CN117327158A - Photo-induced phase change protein element and application thereof - Google Patents

Photo-induced phase change protein element and application thereof Download PDF

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CN117327158A
CN117327158A CN202311158409.1A CN202311158409A CN117327158A CN 117327158 A CN117327158 A CN 117327158A CN 202311158409 A CN202311158409 A CN 202311158409A CN 117327158 A CN117327158 A CN 117327158A
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kctd17
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idr
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郝峻巍
刘亮
文欣玫
徐芳
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Xuanwu Hospital
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Abstract

The invention provides a photoinduction phase change protein element and application thereof. The photoinduced phase change protein element is a polypeptide which comprises KCTD17IDR with an amino acid sequence shown as SEQ ID NO. 3. The polypeptides of the invention can be used as phase change protein elements to form fusion proteins with target proteins (e.g., antigenic proteins, in particular, autoimmune antigenic proteins).

Description

Photo-induced phase change protein element and application thereof
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a photoinduction phase change protein element and application thereof.
Background
Specific antibody detection is one of the important clinical diagnostic criteria in the immune disease laboratory test program. Cell-Based Assays (CBA) are the method of choice for the detection of Cell surface antigens and certain synaptoprotein-related autoantibodies. The method is based on antigen-antibody reaction, known antigen over-expression plasmids are transfected in mammalian cells (HEK 293, hela or Hep2 cell lines) so that target antigens are expressed in cell membranes or cytoplasm, cell matrixes of the over-expression antigens are incubated with body fluid samples of patients, fluorescent coupling antibodies aiming at anti-human immune globulin (IgG 1/IgG2/IgG3/IgG 4/IgM) are used as secondary antibodies for signal amplification and labeling, and finally the results are read through a fluorescent microscope or a flow cytometer.
The cell immunofluorescence method detection of the existing neuroautoimmune antibody mainly utilizes the expression plasmid constructed by the natural full-length gene to transfect mammalian cells such as HEK293, hep2 and the like to prepare the detected material. For example: titin antigen (MGT 30) or GAD65 antigen was constructed on eukaryotic expression vectors for detection of autoantibodies (Anti-Titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma parts. Arch neuron. 2001Jun;58 (6): 885-90; GAD65 biological autoimmune. Muscle and Nerve,2017 56 (1), 15-27.). The intracellular expression mode of the antigen proteins is cytoplasmic diffuse distribution, no specific form exists, and when the autoimmune antibody concentration in a sample is low, a positive signal is weak, so that the positive or negative of the antibody cannot be accurately judged.
In 2017, researchers from the university of Prlington, USA developed a tool called optoDroplets that could modulate multivalent states using blue light to promote or reverse formation of biomolecular aggregates in vivo, they fused light-sensitive protein markers Cry2 with internal disordered regions (intrinsically disordered regions, IDR) that promote protein phase changes, and with blue light, researchers could induce these proteins to shrink into clusters, thereby mimicking the phase change, concentration process that occurs in cells (Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets. Cell.2017Jan 12;168 (1-2): 159-171. E14.). Cry2 has the main function of being capable of being induced to oligomerize by blue light (Structural insights into the photoactivation of Arabidopsis cryptochrome 2.Nat Plants.2020Dec;6 (12): 1432-1438.), cry2 alone cannot aggregate and phase change under the condition of blue light, a key factor is IDR of phase change protein, and Cry2 can only play a synergistic effect under the condition that the IDR exists to promote the occurrence of protein phase change.
The smaller the IDR of the phase change protein is, the easier the modularization is, and the gene operation is convenient. As the IDR of the phase-change protein, there have been reported that the Internal Disorder Region (IDR) of DDX4 (1-236 aa) and HNRNPA1 (186-320 aa) is relatively large, regardless of Fus (1-214 aa).
Thus, there is a need to find a smaller, more efficient IDR phase change protein element.
Disclosure of Invention
The inventors have found a new phase change protein KCTD17.KCTD17 has strong phase transformation capability, can independently generate phase transformation to form a droplet-shaped structure, and can effectively generate protein phase transformation of fusion protein after being coupled with target protein (such as antigen protein, in particular autoimmune antigen protein) to form a solid phase transformation protein structure. To make the phase change more dynamic and steerable, the inventors further screened the Internal Disorder Region (IDR) -KCTD17 IDR of the phase change protein, which has the following advantages: only 54 amino acids, smaller and more advantageous than the various known IDRs (Fus: 214aa; DDX4:236aa; HNRNPA1:135 aa). Further, the present inventors introduced two phase change essential elements on the basis of pEGFP-N1 vector: KCTD17 IDR and Cry2 PHR, finally a simple eukaryotic expression plasmid system-optoDroplet-KCTD 17 IDR with the phase change of the space-time regulatory protein is obtained.
The present inventors have combined the expression of the phase-changing eukaryotic expression plasmid system optoDroplet-KCTD17 IDR with the expression of a target protein (e.g., an antigen protein, in particular, an autoimmune antigen protein), invented a novel protein expression system, inserted the target protein (e.g., an antigen protein, in particular, an autoimmune antigen protein) into the optoDroplet-KCTD17 IDR plasmid, expressed in a coupled manner with KCTD17 IDR, cry2 elements, and induced by blue light to induce the fusion protein expressed by the optoDroplet plasmid to undergo protein phase change inside the cell to form a droplet-like structure, thereby increasing the local concentration of the target protein. After the transfected cells are subjected to PFA/methanol immobilization, the phase change protein aggregates with the droplet-shaped structures can still exist stably. Thus, the present invention combines the newly engineered optoDroplet-KCTD17 IDR with expression plasmids for clinical test proteins (e.g., antigenic proteins, particularly autoimmune antigenic proteins) to alter the expression pattern of a variety of intracellular proteins to form a droplet-like structure. Thus, the invention combines KCTD17 IDR, cry2 element and target protein (such as antigen protein, in particular autoimmune antigen protein) into a new eukaryotic expression plasmid system optoDroplet-KCTD17 IDR, which can aggregate protein (such as antigen protein, in particular autoimmune antigen protein) and enhance the signal. In some embodiments, measuring the signal intensity of an antigen protein on a confocal fluorescence microscope can increase by a factor of ten or more.
Accordingly, in one aspect, the present invention provides a polypeptide comprising KCTD17 IDR having an amino acid sequence as shown in SEQ ID NO. 3. The polypeptides of the invention can be used as phase change protein elements to form fusion proteins with target proteins (e.g., antigenic proteins, in particular, autoimmune antigenic proteins).
In another aspect, the invention provides a nucleic acid molecule comprising a polynucleotide encoding the polypeptide described above.
In some embodiments, the nucleic acid molecule comprises a polynucleotide as set forth in SEQ ID NO. 4.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In some embodiments, the vector may be an expression vector. Further, in some embodiments, the vector may be a prokaryotic expression vector or a eukaryotic expression vector.
In some embodiments, the vector may be a plasmid.
In some embodiments, the expression vector may further comprise a polynucleotide encoding a light-induced multimerization protein domain. The light-induced multimerization protein domain may refer to a domain of a protein capable of dimerizing, or even multimerizing, in response to a light stimulus, examples of which include, but are not limited to, cry2 PHR, the NTE domain of photopigment B (N-terminal extension (NTE)), and the LOV2 domain of photopigment 1 (LOV 2 domain of phototropin 1).
In some embodiments, the expression vector may further comprise a polynucleotide encoding a fluorescent protein tag. The fluorescent protein label can be protein capable of automatically generating fluorescence, and has the functions of fluorescent labeling and tracing. Examples include, but are not limited to, mCherry, GFP, RFP, YFP, dsRed, and the like.
In some embodiments, the expression vector may further comprise a polyclonal restriction site (MCS) to facilitate insertion of various target protein genes into the expression vector.
In some embodiments, the expression vector may comprise, in order from upstream to downstream, a polyclonal restriction site (MCS), a polynucleotide encoding a KCTD17 IDR, a polynucleotide encoding a fluorescent protein tag, a polynucleotide encoding a light-induced multimerization protein domain (particularly a Cry2 PHR), wherein the elements may optionally be linked by a linker.
In some embodiments, the nucleotide sequence of the expression vector optoDroplet-KCTD17 IDR is shown in SEQ ID NO. 26.
In some embodiments, the expression vector may further comprise a target protein gene. The target protein gene may be a gene expressing any protein, including but not limited to, an antigen protein gene, particularly an autoimmune antigen protein gene, examples of which include, but are not limited to, a tin gene, a GAD65 gene, a GFAP gene, an MBP gene, a Hu gene, a Yo gene, a Ri gene, a CV2 gene, an amp phiysin gene, a Ma1 gene, a Ma2 gene, a SOX1 gene, a Zic4 gene, a Recoverin gene, a pkcγ gene.
