fusion protein gene composed of a NONO protein gene and a fluorescent protein gene expressed as a protein in a fertilized egg;
FIG. 3.pcDNA3.1-SRSF1-mCherry vector map;
FIG. 4 shows the result of phase transition of a fusion protein gene composed of SRSF1 protein gene and fluorescent protein gene expressed as protein in fertilized egg;
FIG. 5. pcDNA3.1-mCherry-NLS vector map;
FIG. 6 shows the result that only the fluorescent protein gene is expressed in the fertilized egg and no phase transition occurs.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
The materials, reagents, methods and apparatus used in the following examples, which are not specifically illustrated, are conventional in the art and are commercially available to those skilled in the art.
Example 1. method for determining whether a NONO protein can undergo liquid-liquid phase separation.
The method for judging whether the NONO protein can be subjected to liquid-liquid phase separation is carried out according to the following steps:
(1) constructing an in vitro transcription element of the protein to be detected, wherein the in vitro transcription element is a fusion gene formed by connecting an in vitro transcription promoter, a gene sequence of the protein to be detected and a gene encoding a fluorescent protein, and the specific steps are as follows:
1) constructing a vector containing a gene sequence of NONO protein, wherein the CDS region sequence of the NONO protein is shown as SEQ ID No.1, the gene sequence of the protein to be detected in the vector is connected with a red fluorescent protein gene with a labeling effect to form a fusion gene, the upstream of the fusion gene is connected with an in vitro transcription promoter T7 promoter, the sequence is shown as SEQ ID No.2, the in vitro transcription element of the NONO protein gene is constructed into the vector and is synthesized by Youbao biology in a commercial mode, the vector name is pcDNA3.1-NONO-mCherry, the product number is L2772, and the vector map is shown as FIG. 1;
2) taking the vector in the step (1) as a template, respectively designing and synthesizing PCR primers aiming at a T7 promoter and a bGH poly (A) sequence, wherein the names of the primers are T7-F and bGH poly (A) -R, and the sequences are shown as SEQ ID NO.3 and SEQ ID NO.4, and carrying out PCR amplification to obtain an in vitro transcription element PCR kit 2 x Vazyme LAmp Master Mix (Dye Plus) of the needed protein to be detected; the manufacturer Vazyme; item number P312.
The PCR system was as follows: 10ng of plasmid template; upstream and downstream primers were 0.5. mu.L each; 2 × 10 μ L Vazyme LAmp Master Mix (Dye Plus); water was added to make up to 20 μ L.
PCR conditions were as follows: 2min at 95 ℃; 30 cycles of 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 3 min; 10min at 72 ℃.
(2) In vitro transcription to obtain mRNA of the fusion gene;
using HiScribeTMT7 ARCA mRNA Kit (with labeling) (NEB, E2060) was transcribed in vitro, according to the instructions.
1) The in vitro transcription reaction solution was mixed. Melt 10 × reaction Buffer at room temperature, and the rest must be iced.
TABLE 1 in vitro transcription System
| Measurement of | Composition (I) |
| 10μL | 2×ARCA/NTP Mix |
| 1μg | In vitro transcription element for test protein |
| 2μL | T7 Enzyme Mix |
| to 20μL | Nuclease-free Water |
2) Mix gently and centrifuge briefly. Incubate at 37 ℃ for 30 min.
3) Add 2. mu.L of DNase I and incubate at 37 ℃ for 15 min.
4) The system A was added directly to the above reaction system. (Do not centrifuge mixing)
TABLE 2 in vitro transcription System
| Measurement of | Composition (I) |
| 20μL | In vitro transcription products |
| 5μL | Poly(A)Polymerase |
| 10μL | 10×Poly(A)Polymerase Reaction Buffer |
| 65μL | Nuclease-free Water |
5) And (5) mixing the mixture gently. Incubate at 37 ℃ for 30 min.
6) 50 μ L of LiCl Solution was added.
7) Mix well and incubate at-20 ℃ for 30 min.
8)12000g, 15min at 4 ℃, then washing with 1ml of 70% -80% ethanol.
9) Air-drying the pellet for 15min, and then suspending the RNA pellet with 30. mu.L nuclease-free water
10) Detecting RNA concentration, diluting to 150 ng/. mu.L
11) Subpackaging and storing at-80 deg.C.