In the present invention, the positions of the elements such as KCTD17IDR, photoinduced multimerization protein domain, fluorescent protein tag, etc. are not particularly limited as long as they are capable of forming a fusion protein with the target protein element. In some embodiments, in the fusion protein, the target protein is located at the N-terminus of KCTD17 IDR.
In some embodiments, the expression vector may be obtained by a method comprising the steps of: KCTD17IDR was coupled to a fluorescent protein tag (e.g., mCherry) and a photoinduced multimerization protein domain (e.g., cry2 PHR), constructed into pEGFP-N1 plasmid, and a polyclonal cleavage site was maintained prior to KCTD17IDR and fluorescent tag mCherry for insertion of the target protein gene.
In addition, in the present invention, for other elements which are necessarily present in the vector, particularly the expression vector, elements well known in the art may be used, and a detailed description thereof will be omitted.
In another aspect, the invention provides a cell comprising the vector described above. In some embodiments, the cell may be a HEK293T cell, a Hela cell, a Hep2 cell, examples of which include, but are not limited to, a HEK293T cell, a Hela cell, a Hep2 cell, and the like.
The present invention provides a new phase change IDR protein and develops a smaller optoDroplet tool: optoDroplet-KCTD17 IDR. The invention combines the expression of the tool and the target protein (such as antigen protein, especially neuroautoimmune antigen protein) for the first time, and puts the target protein gene into the tool to prepare the expression plasmid, which can significantly promote the aggregation of the target protein and form a droplet-shaped structure, thus being beneficial to amplifying the target protein signal, for example, in some embodiments, the signal intensity of the target protein can be improved by tens of times when measured on a confocal fluorescence microscope.
The inventive optoDroplet-KCTD17 IDR has its own advantages over previous tools of optodroplets: firstly, the KCTD17 IDR protein becomes smaller, only 54 amino acids; in addition, the phase change capability is strong, and even if Cry2 is not used, the phase change capability can also enable small GFP protein to generate protein phase change; secondly, the original optoDroplet tool adopts pHR-FusN-mCherry-Cry2 plasmid, the plasmid size is 11954bp, and the plasmid contains sequence characteristics of some virus plasmids, such as: LTR, RRE, cPPT and other unnecessary elements, and the invention introduces target protein genes and two other phase change necessary elements based on pEGFP-N1 vector: KCTD17 IDR and Cry2 PHR finally obtain a simple expression plasmid optoDroplet-KCTD17 IDR, and the plasmid size is about 6500bp.
Drawings
FIG. 1 is a diagram showing that fusion proteins of KCTD17 and GFP (GFP-KCTD 17, upper panel) and KCTD17 IDR (209-262 aa) and GFP (GFP-KCTD 17 IDR, lower panel) undergo a phase change in cytoplasm to form a droplet-like structure.
Fig. 2 is a graph showing protein phase transition of KCTD17 protein in vitro. Wherein: A. prokaryotic protein expression plasmid map of KCTD 17: MBP label promotes protein expression and helps protein folding, MBP is followed by an HRV3C protease cleavage site, MBP label can be removed in later stage, then GFP label is removed, whether KCTD17 protein is aggregated in a test tube and under a microscope or not can be observed conveniently, and a droplet-shaped structure is formed; B. prokaryotic expression protein of KCTD17 and protein electrophoretogram after cleavage with HRV3C (coomassie brilliant blue staining); C. after removing the MBP label, KCTD17 undergoes protein phase change under different protein concentrations under a fluorescent microscope.
FIG. 3 is a graph showing that full length KCTD17 promotes phase transition of Titin/GAD65 fusion protein.
FIG. 4 is a schematic representation (B) of the optoDroplet-KCTD17 IDR expression plasmid of the invention and a schematic representation (A) of the original optoDroplets tool using pHR-FusN-mCherry-Cry2 plasmid.
FIG. 5 is a diagram showing the in vitro protein phase transition of the optoDroplet-KCTD17 IDR-Titin.
FIG. 6 is a diagram showing the in vitro protein phase change of the optoDroplet-KCTD17 IDR-GAD 65.
FIG. 7 is a graph showing that the optoDroplet-KCTD17 IDR system can withstand the effects of cell fixation.
Detailed Description
Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 discovery of phase change protein KCTD17
The forces between the protein and the protein or RNA molecules can cause them to separate or aggregate, and when the molecules reach a certain concentration, "phase separation" occurs, similar components aggregate together, and extraneous molecules are isolated. Phase change proteins generally have an important feature, namely having an Internal Disordered Region (IDR). IDR is a common domain in phase separated proteins and is characterized by a low complexity sequence region, such as a single amino acid repeat sequence, e.g., arginine-rich, etc.
The invention discovers a protein KCTD17 with the IDR at the C terminal through screening, the IDR sequence is 209-262aa, and the amino acid sequence is shown in SEQ ID NO. 3. In order to verify that the KCTD17 protein can generate protein phase transition, the invention makes an intracellular verification experiment and an in vitro verification experiment.
Intracellular experiments:
the GFP fusion expression plasmid of KCTD17 was constructed, and the expression of GFP fusion protein KCTD17 was observed in HEK293T cells, and a droplet-like structure was formed.
1. Main reagents, instruments and sources:
PCR enzyme:max DNA Polymerase (Takara, cat# R045A);
NEB restriction enzyme: ecoRI-HF and BamHI-HF;
cell line: HEK293T cells;
glue recovery kit: omega Gel Extraction Kit;
plasmid: pEGFP-C1 was purchased from Clontech; plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Shandong Vietnam Europe Biotechnology (CH 873972);
PEI linear transfection reagent PEI MAX (24765-1) was purchased from Polysciences;
plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；
Pipetting: eppendorf;
PCR instrument: bio-Rad T100.
The KCTD17 gene can be subcloned into pEGFP-C1 plasmid by the following steps with the above reagents and consumables. In this example, KCTD17 gene was amplified by PCR and inserted into pEGFP-C1 to obtain eukaryotic expression plasmid with correct expression.
2. Experimental method
1. Primer design:
the KCTD17 gene was constructed on plasmid pEGFP-C1 with GFP tag. PCR amplification primer: the sequence of the upstream primer KCTD17-F (SEQ ID NO: 5) and the sequence of the downstream primer KCTD17-R (SEQ ID NO: 6) of the KCTD17 gene are as follows, wherein the upstream primer comprises a homologous arm sequence before the insertion position on the target vector pEGFP-C1 and a KCTD17 gene 5 '-end specific primer, and the downstream primer comprises a homologous arm reverse complementary sequence after the insertion position on the pEGFP-C1 and a KCTD17 gene 3' -end specific primer:
primer(s) Sequence(s)
KCTD17-F 5’-CTCGAGCTCAAGCTTCGAATTCTATGCAGACGCCGCGGCCG-3’
KCTD17-R 5’-CAGTTATCTAGATCCGGTGGATCCGATGGGAACCCCAAGTCCCTGGAGGTG-3’
Primers were synthesized by Jin Wei Intelligence company.
2. And (3) PCR reaction:
the PCR system is as follows:
uniformly mixing the PCR reaction system, and starting PCR amplification after transient instantaneous centrifugation;
the PCR reaction procedure is as follows:
pre-denaturation: 94℃,2min.
denaturation: 98℃,10sec.
annealing: 63℃,10sec.
extension: 72℃,20s(1Kb/10s)
cycle number: 35 cycles
Extension: 72℃,2min
preservation conditions: 10℃
3. after the PCR is completed, electrophoresis runs and cuts gel to recover the gene product.
4. Linearization of pEGFP-C1 vector: pEGFP-C1 was digested with EcoRI and BamHI to obtain linearized vector fragments, the reaction system was as follows:
and incubating at 37 ℃ for enzyme digestion for 2 hours, carrying out Agarose gel electrophoresis, and cutting gel to recover large carrier fragments.
5. Seamless connection
The reaction system is as follows:
reaction components Volume of
Large fragment of pEGFP-C1 plasmid 3μl
KCTD17 gene fragment 2μl
NEBuilder HiFi DNA Assembly Master Mix 10μl
ddH 2 O 5μl
Total volume of 20μl
The reaction system is evenly mixed and then placed at 50 ℃ for reaction for 5-15 minutes.
6. Conversion: mu.l of the homologous recombination product was used to transform E.coli competent cells, which were recovered and plated on LB solid culture plates resistant to kanamycin (Kan) for cultivation.
7. Single colonies are picked, added into LB culture medium containing Kan antibiotics, and cultured at 37 ℃ by shaking overnight.
8. And (3) extracting recombinant plasmid pEGFP-C1-KCTD17, and screening recombinants with correct sequencing results after electrophoresis is correctly screened (the migration speed of the recombinant plasmid is slower compared with that of a template empty plasmid due to the fact that the molecular weight is increased).
9. And (3) taking the correct pEGFP-C1-KCTD17 plasmid, and transfecting the cultured HEK293 cells according to a PEI transfection reagent transfection method to obtain HEK293 cells expressing the pEGFP-C1-KCTD17 gene.
10. And (3) fixing and tabletting the transfected cells for 24 hours by using a fixing solution to prepare the cell climbing tablet. The fixing liquid is fixing agents such as acetone, formaldehyde, paraformaldehyde, methanol, ethanol and the like. The immobilized cell slide was directly observed under a microscope and photographed.
As shown in the upper graph of fig. 1, the fusion of KCTD17 gene and GFP resulted in a significant change in the morphology of GFP protein, and the green fluorescent protein aggregated in almost all cells and exhibited a droplet-like structure.
In vitro experiments:
the invention constructs a procaryotic expression plasmid of KCTD17, obtains KCTD17 protein through escherichia coli expression, and observes that fusion protein KCTD17 can form a liquid drop-shaped structure in a test tube.
1. Construction of KCTD17 prokaryotic expression plasmid
1. Main reagents, instruments and sources:
PCR enzyme:max DNA Polymerase (Takara, cat# R045A);
NEB restriction enzyme: ecoRI-HF and BamHI-HF;
cell line: HEK293T cells;
glue recovery kit: omega Gel Extraction Kit;
plasmid: pET-22b was purchased from sigma,69744; pMal-c2x is purchased from Addgene,75286; pEGFP-C1 was purchased from Clontech, plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Shandong Vietnam Europe Co (CH 873972);
plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；
Pipetting: eppendorf;
Ni-NTA agarose：QIAGEN 30210
PCR instrument: bio-Rad T100.
The KCTD17 gene can be subcloned into pET22b plasmid by the following steps with the reagent and consumable materials.
2. Experimental method
In this example, the KCTD17 gene was PCR amplified and other elements were added such as: MBP and GFP tags are inserted into pET22b to obtain a procaryotic expression plasmid of KCTD17, and BL21 (DE 3) competent cells are transformed to obtain a procaryotic expression strain with high KCTD17 expression as shown in figure 2A.
1) Primer design:
in order to construct KCTD17 gene, MBP gene (from pMal-C2 x) and GFP tag (from pEGFP-C1 plasmid) onto prokaryotic expression plasmid pET22b, a set of primers for gene assembly was designed, which contained MBP gene upstream primer MBP-Nde1-F (SEQ ID NO: 7), MBP gene downstream primer MBP-R (SEQ ID NO: 8), EGFP gene upstream primer EGFP-F (SEQ ID NO: 9), EGFP gene downstream primer EGFP-R (SEQ ID NO: 10), KCTD17 gene upstream primer pET22b-KCTD17-F (SEQ ID NO: 11), KCTD17 gene downstream primer pET22b-KCTD17-R (SEQ ID NO: 12). The upstream primer comprises a fragment sequence of the upstream vector or the gene overlap, a gene and a self-specific primer sequence at the 5 'end of the element, and the downstream primer comprises a reverse complementary sequence of the fragment sequence of the downstream vector or the gene overlap, and a self-specific primer sequence at the 3' end of the gene and the element. Primers were synthesized by Jin Wei Intelligence company.
2) And (3) PCR amplification and recovery:
the complete set of primers and corresponding DNA templates were selected and amplified using PrimeSTAR (Takara, R045A) system to obtain MBP, EGFP and KCTD17 genes, and after PCR was completed, the gene products were recovered by running and cutting gel.
3) Linearization of pET22b vector:
pET22b was double digested with NdeI and XhoI to obtain a linearized vector fragment, the reaction system was as follows:
reaction components Volume of
pET22b plasmid 1μg
rCutSmart buffer 5μl
NEB restriction endonuclease NdeI 1μl
NEB restriction enzyme XhoI 1μl
ddH 2 O Make up to 50. Mu.l
After the reaction system is evenly mixed, the mixture is placed in a 37 ℃ incubator for incubation for 2 hours, electrophoresis is carried out, and the carrier large fragment is recovered after gel cutting.
4) Seamless connection:
the recovered MBP, EGFP tag, KCTD17 antigen gene and linearized pET22b were subjected to a multi-fragment seamless ligation using NEBuilder HiFi DNA Assembly Master Mix, the reaction system was as follows:
reaction components Volume of
pET22b plasmid large fragment 3μl
MBP fragment 1μl
KCTD17 gene fragment 1μl
EGFP fragment 1μl
NEBuilder HiFi DNA Assembly Master Mix 10μl
ddH 2 O 4μl
Total volume of 20μl
The reaction system is evenly mixed and then placed at 50 ℃ for reaction for 5-15 minutes.
5) Conversion:
mu.l of the homologous recombination product was used to transform E.coli competent cells, which were resuscitated and plated onto ampicillin (Amp) -resistant LB solid culture plates.
6) Single colonies are picked, added into LB culture medium containing Amp antibiotics, and cultured at 37 ℃ by shaking overnight.
7) And (3) extracting recombinant plasmid pET22b-MBP-EGFP-KCTD17, sequencing, and screening recombinants with correct sequencing results after electrophoresis primary screening is correct (compared with a template empty plasmid, the plasmid with successful recombination is slower in migration speed due to the fact that the molecular weight is increased).
2. KCTD17 prokaryotic expression and purification
After preliminary trial expression, KCTD17 protein was abundantly expressed, 0.5mM IPTG,19℃and 220rpm, induced overnight, resuspended in buffer (25mM Tris,0.5M NaCl,5% glycerol, 0.05%DDM,20mM Imidazole,pH 8.0), crushed for 5min at high pressure and centrifuged at high speed (15000 rpm,40min,4 ℃). The following steps were employed for the respective purifications.
(1) His column affinity purification
Washing Buffer:25mM Tris,150mM NaCl,5% glycerol, 0.05% DDM, pH 8.0;
imidazole gradient eluent: 25mM Tris,150mM NaCl,5% glycerol, 0.05% DDM, pH 8.0 containing imidazole at different concentrations of 20mM,50mM,100mM,250mM,500mM (5-10 column volumes).
The steps are as follows:
1) Balancing 2-5 column bed volumes by using a buffer solution 1, wherein the flow rate is 2mL/min;
2) Filtering the cell disruption solution (50mM PBS,pH7.4,0.5M NaCl) with a 0.45 μm filter membrane, and loading the sample at a flow rate of 1mL/min;
3) Washing 2-5 column bed volumes by using a Washing Buffer, wherein the flow rate is 2mL/min;
4) Performing stage elution with gradient eluents containing 20, 50, 100, 250 and 500mM Imidazole respectively at a flow rate of 2mL/min, collecting elution peaks at each stage, and detecting the molecular weight and purity of the fusion protein by SDS-PAGE;
(2) Molecular sieve purification
Molecular sieve Buffer:25mM Tris,150M NaCl,pH 8.0;
Column：SD200 increase。
the steps are as follows:
1) Filtering the sample with 0.2 μm pore size filter membrane or centrifuging 10000g for 5min before loading, and removing residue;
2) Concentrating the protein sample to 0.5-1.0ml, and injecting the sample into a protein purifier by using a sample injector;
3) Running a molecular sieve program;
4) And collecting different fractions according to the peak diagram of the molecular sieve, and concentrating to obtain the target protein.
Through the above purification steps, a recombinant expressed KCTD17 protein was obtained, as shown in FIG. 2B, using HRV3C
Proteases are able to cleave MBP tags.
3. KCTD17 protein in vitro phase change experiment
Protein phase separation was tested in the presence or absence of a crowding agent (10% dextran) and protein dilutions were 25mM Tris-HCl7.4, 150mM KCl, 2.5% glycerol and 0.5mM DTT in buffer. The proteins were diluted to 1-20. Mu.M each and the total solution volume was 20. Mu.L, and HRV3C protease was added to the diluted proteins in a 1:50 ratio. The sample is added into a 96-well microplate, an image is taken after all liquid drops are settled to the bottom of the dish, as shown in a figure 2B, the KCTD17 fusion protein is digested by HRV3C protease to release an MBP label (inhibit protein from changing phase), as shown in a figure 2C, the KCTD17 protein is easier to form green liquid drop shapes in the solution along with the increase of the protein concentration, and the liquid drop shapes formed by the KCTD17 protein can be fused in vitro, so that the protein is in a flowing state and the protein phase change phenomenon occurs. Thus in vitro experiments indicate that KCTD17 is capable of protein phase change.
The expression of KCTD17 protein in-vivo and in-vitro experiments is synthesized, and the fact that the invention provides a novel phase change protein is confirmed.
Example 2KCTD17 promotes phase transition of autoimmune antigen protein
The existing CBA method detection of the neuroautoimmune antibody mainly utilizes the expression plasmid constructed by the natural full-length gene to transfect 293 and hep2 cells to prepare the detected material. For example: titin antigen (MGT 30) or GAD65 antigen gene is constructed on eukaryotic expression vector for detection of autoantibody. The Titin/GAD65 antigen is expressed in cytoplasm and distributed in disperse mode without specific form. The invention designs a new recombinant expression plasmid which is constructed by adding antigen Titin/GAD65 to the N end of pEGFP-C1-KCTD17 recombinant plasmid KCTD17 gene and expresses fusion protein of Titin/GAD65 and KCTD 17. After cell transfection, it was found that the introduction of the phase change protein KCTD17 was able to change the diffuse distribution of the Titin/GAD65 protein, but rather to form a certain aggregation state. The KCTD17 is shown to promote protein phase change of the fusion protein of Titin/GAD65 to form a denser aggregate.