(3) Injecting the mRNA obtained in the step (2) into a fertilized egg;
the ICR mice of 6-8 weeks were used for superovulation treatment, and the mice were sacrificed at the human site 25 hours after the injection of human chorionic gonadotropin (hCG) to obtain fertilized eggs in vivo. Injecting by using an eppendorf FemtoJet 4i injection instrument, wherein the concentration of injected mRNA is 150 ng/mu l, adjusting injection parameters to injection pressure (pi), 80hpa, the injection time (ti) is 0.1s, and the compensation pressure (pc) is 300hpa, then inserting an injection needle into a needle holder, erecting the injection needle in an operating dish, clicking a clean key of the injection instrument, observing under an inverted microscope, performing fracture of a needle opening by using a physical needle-knocking mode (namely the injection needle touches a fixed needle), judging the injection strength by judging the flow rate of the liquid flow, then adjusting the injection pressure in an injection pressure interval to a reasonable index according to visual field judgment, and then performing prokaryotic injection of fertilized eggs.
(4) Culturing the fertilized eggs after injection for 15 hours, and judging whether the protein to be measured has liquid-liquid phase separation.
When observed by a fluorescence microscope, since liquid-liquid phase separation is droplet formation and spherical, when observed under a confocal laser microscope, if the formed fluorescence signals are gathered into a regular circle, it is proved that liquid-liquid phase separation occurs, otherwise, liquid-liquid phase separation does not occur.
FIG. 2 shows the result of phase transition of fusion protein gene composed of NONO protein gene and fluorescent protein gene expressed as protein in fertilized egg, and the observed red fluorescence shows regular circle, which indicates that the NONO protein can undergo liquid-liquid phase separation.
Example 2. method for determining whether a SRSF1 protein can undergo liquid-liquid phase separation.
(1) Constructing an in vitro transcription element of a protein to be detected, wherein the element is a fusion gene formed by connecting an in vitro transcription promoter, a gene sequence of the protein to be detected and a gene encoding a fluorescent protein, the specific operation is the same as that in example 1, the difference is that the gene to be judged whether liquid-liquid phase separation can occur is SRSF1, the CDS region of SRSF1 is shown as SEQ ID No.5, a vector containing the SRSF1 in vitro transcription element is commercially synthesized by Youbao organisms, the name of the vector is pcDNA3.1-SRSF1-mCherry, and the vector map is shown as figure 3;
(2) in vitro transcription to obtain mRNA of the fusion gene; the specific operation was the same as in example 1.
(3) Injecting the mRNA obtained in the step (2) into a fertilized egg; the procedure was the same as in example 1, except that the concentration of mRNA injected was adjusted to 50 ng/. mu.l, the prokaryotic injection pressure (pi) was 200hpa, the injection time (ti) was 0.1s, and the offset pressure (pc) was 100 hpa.
(4) Culturing the fertilized eggs after injection for 16 hours, and judging whether the protein to be measured has liquid-liquid phase separation. The specific operation was the same as in example 1.
FIG. 4 shows the result of phase transition of fusion protein gene composed of SRSF1 protein gene and fluorescent protein gene expressed as protein in fertilized egg, and the observed red fluorescence shows regular circle, which indicates that the SRSF1 protein can generate liquid-liquid phase separation.
Example 3. method for determining whether a NONO protein can undergo liquid-liquid phase separation.
The procedure of example 1 was repeated, except that the concentration of injected mRNA was adjusted to 300 ng/. mu.l, the injected fertilized egg was injected into the cell, cultured for 20 hours, and then it was judged whether or not the protein to be measured had liquid-liquid phase separation.
Similar results to those in example 1 were obtained by following the procedure of example 3, which indicates that the NONO protein was capable of liquid-liquid phase separation.
Example 4 comparative example to example 1.
The embodiment 4 is the same as the embodiment 1 except that the vector constructed in the step (1) does not contain a gene of a target protein to be detected, is synthesized commercially by a Taobao organism, has the name of pcDNA3.1-mCherry-NLS, has a vector map shown in figure 5, and has a sequence shown in SEQ ID No. 6.