1. Main reagents, instruments and sources:
PCR enzyme:max DNA Polymerase (Takara, cat# R045A);
NEB restriction enzyme: ecoRI-HF and BamHI-HF
Cell line: HEK293T cells;
glue recovery kit: omega Gel Extraction Kit;
plasmid: pEGFP-C1-KCTD17 (obtained in example 1); the plasmid pEGFP-N1-Titin/GAD65 containing Titin gene and GAD65 gene was synthesized by general biosystems (Titin gene and GAD65 gene were subcloned into pEGFP-N1, respectively, and inserted into Nhe1 and Xho1 sites).
Plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；
Pipetting: eppendorf;
PCR instrument: bio-Rad T100;
the Titin/GAD65 gene can be subcloned into pEGFP-C1-KCTD17 plasmid by the following steps with the reagent and the consumable.
2. Experimental method
1. Construction of pEGFP-C1-tin/GAD 65-KCTD17 plasmid:
the Titin gene, GAD65 gene, was inserted into pEGFP-C1-KCTD17 by a 2-step procedure.
1) Primer design:
Titin/GAD65 gene was constructed into pEGFP-C1-KCTD 17.
The first round PCR amplification primers Titin-F (SEQ ID NO: 13), titin-R (SEQ ID NO: 14), GAD65-F (SEQ ID NO: 15), GAD65-R (SEQ ID NO: 14) were designed.
The primer sequences are shown in the following table, the upstream primer comprises a homologous arm sequence before the insertion position on the target vector pEGFP-C1-KCTD17 and a Titin/GAD65 gene specific upstream primer, and the downstream primer comprises a homologous arm reverse complement sequence after the insertion position on the target vector pEGFP-C1-KCTD17 and a Titin/GAD65 gene vector specific downstream primer.
Primers were synthesized by Jin Wei Intelligence company.
2) First round PCR reaction
First, diluting the first round PCR amplification primer to a final concentration of 10 mu M according to the concentration;
the PCR system is as follows:
the PCR reaction system is uniformly mixed, and PCR amplification is started after transient instantaneous centrifugation;
the PCR reaction procedure is as follows:
after the PCR is completed, electrophoresis runs and cuts gel to recover the gene product.
The PCR products were recovered according to the protocol of the gel recovery kit, with the concentration of the first round of PCR recovery products being approximately 50-100 ng/. Mu.l.
3) Second round PCR reaction
The reaction system is as follows:
reagent(s) Volume (mul)
KOD buffer 5
2mM dNTPs 5
25mM MgSO 4 2
First round PCR recovery of product 16
50 ng/. Mu.l pEGFP-C1-KCTD17 plasmid 1
KOD DNA Polymerase 1
ddH 2 O 20
Total volume of 50
The PCR reaction system is uniformly mixed, and PCR amplification is started after transient instantaneous centrifugation; the PCR reaction procedure is as follows:
pre-denaturation: 94℃,2min.
denaturation: 98℃,10sec.
annealing: 55℃,20sec.
extension: 68℃,7min30s
cycle number 13 cycles
Extension: 68℃,10min
preservation conditions: 10℃
4) Dpn1 digestion:
after the second round of PCR reaction is completed, 1 μl of Dpn1 enzyme is added into the reaction tube to react for 1-2 hours at 37 ℃, and the template plasmid in the PCR reaction system is thoroughly removed.
5) Conversion:
the PCR product after digestion with 10. Mu.l of Dpn1 was used to transform E.coli competent cells, which were resuscitated and plated onto Kan-resistant LB solid culture plates.
6) Single colonies are picked, added into LB culture medium containing Kan antibiotics, and cultured at 37 ℃ by shaking overnight.
7) And (3) extracting recombinant plasmids, sequencing after the electrophoresis primary screening is correct, and screening recombinant plasmids pEGFP-C1-tin/GAD 65-KCTD17 with correct sequencing results.
2. Intracellular observation of protein phase transition experiments
And (3) taking the correct pEGFP-C1-tin/GAD 65-KCTD17 plasmid, and transfecting the cultured HEK293 cells according to a PEI transfection reagent transfection method to obtain HEK293 cells expressing the pEGFP-C1-tin/GAD 65-KCTD17 gene.
And (3) fixing and tabletting the transfected cells for 24 hours by using a fixing solution to prepare the cell climbing tablet. The fixing liquid is fixing agents such as acetone, formaldehyde, paraformaldehyde, methanol, ethanol and the like. The immobilized cell slide was directly observed under a microscope and photographed.
As shown in FIG. 3, the fusion of KCTD17 gene and Titin/GAD65 antigen gene results in obvious change of Titin/GAD65 protein morphology, and aggregation of Titin/GAD65 protein in almost all cells occurs.
Example 3 determination of KCTD17 IDR sequence
Phase change proteins typically have an important structural Internal Disorder Region (IDR), which generally determines the phase change of the protein.
A series of plasmids of KCTD 17C-terminal protein are constructed, and a section of IDR region (209-262 aa) which is the most core in KCTD17 is screened through an intracellular experiment and is named KCTD17 IDR.
As shown in the lower panel of FIG. 1, KCTD17 IDR alone (209-262 aa) was able to cause the coupled GFP protein to exhibit a drip-like structure as determined by the following experiment.
1. Main reagents, instruments and sources:
PCR enzyme:max DNA Polymerase (Takara, cat# R045A);
NEB restriction enzyme: ecoRI-HF and BamHI-HF;
cell line: HEK293T cells;
glue recovery kit: omega Gel Extraction Kit;
plasmid: pEGFP-C1 (available from Clontech); plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Shandong Vietnam Europe Biol Co (CH 873972);
plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；/>
Pipetting: eppendorf;
PCR instrument: bio-Rad T100.
The KCTD17 gene can be subcloned into pEGFP-C1 plasmid by the following steps with the above reagents and consumables. In this example, KCTD17 IDR gene was amplified by PCR and inserted into pEGFP-C1 to obtain eukaryotic expression plasmid with correct expression.
2. Construction of pEGFP-C1-KCTD17 IDR plasmid
1. Primer design:
The KCTD17 IDR gene was constructed on plasmid pEGFP-C1 with GFP tag.
PCR amplification primer: the primer sequences of the upstream primer KCTD17-F1 (SEQ ID NO: 16) and the downstream primer KCTD17-R1 (SEQ ID NO: 17) of the KCTD17 IDR gene are shown in the following table. The upstream primer comprises a homologous arm sequence before the insertion position on the target vector pEGFP-C1 and a KCTD17 IDR gene 5 'end specific primer, and the downstream primer comprises a homologous arm reverse complement sequence after the insertion position on the pEGFP-C1 and a KCTD17 IDR gene 3' end specific primer:
primers were synthesized by Jin Wei Intelligence company.
2. And (3) PCR reaction:
the PCR system is as follows:
the PCR reaction system is uniformly mixed, and PCR amplification is started after transient instantaneous centrifugation;
the PCR reaction procedure is as follows:
pre-denaturation: 94℃,2min.
denaturation: 98℃,10sec.
annealing: 63℃,10sec.
extension: 72℃,20s(1Kb/10s)
cycle number: 35 cycles
Extension: 72℃,2min
preservation conditions: 10℃
3. after the PCR is completed, electrophoresis runs and cuts gel to recover the gene product.
4. Linearization of pEGFP-C1 vector: pEGFP-C1 was digested with EcoRI and BamHI to obtain linearized vector fragments, the reaction system was as follows:
reaction components Volume of
pEGFP-C1 plasmid Total 2. Mu.g
rcutsmart buffer 5μl
NEB restriction endonuclease EcoRI-HF 1μl
NEB restriction enzyme BamHI-HF 1μl
ddH 2 O Up to 50μl
Total volume of 50μl
And incubating at 37 ℃ for enzyme digestion for 2 hours, carrying out Agarose gel electrophoresis, and cutting gel to recover large carrier fragments.
5. Seamless connection
The reaction system is as follows:
reaction components Volume of
Large fragment of pEGFP-C1 plasmid 3μl
KCTD17IDR gene fragment 2μl
NEBuilder HiFi DNA Assembly Master Mix 10μl
ddH2O 5μl
Total volume of 20μl
The reaction system is evenly mixed and then placed at 50 ℃ for reaction for 5-15 minutes.
6. Conversion: mu.l of the homologous recombination product was used to transform E.coli competent cells, which were then plated on Kan-resistant LB solid culture plates for cultivation after resuscitation.
7. Single colonies are picked, added into LB culture medium containing Kan antibiotics, and cultured at 37 ℃ by shaking overnight.
8. Extracting recombinant plasmid pEGFP-C1-KCTD17 IDR, sending to sequencing, screening recombinant with correct sequencing result.
3. Intracellular experiments
And (3) taking the correct pEGFP-C1-KCTD17 IDR plasmid, and transfecting the cultured HEK293 cells according to a PEI transfection reagent transfection method to obtain HEK293 cells expressing the pEGFP-C1-KCTD17 IDR gene.
And (3) fixing and tabletting the transfected cells for 24 hours by using a fixing solution to prepare the cell climbing tablet. The fixing liquid is fixing agents such as acetone, formaldehyde, paraformaldehyde, methanol, ethanol and the like. The immobilized cell slide was directly observed under a microscope and photographed.
The experimental results are shown in the lower graph of fig. 1, and the fusion of the independent KCTD17IDR gene and GFP results in a significant change in the morphology of GFP protein, and almost all green fluorescent proteins in cells aggregate, indicating that the KCTD17IDR sequence does have a certain phase change capability.
Example 4 new optoDroplet tool: design and construction of an expression plasmid of an optoDroplet-KCTD17 IDR
Although KCTD17IDR can promote GFP to generate phase change in vivo, KCTD17IDR can not cause Titin/GAD65-GFP to generate a phase change phenomenon similar to full length KCTD17 to bring about obvious fusion protein, and the phase change capability of KCTD17IDR is weakened compared with that of full length KCTD17, so that KCTD17IDR plus Cry2 elements are designed, the phase change capability of the KCTD17IDR is similar to that of full length KCTD17, the phase change process is controlled by light, and the fusion protein can generate obvious protein phase change to be aggregated only when blue light is irradiated, cry2 plays a role. Thus, the present invention contemplates an optoDroplet-KCTD17 IDR expression system.
Original tools of optodraglets use pHR-FusN-mCherry-Cry2 plasmid, the size of which is 11954bp, and which contains the sequence features of some viral plasmids, such as: LTR, RRE, cPPT, and the like (as shown in FIG. 4A).
To simplify the optoDroplets plasmid, the inventors introduced an antigenic gene and two additional phase change essential elements on the basis of pEGFP-N1 vector: KCTD17IDR and Cry2 PHR, mCherry and Cry2 were obtained from PCR amplification of pHR-FusN-mCherry-Cry 2. The KCTD17IDR and Cry2 PHR phase change elements are connected in series, an antigen gene is introduced into the N end, a linker is added between the antigen gene and the phase change elements, and finally a novel simple optoDroplets expression plasmid is obtained: the plasmid size of the optoDroplet-KCTD17 IDR (shown as B in FIG. 4, SEQ ID NO: 26) was 6504bp. In addition, the invention reserves the polyclonal enzyme cutting site, and is convenient for inserting various antigen genes into expression plasmids.
1. Main reagents, instruments and sources:
PCR enzyme:max DNA Polymerase (Takara, cat# R045A);
NEB restriction enzyme: notI-HF and BamHI-HF;
cell line: HEK293T cells;
glue recovery kit: omega Gel Extraction Kit;
plasmid: pEGFP-N1 was purchased from Clontech; plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Shandong Vietnam Europe Biotechnology (CH 873972); pHR-FusN-mCherry-Cry2 is purchased from Addgene 101221;
plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；
Pipetting: eppendorf;
PCR instrument: bio-Rad T100.
KCTD17 IDR, cry2 and antigen genes can be subcloned into pEGFP-N1 plasmid using the reagents and consumables described above as follows.
Experimental method
1. Primer design:
KCTD17 IDR, mCherry (from pHR-FusN-mCherry-Cry 2) and Cry2 (pHR-FusN-mCherry-Cry 2) were subcloned onto pEGFP-N1 plasmid. PCR amplification primer: the KCTD17 IDR gene upstream primer KCTD17 IDR-F (SEQ ID NO: 18), EGFP gene downstream primer KCTD17 IDR-R (SEQ ID NO: 19), mCherry and Cry2 genes were amplified from pHR-FusN-mCherry-Cry2, upstream primer mCherry-Cry2-F (SEQ ID NO: 20), downstream primer mCherry-Cry2-R (SEQ ID NO: 21). The sequence of the primers is as follows, the upstream primer comprises a homologous arm sequence before the insertion position on the target vector pEGFP-N1 and a KCTD17 gene 5 'end specific primer, and the downstream primer comprises a homologous arm reverse complement sequence after the insertion position on the pEGFP-N1 and a KCTD17 gene 3' end specific primer:
Primers were synthesized by Jin Wei Intelligence company.
2. And (3) PCR amplification and recovery: the set of primers and corresponding DNA templates were selected and amplified using PrimeSTAR (Takara, R045A) system to obtain the MBP, EGFP and KCTD17 genes.
3. After completion of PCR, the gene products KCTD17 IDR and mCherry-Cry2 were recovered by electrophoresis run and cut.
4. Linearizing a carrier: pEGFP-N1 was digested with NheI and NotI to obtain a linearized vector fragment, the reaction system was as follows:
reaction components Volume of
pEGFP-N1 plasmid Total 2ug
rcutsmart buffer 5μl
BamHI-HF 1μl
NotI-HF 1μl
ddH2O Up to 50μl
Total volume of 50μl
And incubating at 37 ℃ for enzyme digestion for 2 hours, carrying out Agarose gel electrophoresis, and cutting gel to recover large carrier fragments.
5. Seamless connection
The reaction system is as follows:
the reaction system was mixed and then allowed to react at 50℃for 30 minutes.
6. Conversion: mu.l of the homologous recombination product was used to transform E.coli competent cells, which were then plated on Kan-resistant LB solid culture plates for cultivation after resuscitation.
7. Single colonies are picked, added into LB culture medium containing Kan antibiotics, and cultured at 37 ℃ by shaking overnight.
8. Extracting recombinant plasmid optoDroplet-KCTD17 IDR, sequencing, and screening the recombinant with correct sequencing result.
EXAMPLE 5 construction of the vector for the optoDroplet-KCTD17 IDR-Titin and Titin protein expression
Anti-striated muscle antibodies are characteristic of systemic myasthenia gravis, while tin is the primary autoantigen recognized by anti-striated muscle antibodies. Although the detection method commonly used for Titin is ELISA, the CBA method is widely used at present, and the key material of CBA is plasmid capable of efficiently expressing antigen, so the invention designs the photoinduction phase change expression plasmid of Titin.
1. Main reagents, instruments and sources:
PCR enzyme:max DNA Polymerase (Takara, cat# R045A);
NEB restriction enzyme: nheI-HF and XhoI-HF;
cell line: HEK293T cells;
glue recovery kit: omega Gel Extraction Kit;
plasmid: the optoDroplet-KCTD17IDR (from example 4); plasmid pEGFP-N1-Titin containing Titin gene was synthesized by general biosystems (subcloning Titin gene into pEGFP-N1);
plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；
Pipetting: eppendorf;
PCR instrument: bio-Rad T100.
Titin and antigen genes can be subcloned into the optoDroplet-KCTD17IDR plasmid by using the reagent and the consumable material according to the following steps.
2. Vector construction of optoDroplet-KCTD17 IDR-Titin
1) Primer design:
the primer comprises a Titin-NheI-F (SEQ ID NO: 22) primer upstream of the Titin gene and a Titin-XhoI-R (SEQ ID NO: 23) primer downstream of the Titin gene.
Primer(s) Sequence(s)
Titin-NheI-F 5’-GTGAACCGTCAGATCCGCTAGCATGAGGTGCGAGGAGGGCAAAGATAATT-3’
Titin-XhoI-R 5’-CAGAATTCGAAGCTTGAGCTCGAGAGCCGCCGTCATTCTTGGGAG-3’
2) And (3) PCR amplification and recovery:
the set of primers and the corresponding DNA template (pEGFP-N1-tin) were selected, and the Titin gene was amplified using PrimeSTAR (Takara, R045A) system, and after PCR was completed, the gene product was recovered by running electrophoresis and cutting.
3) Linearizing a carrier:
The optoDroplet-KCTD17 IDR was digested with NheI and XhoI to obtain linearized vector fragments, the reaction system was as follows:
reaction components Volume of
optoDroplet-KCTD17 IDR plasmid Total 2ug
rcutsmart buffer 5μl
NheI-HF 1μl
XhoI-HF 1μl
ddH 2 O Up to 50μl
Total volume of 50μl
And incubating at 37 ℃ for enzyme digestion for 2 hours, performing Agarose gel electrophoresis, cutting gel to recover large fragments of the carrier, and recovering DNA according to the specification of the gel recovery kit.