As shown in FIG. 6, the control group injected mRNA expressed only by the fluorescent protein, and the result showed that the fluorescent protein of the control group could not undergo liquid-liquid phase separation, and the red fluorescent signal appeared to be dispersed.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Nucleotide sequence listing
<110> northeast university of agriculture
<120> a method for judging whether a protein can undergo liquid-liquid phase separation
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1422
<212> DNA
<213> CDS region of NONO
<400> 1
atgcagagca ataaagcctt taacttggag aagcagaatc atactccaag gaagcatcat 60
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ccaccaatac ctgcaaatgg ccagcaggcc agcagccaga atgaaggctt gactattgac 180
ctgaagaatt ttaggaaacc aggagagaag acctttacac agcgtagccg tctctttgtg 240
ggcaatcttc cccctgatat cactgaggag gaaatgagga aactatttga gaaatatgga 300
aaagcaggcg aagttttcat tcataaggat aaaggctttg gctttattcg cttggaaaca 360
cgaaccctag cggaaattgc caaagtggag ctggacaaca tgcccctccg tgggaagcag 420
ctgcgagtgc gctttgcctg tcacagtgca tcccttacag tccgcaacct tcctcagtac 480
gtgtcgaacg aactgctgga agaagccttt tctgtgttcg gccaggtgga gagggctgta 540
gtcattgtgg atgaccgagg aaggccctca gggaaaggca ttgttgagtt ctcagggaag 600
ccagctgctc ggaaagctct ggacagatgc agtgaaggct ccttcttgct gactacattt 660
cctcggcctg tgactgtgga gcctatggac cagttagatg atgaagaggg acttccagag 720
aaactggtta taaaaaacca gcaattccac aaggagagag aacagccacc cagatttgca 780
caacctggct cctttgagta tgagtatgcc atgcgctgga aggcactcat tgagatggag 840
aagcaacagc aggatcaagt ggatcggaac atcaaggagg ctcgtgagaa gctggagatg 900
gagatggagg ctgcacgtca tgagcaccag gttatgctaa tgaggcagga tttgatgaga 960
cgtcaagaag agcttcggag aatggaggag ctgcataacc aagaggttca gaagcgaaag 1020
cagttagaac tcaggcagga agaggaacgc aggcgccgtg aggaagagat gcggcgacag 1080
caagaggaaa tgatgcgccg acagcaggaa ggattcaagg gaaccttccc tgatgcgaga 1140
gaacaagaga tacggatggg ccaaatggct atgggaggtg ctatgggcat aaacaataga 1200
ggcgcgatgc cccctgctcc tgtgccacct ggtactccag ctcctccagg acctgccact 1260
atgatgccag atggaaccct tggattgacc ccaccaacaa ctgaacgttt tggccaagct 1320
gcaacaatgg aaggaattgg agcaattggt ggaactcctc ctgcattcaa ccgtccagct 1380
ccgggagctg aatttgctcc aaataaacgc cgccgatatt ag 1422
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<213> T7 promoter
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<212> DNA
<213> bGH poly(A)-R
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acaacagatg gctggcaact agaa 24
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<213> CDS region of SRSF1
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ccaccaatac ctgcaaatgg ccagcaggcc agcagccaga atgaaggctt gactattgac 180
ctgaagaatt ttaggaaacc aggagagaag acctttacac agcgtagccg tctctttgtg 240
ggcaatcttc cccctgatat cactgaggag gaaatgagga aactatttga gaaatatgga 300
aaagcaggcg aagttttcat tcataaggat aaaggctttg gctttattcg cttggaaaca 360
cgaaccctag cggaaattgc caaagtggag ctggacaaca tgcccctccg tgggaagcag 420
ctgcgagtgc gctttgcctg tcacagtgca tcccttacag tccgcaacct tcctcagtac 480
gtgtcgaacg aactgctgga agaagccttt tctgtgttcg gccaggtgga gagggctgta 540
gtcattgtgg atgaccgagg aaggccctca gggaaaggca ttgttgagtt ctcagggaag 600
ccagctgctc ggaaagctct ggacagatgc agtgaaggct ccttcttgct gactacattt 660
cctcggcctg tgactgtgga gcctatggac cagttagatg atgaagaggg acttccagag 720
aaactggtta taaaaaacca gcaattccac aaggagagag aacagccacc cagatttgca 780
caacctggct cctttgagta tgagtatgcc atgcgctgga aggcactcat tgagatggag 840
aagcaacagc aggatcaagt ggatcggaac atcaaggagg ctcgtgagaa gctggagatg 900
gagatggagg ctgcacgtca tgagcaccag gttatgctaa tgaggcagga tttgatgaga 960
cgtcaagaag agcttcggag aatggaggag ctgcataacc aagaggttca gaagcgaaag 1020
cagttagaac tcaggcagga agaggaacgc aggcgccgtg aggaagagat gcggcgacag 1080
caagaggaaa tgatgcgccg acagcaggaa ggattcaagg gaaccttccc tgatgcgaga 1140
gaacaagaga tacggatggg ccaaatggct atgggaggtg ctatgggcat aaacaataga 1200
ggcgcgatgc cccctgctcc tgtgccacct ggtactccag ctcctccagg acctgccact 1260
atgatgccag atggaaccct tggattgacc ccaccaacaa ctgaacgttt tggccaagct 1320
gcaacaatgg aaggaattgg agcaattggt ggaactcctc ctgcattcaa ccgtccagct 1380
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<213> pcDNA3.1-mCherry-NLS sequence
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gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
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cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 900
taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca 960
gcacagtggc ggccgctcga gtctagaggg cccttcgggg gtggaggctc tatggtgagc 1020
aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg 1080
gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac 1140
gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg 1200
gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac 1260
atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac 1320
ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc 1380
atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag 1440
aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag 1500
ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag 1560
accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag 1620
ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag 1680
ggccgccact ccaccggcgg catggacgag ctgtacaagc caaaaaagaa gagaaaggta 1740
ccaaaaaaga agagaaaggt accaaaaaag aagagaaagg tatagtttaa acccgctgat 1800
cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 1860
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 1920
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 1980
gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 2040
aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 2100
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 2160
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 2220
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 2280
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 2340
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 2400
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 2460
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 2520
tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 2580
catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 2640
aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 2700
catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 2760
ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 2820
aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 2880
cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 2940
cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 3000
aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 3060
tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 3120
gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 3180
aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 3240
tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 3300
ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 3360
ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 3420
cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 3480
tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 3540
ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 3600
tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 3660
tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 3720
ctggggttcg cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 3780
ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 3840
tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 3900
cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 3960
tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 4020
taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 4080
attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 4140
ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 4200
agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 4260
gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 4320
ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 4380
gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 4440
aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 4500
gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 4560
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 4620
cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 4680
cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 4740
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 4800
cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 4860
agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 4920
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 4980
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 5040
gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 5100
cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 5160
attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 5220
accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 5280
ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 5340
gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 5400
agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 5460
ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 5520
ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 5580
gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 5640
ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 5700
tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 5760
tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 5820
cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 5880
tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 5940
gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 6000
tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 6060
ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 6120
attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 6180
cgcgcacatt tccccgaaaa gtgccacctg acgtc 6215
Claims (8)
1. A method for determining whether a protein is capable of liquid-liquid phase separation, comprising the steps of:
(1) constructing an in vitro transcription element of the protein to be detected, wherein the in vitro transcription element is a fusion gene formed by connecting an in vitro transcription promoter, a gene sequence of the protein to be detected and a gene encoding the fluorescent protein;
(2) in vitro transcription to obtain mRNA of the fusion gene;
(3) injecting the mRNA obtained in the step (2) into a fertilized egg;
(4) culturing the injected fertilized eggs for 15-20 hours, and judging whether the protein to be determined is subjected to liquid-liquid phase separation or not through a fluorescence signal;
and (4) judging whether the liquid-liquid phase separation of the protein to be determined occurs or not is observed through a fluorescence microscope, if the formed fluorescence signal is circular, the liquid-liquid phase separation occurs, and if the formed fluorescence signal is in dispersive distribution, the liquid-liquid phase separation does not occur.
2. The method for determining whether a protein can undergo liquid-liquid phase separation according to claim 1, wherein the in vitro transcription promoter in step (1) is a T7 promoter, and the sequence is shown in SEQ ID No. 2.
3. The method of claim 1, wherein the step of injecting the mRNA into the fertilized egg comprises injecting the mRNA into cytoplasm or nucleus of the fertilized egg.
4. The method for determining whether a protein can undergo liquid-liquid phase separation according to claim 1, wherein the injected concentration of mRNA in the step (3) is 50 to 300ng/μ l.
5. The method of claim 1, wherein the injected concentration of mRNA in step (3) is 150 ng/. mu.l.
6. The method as claimed in claim 1, wherein the mRNA injection parameters in step (3) are injection pressure of 80-200hpa, injection time of 0.1s, and compensation pressure of 100 hpa.
7. The method for determining whether a protein can undergo liquid-liquid phase separation according to claim 1, wherein the fertilized egg of step (3) is a mouse fertilized egg.
8. The method for determining whether a protein can undergo liquid-liquid phase separation according to claim 7, wherein the fertilized egg culture condition in the step (1) is 37 ℃ and CO2The concentration of (2) is 5%.
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