4) Seamless connection:
the recovered tin gene and linearized optoDroplet-KCTD17 IDR were seamlessly connected using NEBuilder HiFi DNA Assembly Master Mix, and the reaction system was as follows:
reaction components Volume of
optoDroplet-KCTD17 IDR plasmid skeleton 3μl
Titin gene 1μl
NEBuilder HiFi DNA Assembly Master Mix 10μl
ddH 2 O 4μl
Total volume of 20μl
5) Recombinant clone screening:
mu.l of the ligation product was used to transform E.coli competent cells, which were then plated onto Kan-resistant LB solid culture plates for cultivation after resuscitation. Picking single colony, adding into LB culture medium containing Kan antibiotics, shaking at 37deg.C for overnight culture, extracting plasmid, sequencing and screening correct recombinant optoDroplet-KCTD17 IDR-tin.
3. Expression of optoDroplet-KCTD17 IDR-Titin
1. Cell transfection:
treating the cultured Hep2 cells with pancreatin digestion, stopping digestion with a complete DMEM medium containing 10% serum, sucking the digested cells into a centrifuge tube, centrifuging at 800-1000 rpm for 3min, pouring out the supernatant, adding the complete DMEM medium containing 10% serum, and lightly blowing and mixing with a pipette to prepare a cell suspension.
Placing sterilized glass slide in cell culture plate, treating with Polylysine (PDL), rinsing for 3 times, air drying, adding prepared cell suspension into cell culture plate, gently mixing, placing at 37deg.C, 5% CO 2 The incubator was cultured overnight. The cells are observed the next day, and the cells can be transfected when the density reaches 40-50%.
Mixing the prepared optoDroplet-KCTD17 IDR-Titin plasmid and a transfection reagent PEI in a mass-volume ratio of 1:3, vortexing, standing for 10min, and respectively transfecting to the prepared cells at 37 ℃ and 5% CO 2 Culturing for 48h.
Blue light excitation of the optoDroplet-KCTD17 IDR-tin transfected cells: placing the pore plate or the culture dish on a confcol, and exciting for 5-120S by 405nm blue light;
after the cells are excited by blue light, observing the fluorescence conditions of the Titin in the optoDroplet-KCTD17 IDR-Titin and the conventional plasmid pEGFP-N1-Titin under a red channel, before the blue light is not excited, the Titin fusion protein expressed by the optoDroplet-KCTD17 IDR-Titin is consistent with the pEGFP-N1-Titin, and presents cytoplasmic dispersion distribution, and obviously aggregated red fluorescence signals appear in the cells transfected by the optoDroplet-KCTD17 IDR-Titin after 30s of blue light excitation, and larger droplet-shaped forms can be seen after 120s of excitation. Compared with the Titin fusion protein before blue light excitation, the Titin fusion protein after excitation is aggregated due to phase change, and the fluorescence intensity is improved by 9 times.
Example 6 vector construction of optoDroplet-K10C-GAD65 and protein expression
Autoimmune encephalitis (autoimmune encephalitis, AE) is an autoimmune neurological disease mediated by autoantibodies, and may have symptoms such as cognitive dysfunction, behavioral abnormalities, seizure, mental disorders, involuntary movements, and autonomic nerve dysfunction. GAD65 antibody detection is of great importance for diagnosis of autoimmune encephalitis. Currently, the detection methods commonly used for GAD65 are ELISA and CBA. The key material of CBA is plasmid capable of expressing antigen effectively, so that the invention designs the optoDroplet-KCTD17 IDR-GAD65 expression plasmid.
1. Vector construction of optoDroplet-KCTD17 IDR-GAD65
1) Primer design:
the primers contained GAD65-Nhe1-F (SEQ ID NO: 24) as the upstream primer of the GAD65 gene and GAD65-Xho1-R (SEQ ID NO: 25) as the downstream primer of the GAD65 gene.
2) And (3) PCR amplification and recovery:
GAD65-Nhe1-F, GAD-Xho 1-R primers and corresponding DNA templates (plasmid pEGFP-N1-GAD65 containing the GAD65 gene was synthesized by general biosystems (subcloning GAD65 gene into pEGFP-N1)), amplified using PrimeSTAR (Takara, R045A) system to give GAD65 gene, and after PCR was completed, the gene product was recovered by running and cutting.
3) Double enzyme cutting of the carrier:
the optoDroplet-KCTD17 IDR was digested with NheI and XhoI to obtain large linearized vector fragments as in example 5; and incubating at 37 ℃ for enzyme digestion for 2 hours, performing Agarose gel electrophoresis, cutting gel to recover large fragments of the carrier, and recovering DNA according to the specification of the gel recovery kit.
4) Seamless connection:
the recovered GAD65 gene and the linearized large fragment of the optoDroplet-KCTD17 IDR were subjected to multi-fragment seamless connection by using NEBuilder, and the reaction system is as follows:
reaction components Volume of
optoDroplet-KCTD17 IDR plasmid skeleton 3μl
GAD65 gene 1μl
NEBuilder HiFi DNA Assembly Master Mix 10μl
ddH 2 O 6μl
Total volume of 20μl
5) Recombinant clone screening:
mu.l of the ligation product was used to transform E.coli competent cells, which were then plated onto Kan-resistant LB solid culture plates for cultivation after resuscitation. Picking single colony, adding into LB culture medium containing Kan antibiotics, shaking at 37deg.C for overnight culture, extracting plasmid, sequencing, and screening correct recombinant optoDroplet-KCTD17 IDR-GAD65.
2. Expression of optoDroplet-KCTD17 IDR-GAD65
1. Cell transfection:
treating the cultured Hep2 cells with pancreatin digestion, stopping digestion with a complete DMEM medium containing 10% serum, sucking the digested cells into a centrifuge tube, centrifuging at 800-1000 rpm for 3min, pouring out the supernatant, adding the complete DMEM medium containing 10% serum, and lightly blowing and mixing with a pipette to prepare a cell suspension.
Placing sterilized glass slide in cell culture plate, treating with Polylysine (PDL), rinsing for 3 times, air drying, adding prepared cell suspension into cell culture plate, gently mixing, placing at 37deg.C, 5% CO 2 The incubator was cultured overnight. The cells are observed the next day, and the cells can be transfected when the density reaches 40-50%.
Mixing the prepared optoDroplet-K10C-GAD65 plasmid and a transfection reagent PEI in a mass-volume ratio of 1:3, vortexing, standing for 10min, and respectively transfecting to the prepared cells at 37 ℃ and 5% CO 2 Culturing for 48h.
Blue light excitation of the optoDroplet-K10C-GAD65 transfected cells: placing the pore plate on Confocol, and exciting for 5-120s under a 405nm blue light channel;
after the cells are excited by blue light, the fluorescence condition of GAD65 in the optoDroplet-KCTD17 IDR-GAD65 is observed under a red channel, the GAD65 fusion protein expressed by the optoDroplet-KCTD17 IDR-GAD65 is consistent with pEGFP-N1-GAD65 before the blue light is not excited, the cytosolic dispersion distribution is presented, and obviously aggregated red fluorescence signals appear in the cells transfected by the optoDroplet-KCTD17 IDR-GAD65 after 30s of blue light excitation, and the more enlarged droplet-shaped morphology can be seen after 120s of excitation. The stimulated GAD65 fusion protein aggregated due to phase change, and its fluorescence intensity was 11-fold improved compared to that before blue light stimulation (FIG. 6).
Example 7 aggregation of the antigen of the otoDroplet-KCTD 17 IDR System resistant to methanol fixation
The phenomenon of phase change was observed in living cells before optodroplets, but it is unknown whether the phase change of this protein can withstand the effects of cell fixation procedures. According to the invention, after the cells transfected with the otoDroplet-KCTD 17 IDR are excited by blue light, the cells are rapidly fixed, and then the condition of phase change proteins is observed, so that the phase edge structure of the proteins induced by the otoDroplet-KCTD 17 IDR blue light can be reserved both in methanol and Paraformaldehyde (PFA).
1. Main reagents, instruments and sources:
cell line: HEK293T cells;
plasmid: optoDroplet-KCTD17 IDR-tin/GDA 65 (from examples 5 and 6);
plasmid extraction kit: omega plasmid mini kit;
pure water meter: millipore5UV Water Purification System；
Pipetting: eppendorf;
blue light viewer: six biotechnology companies, WD-9403X, beijing;
the following steps can be carried out by using the reagent and the consumable to observe whether the Titin and the GDA65 antigen expressed by the optoDroplet-KCTD17 IDR system can tolerate cell fixation.
2. Cell transfection:
1. treating the cultured Hep2 cells with pancreatin digestion, stopping digestion with a complete DMEM medium containing 10% serum, sucking the digested cells into a centrifuge tube, centrifuging at 800-1000 rpm for 3min, pouring out the supernatant, adding the complete DMEM medium containing 10% serum, and lightly blowing and mixing with a pipette to prepare a cell suspension.
2. Placing sterilized glass slide in cell culture plate, treating with Polylysine (PDL), rinsing for 3 times, air drying, adding prepared cell suspension into cell culture plate, gently mixing, placing at 37deg.C, 5% CO 2 The incubator was cultured overnight. The cells are observed the next day, and the cells can be transfected when the density reaches 40-50%.
3. Mixing the prepared optoDroplet-KCTD17 IDR-tin and optoDroplet-KCTD17 IDR-GAD65 plasmids with a transfection reagent PEI in a mass-volume ratio of 1:3, vortexing, standing for 10min, and respectively transfecting the mixture into prepared cells at 37 ℃ and 5% CO 2 Culturing for 48h.
Fixation of transfected cells with optoDroplet-KCTD17 IDR-tin and optoDroplet-KCTD17 IDR-GAD 65:
(1) Placing the pore plate on a blue light observer, and exciting the pore plate by blue light for 1-5 minutes;
(2) Cells were fixed with acetone/ice methanol for 10min and washed 2 times with PBS;
after the cells were fixed, fluorescent conditions of the tin and GAD65 proteins in the optoDroplet-KCTD17 IDR system were observed in the red channel, and the Titin and GAD65 fusion proteins expressed by the optoDroplet-KCTD17 IDR system in the transfected cells were significantly aggregated and exhibited a droplet-like morphology, indicating that the optoDroplet-KCTD17 IDR expressed proteins were able to withstand the effects of fixing reagents such as methanol and PFA after undergoing phase change (FIG. 7).
Sequence:
KCTD17 amino acid sequence (SEQ ID NO: 1):
MQTPRPAMRMEAGEAAPPAGAGGRAAGGWGKWVRLNVGGTVFLTTRQTLCREQKSFLSRLCQG
EELQSDRDETGAYLIDRDPTYFGPILNFLRHGKLVLDKDMAEEGVLEEAEFYNIGPLIRIIKDRMEE
KDYTVTQVPPKHVYRVLQCQEEELTQMVSTMSDGWRFEQLVNIGSSYNYGSEDQAEFLCVVSKE
LHSTPNGLSSESSRKTKSTEEQLEEQQQQEEEVEEVEVEQVQVEADAQEKGSRPHPLRPEAELAVR
ASPRPLARPQSCHPCCYKPEAPGCEAPDHLQGLGVPI
KCTD17 nucleotide sequence (SEQ ID NO: 2):
ATGCAGACGCCGCGGCCGGCGATGAGGATGGAGGCCGGGGAGGCAGCGCCGCCGGCGGGGGCGGGCGGCCGCGCCGCAGGCGGCTGGGGCAAGTGGGTGCGGCTCAACGTGGGGGGCACGGTGTTCCTGACCACCCGGCAGACGCTGTGCCGCGAGCAGAAGTCCTTCCTCAGCCGCCTGTGCCAGGGGGAAGAGCTGCAGTCGGACCGGGATGAGACCGGGGCCTACCTCATTGACCGTGACCCCACCTACTTCGGGCCCATCCTGAACTTCCTCCGGCATGGCAAGCTGGTGCTGGACAAGGACATGGCTGAGGAGGGGGTCCTGGAGGAAGCCGAGTTCTACAACATCGGCCCGCTGATCCGCATCATCAAAGACCGGATGGAAGAGAAGGACTACACGGTCACCCAGGTCCCACCCAAGCACGTGTACCGCGTGCTGCAGTGCCAGGAGGAGGAGCTCACGCAAATGGTCTCCACCATGTCTGATGGCTGGCGCTTCGAGCAGCTGGTGAACATCGGCTCCTCCTACAACTACGGCAGCGAGGACCAGGCAGAGTTCCTGTGTGTGGTGTCCAAGGAGCTCCACAGCACCCCAAACGGGCTGAGCTCAGAGTCCAGCCGCAAAACCAAGAGCACGGAGGAGCAGCTGGAGGAGCAGCAGCAGCAGGAGGAGGAGGTGGAGGAGGTGGAGGTGGAACAGGTGCAGGTGGAGGCAGATGCACAGGAGAAAGGTTCCCGTCCGCACCCTCTCAGACCTGAGGCTGAGCTTGCAGTGAGGGCTTCTCCTCGGCCCCTCGCCCGCCCCCAGAGCTGCCATCCCTGCTGTTACAAGCCAGAGGCACCCGGATGTGAGGCCCCAGATCACCTCCAGGGACTTGGGGTTCCCATC
KCTD17 IDR amino acid sequence (SEQ ID NO: 3):
KTKSTEEQLEEQQQQEEEVEEVEVEQVQVEADAQEKGSRPHPLRPEAELAVRAS
KCTD17 IDR nucleotide sequence (SEQ ID NO: 4):
AAAACCAAGAGCACGGAGGAGCAGCTGGAGGAGCAGCAGCAGCAGGAGGAGGAGGTGGAGGAGGTGGAGGTGGAACAGGTGCAGGTGGAGGCAGATGCACAGGAGAAAGGTTCCCGTCCGCACCCTCTCAGACCTGAGGCTGAGCTTGCAGTGAGGGCTTCT
the complete sequence of the optoDroplet-KCTD17 IDR vector (SEQ ID NO: 26):
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCATGAAAACCAAGAGCACGGAGGAGCAGCTGGAGGAGCAGCAGCAGCAGGAGGAGGAGGTGGAGGAGGTGGAGGTGGAACAGGTGCAGGTGGAGGCAGATGCACAGGAGAAAGGTTCCCGTCCGCACCCTCTCAGACCTGAGGCTGAGCTTGCAGTGAGGGCTTCTGGCGGCGGATCTGCCCGGGATCCACCGGTCGCCACCatggtgtctaaaggcgaggaggataacatggctataatcaaggaatttatgaggttcaaggtgcatatggagggatctgtgaacggtcacgagttcgaaatcgagggcgaaggcgaggggcgaccctatgaagggacacagacggccaaactgaaggtgaccaagggtggccccctgcctttcgcctgggacatcctgtcaccccagtttatgtacggctctaaggcttacgttaagcaccctgccgatattccggactacttgaaactgagcttccctgaagggttcaagtgggaacgggtaatgaattttgaggatggcggtgtagtcacagttacccaggacagctctcttcaggacggagaatttatctataaggttaaacttcggggcactaacttcccatcagacggccccgtgatgcagaagaaaactatgggctgggaagccagctcagaacgcatgtatcccgaggacggagcactgaagggagaaatcaagcagcgactcaagctgaaggatgggggacattatgatgctgaggtcaaaaccacctataaggccaagaaacccgttcagcttcctggggcatacaacgtgaatatcaaactggatattacatcccataacgaagactataccatcgtggaacagtacgagcgggccgagggcagacatagcacagggggcatggatgaattgtacaaggcggccacgcgtatgaagatggacaaaaagactatagtttggtttagaagagacctaaggattgaggataatcctgcattagcagcagctgctcacgaaggatctgtttttcctgtcttcatttggtgtcctgaagaagaaggacagttttatcctggaagagcttcaagatggtggatgaaacaatcacttgctcacttatctcaatccttgaaggctcttggatctgacctcactttaatcaaaacccacaacacgatttcagcgatcttggattgtatccgcgttaccggtgctacaaaagtcgtctttaaccacctctatgatcctgtttcgttagttcgggaccataccgtaaaggagaagctggtggaacgtgggatctctgtgcaaagctacaatggagatctattgtatgaaccgtgggagatatactgcgaaaagggcaaaccttttacgagtttcaattcttactggaagaaatgcttagatatgtcgattgaatccgttatgcttcctcctccttggcggttgatgccaataactgcagcggctgaagcgatttgggcgtgttcgattgaagaactagggctggagaatgaggccgagaaaccgagcaatgcgttgttaactagagcttggtctccaggatggagcaatgctgataagttactaaatgagttcatcgagaagcagttgatagattatgcaaagaacagcaagaaagttgttgggaattctacttcactactttctccgtatctccatttcggggaaataagcgtcagacacgttttccagtgtgcccggatgaaacaaattatatgggcaagagataagaacagtgaaggagaagaaagtgcagatctttttcttaggggaatcggtttaagagagtattctcggtatatatgtttcaacttcccgtttactcacgagcaatcgttgttgagtcatcttcggtttttcccttgggatgctgatgttgataagttcaaggcctggagacaaggcaggaccggttatccgttggtggatgccggaatgagagagctttgggctaccggatggatgcataacagaataagagtgattgtttcaagctttgctgtgaagtttcttctccttccatggaaatggggaatgaagtatttctgggatacacttttggatgctgatttggaatgtgacatccttggctggcagtatatctctgggagtatccccgatggccacgagcttgatcgcttggacaatcccgcgttacaaggcgccaaatatgacccagaaggtgagtacataaggcaatggcttcccgagcttgcgagattgccaactgaatggatccatcatccatgggacgctcctttaaccgtactcaaagcttctggtgtggaactcggaacaaactatgcgaaacccattgtagacatcgacacagctcgtgagctactagctaaagctatttcaagaacccgtgaagcacagatcatgatcggagcagcagcccgggatccaccggtcgccaccggttcagggagcggatccggctcagggagtgcggccgcaactcccacctgcaacatgcgtgactgaGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT
titin nucleotide sequence (SEQ ID NO: 27)
ATGAGGTGCGAGGAGGGCAAAGATAATTGGATTAGATGCAATATGAAGCTGGTGCCTGAGCTGACCTACAAAGTGACCGGCCTGGAGAAGGGCAACAAGTATCTGTATAGAGTGAGCGCCGAGAACAAAGCCGGCGTGAGCGATCCTAGCGAGATCCTGGGCCCCCTGACCGCCGATGACGCCTTCGTGGAGCCTACCATGGACCTGAGCGCCTTCAAAGACGGCCTGGAGGTCATTGTGCCTAATCCTATCACCATTCTGGTGCCTAGCACCGGCTACCCCAGACCCACCGCCACCTGGTGTTTTGGCGACAAAGTGCTGGAGACCGGCGATAGAGTGAAGATGAAAACCCTGTCCGCCTACGCCGAGCTGGTCATTTCCCCCTCCGAGAGATCCGATAAGGGCATCTATACCCTGAAGCTGGAGAATAGAGTGAAGACCATTTCCGGCGAGATTGACGTGAACGTGATCGCCAGACCCTCCGCCCCCAAGGAGCTGAAGTTTGGCGATATTACCAAGGACTCCGTGCACCTGACCTGGGAGCCCCCCGATGATGATGGCGGCAGCCCCCTGACCGGCTATGTGGTGGAGAAAAGGGAGGTGAGCAGGAAAACCTGGACCAAGGTCATGGATTTTGTGACCGATCTGGAGTTTACCGTGCCTGATCTGGTGCAGGGCAAAGAGTACCTGTTTAAAGTGTGTGCCAGGAATAAGTGCGGCCCTGGCGAGCCCGCCTACGTGGATGAGCCCGTGAATATGAGCACCCCCGCCACCGTGCCCGACCCACCTGAAAATGTGAAATGGAGGGACAGAACCGCCAATTCCATCTTTCTGACCTGGGATCCTCCCAAGAATGACGGCGGC
GAD65 nucleotide sequence (SEQ ID NO: 28)
ATGGCCAGCCCCGGATCCGGATTTTGGAGCTTTGGCTCCGAGGATGGCTCCGGCGATTCCGAGAATCCCGGCACCGCCAGAGCCTGGTGCCAGGTTGCCCAGAAATTCACCGGCGGCATTGGCAATAAGCTGTGCGCCCTGCTGTACGGCGACGCCGAGAAACCTGCCGAGAGCGGCGGCAGCCAGCCCCCTAGGGCTGCTGCTAGGAAAGCCGCCTGCGCCTGTGATCAGAAGCCCTGCAGCTGCTCCAAAGTGGACGTGAACTACGCCTTCCTGCACGCCACCGATCTGCTGCCTGCCTGCGATGGCGAGAGGCCCACCCTGGCTTTTCTGCAGGATGTGATGAACATTCTGCTGCAGTATGTGGTGAAGAGCTTTGACAGATCCACCAAAGTGATTGACTTTCACTATCCCAACGAGCTGCTGCAGGAGTACAATTGGGAGCTGGCCGATCAGCCTCAGAATCTGGAGGAGATCCTGATGCACTGCCAGACCACCCTGAAGTACGCCATTAAGACCGGCCACCCCAGATATTTCAATCAGCTGTCCACCGGCCTGGATATGGTGGGCCTGGCCGCCGATTGGCTGACCAGCACCGCCAACACCAACATGTTTACCTATGAGATCGCCCCCGTGTTTGTGCTGCTGGAGTACGTGACCCTGAAAAAGATGAGAGAGATTATCGGCTGGCCTGGCGGCTCCGGCGACGGAATTTTTAGCCCTGGCGGCGCCATCTCCAATATGTACGCCATGATGATCGCCAGATTCAAAATGTTTCCTGAGGTGAAGGAGAAGGGCATGGCCGCCCTGCCTAGGCTGATCGCCTTCACCAGCGAGCACTCCCACTTTTCCCTGAAGAAAGGCGCCGCCGCCCTGGGCATTGGCACCGACTCTGTGATCCTGATTAAGTGCGATGAGAGGGGCAAGATGATTCCTTCCGATCTGGAGAGAAGAATCCTGGAGGCCAAACAGAAGGGCTTTGTGCCTTTTCTGGTGTCCGCCACCGCCGGCACCACCGTTTACGGCGCTTTCGACCCCCTGCTGGCCGTGGCTGATATTTGCAAGAAATACAAGATCTGGATGCACGTGGACGCCGCCTGGGGCGGAGGACTGCTGATGAGCAGAAAACACAAGTGGAAACTGAGCGGCGTGGAGAGAGCCAATAGCGTGACCTGGAATCCTCACAAGATGATGGGCGTGCCTCTGCAGTGTAGCGCCCTGCTGGTGAGAGAGGAGGGCCTGATGCAGAATTGTAATCAGATGCACGCCAGCTACCTGTTCCAGCAGGACAAACACTACGACCTGTCCTATGATACCGGCGATAAGGCCCTGCAGTGTGGCAGACACGTGGACGTGTTCAAGCTGTGGCTGATGTGGAGGGCCAAGGGCACCACCGGCTTTGAGGCCCACGTGGACAAGTGTCTGGAGCTGGCCGAGTACCTGTACAACATTATTAAGAATAGGGAGGGCTATGAGATGGTGTTCGACGGCAAACCCCAGCACACCAACGTGTGCTTCTGGTATATCCCCCCCTCCCTGAGAACCCTGGAGGACAATGAGGAGAGAATGAGCAGACTGTCCAAAGTGGCCCCCGTGATCAAAGCCAGAATGATGGAGTACGGCACCACCATGGTGAGCTATCAGCCCCTGGGCGATAAGGTGAACTTCTTTAGAATGGTCATTAGCAACCCCGCCGCCACCCACCAGGACATTGATTTCCTGATCGAGGAGATTGAGAGGCTGGGCCAGGATCTG

Claims (10)

1. A polypeptide comprising KCTD17 IDR having an amino acid sequence as shown in SEQ ID NO. 3.
2. A nucleic acid molecule comprising a polynucleotide encoding the polypeptide of claim 1, preferably comprising a polynucleotide as set forth in SEQ ID No. 4.
3. A vector comprising the nucleic acid molecule described above.
4. A vector according to claim 3, wherein the vector is an expression vector, preferably the vector is a prokaryotic or eukaryotic expression vector, preferably the vector is a plasmid.
5. The vector of claim 4, wherein the expression vector further comprises
A polynucleotide encoding a light-induced multimerization protein domain, preferably a domain of a protein capable of dimerizing or even multimerizing in response to a light stimulus, more preferably the light-induced multimerization protein domain is selected from the group consisting of a Cry2 PHR, an NTE domain of photopigment B and a LOV2 domain of photopigment 1; and/or
A polynucleotide encoding a fluorescent protein tag, preferably a protein capable of spontaneously generating fluorescence, having a fluorescent labeling and tracing function, more preferably the fluorescent protein tag is selected from mCherry, GFP, RFP, YFP, dsRed; and/or
Multiple cloning restriction sites (MCS).
6. The carrier according to claim 4 or 5,
wherein the expression vector comprises a polyclonal enzyme cutting site (MCS), a polynucleotide for encoding KCTD17IDR, a polynucleotide for encoding a fluorescent protein label and a polynucleotide for encoding a light-induced multimerization protein structural domain from upstream to downstream in sequence, and preferably the expression vector is an optoDroplet-KCTD17 IDR shown as SEQ ID NO. 26.
7. The vector according to claim 4 or 5, wherein the expression vector further comprises a target protein gene, preferably the target protein gene is selected from the group consisting of an antigen protein gene, in particular an autoimmune antigen protein gene, more preferably the target protein gene is selected from the group consisting of a tin gene, a GAD65 gene, a GFAP gene, an MBP gene, a Hu gene, a Yo gene, a Ri gene, a CV2 gene, an amphhysin gene, a Ma1 gene, a Ma2 gene, a SOX1 gene, a Zic gene, a Recoverin gene, a pkcγ gene.
8. The vector of claim 7, wherein the target protein is located at the N-terminus of KCTD17 IDR.
9. The vector according to claim 4 or 5, wherein the expression vector is obtained by a method comprising the steps of: the KCTD17 IDR was conjugated with a fluorescent protein tag and a photoinduced multimerization protein domain, constructed into pEGFP-N1 plasmid, and a polyclonal cleavage site was reserved before KCTD17 IDR and fluorescent protein tag to insert the target protein gene.
10. A cell comprising the vector of any one of claims 4-9, preferably said cell is selected from HEK293T cells, hela cells, hep2 cells.
CN202311158409.1A 2023-09-08 2023-09-08 Photo-induced phase change protein element and application thereof Pending CN117327158A (en)

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