A light-induced phase change protein element and its application

Abstract

The present invention provides a light-induced phase transition protein element and its application. The light-induced phase transition protein element of the present invention is a polypeptide, which contains KCTD17 IDR with the amino acid sequence shown in SEQ ID NO: 3. The polypeptide of the present invention can be used as a phase transition protein element to form a fusion protein with a target protein (for example, an antigen protein, especially an autoimmune antigen protein).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more particularly, relates to a light-induced phase change protein element and its application. Background Art

[0002] In the laboratory tests for immune diseases, the detection of specific antibodies is one of the important clinical diagnostic criteria. Cell-based assays (CBA) are the preferred method for detecting cell surface antigens and certain synaptic protein-related autoantibodies. This method is based on the antigen-antibody reaction. Known antigen overexpression plasmids are transfected into mammalian cells (HEK293, Hela or Hep2 cell lines), so that the target antigen is expressed on the cell membrane or in the cytoplasm. The cell matrix overexpressing the antigen is incubated with the patient's body fluid sample, and then a fluorescently conjugated antibody against human immunoglobulin (IgG1 / IgG2 / IgG3 / IgG4 / IgM) is used as the secondary antibody for signal amplification and labeling. Finally, the results are read by a fluorescence microscope or a flow cytometer.

[0003] Currently, the cell-based immunofluorescence assay for detecting neurological autoantibodies mainly uses expression plasmids constructed with natural full-length genes, and mammalian cells such as HEK293 and Hep2 are transfected to prepare the detection materials. For example, the Titin antigen (MGT30) or the GAD65 antigen is constructed into a eukaryotic expression vector for the detection of autoantibodies (Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients. Arch Neurol. 2001 Jun; 58(6):885-90.; GAD65 neurological autoimmunity. Muscle and Nerve, 2017 56(1), 15-27.). The expression pattern of these antigen proteins in the cell is cytoplasmic diffuse distribution without a specific morphology. When the concentration of autoantibodies in the sample is low, the positive signal may be weak and it may be impossible to accurately judge whether the antibody is positive or negative.

[0004] In 2017, researchers from Princeton University in the United States developed a tool called optoDroplets, which can use blue light to regulate multivalence to promote or reverse the formation of biomolecular condensates in vivo. They fused the light-sensitive protein tag Cry2 with intrinsically disordered regions (IDRs) that promote protein phase transitions. Using blue light, the researchers were able to induce these proteins to clump together, thus mimicking the phase transition and condensation processes that occur to proteins in cells (Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets. Cell. 2017 Jan 12;168(1-2):159-171.e14.). The main function of Cry2 is that it can be induced to oligomerize by blue light (Structural insights into the photoactivation of Arabidopsis cryptochrome 2. Nat Plants. 2020 Dec;6(12):1432-1438.). Alone, Cry2 cannot aggregate and undergo a phase transition under blue light. The key factor is the IDR of the phase transition protein. Only in the presence of the IDR can Cry2 play its synergistic role in promoting protein phase transitions.

[0005] The smaller the IDR of the phase transition protein, the easier it is to modularize and convenient for genetic manipulation. As the IDRs of phase transition proteins, whether Fus (1-214aa), DDX4 (1-236aa), and HNRNPA1 (186-320aa) in previous reports, their intrinsically disordered regions (IDRs) in the proteins are relatively large.

[0006] Therefore, it is necessary to find a smaller and more effective IDR phase transition protein element. Summary of the Invention

[0007] The present inventor has found a new phase transition protein, KCTD17. KCTD17 has a strong ability to undergo phase transition. It can undergo phase transition on its own and form droplet-like structures. After coupling with a target protein (e.g., antigen protein, especially autoimmune antigen protein), it can also effectively cause the fusion protein to undergo protein phase transition and form a solid-phase phase transition protein structure. To make the phase transition more dynamic and controllable, the inventor further screened the intrinsically disordered region (IDR) of the phase transition protein - KCTD17 IDR, which has the following advantages: it has only 54 amino acids, smaller and more advantageous than various known IDRs (Fus: 214aa; DDX4: 236aa; HNRNPA1: 135aa). Further, on the basis of the pEGFP-N1 vector, the present inventor introduced two essential elements for phase transition: KCTD17 IDR and Cry2 PHR, and finally obtained a simple eukaryotic expression plasmid system for spatio-temporal regulation of protein phase transition - optoDroplet-KCTD17 IDR.

[0008] The present inventor combined the eukaryotic expression plasmid system for phase transition optoDroplet-KCTD17 IDR with the expression of a target protein (e.g., antigen protein, especially autoimmune antigen protein), and invented a new protein expression system. The target protein (e.g., antigen protein, especially autoimmune antigen protein) was inserted into the optoDroplet-KCTD17 IDR plasmid and expressed by coupling with KCTD17 IDR and Cry2 elements. After transfection of cells, blue light induction was used to promote the protein phase transition of the fusion protein expressed by the optoDroplet plasmid inside the cells, forming droplet-like structures and increasing the local concentration of the target protein. After the transfected cells were fixed with PFA / methanol, the phase transition protein aggregates in these droplet-like structures could still stably exist. Therefore, the present invention combines the newly modified optoDroplet-KCTD17 IDR with the expression plasmid of a clinical detection protein (e.g., antigen protein, especially autoimmune antigen protein), which can change the expression patterns of various intracellular proteins and form droplet-like structures. Therefore, the present invention combines KCTD17 IDR, Cry2 elements and a target protein (e.g., antigen protein, especially autoimmune antigen protein) to form a new eukaryotic expression plasmid system optoDroplet-KCTD17 IDR, which can cause the protein (e.g., antigen protein, especially autoimmune antigen protein) to aggregate and enhance its signal. In some embodiments, the signal intensity of the antigen protein measured on a confocal fluorescence microscope can be increased by more than ten times.

[0009] Thus, on the one hand, the present invention provides a polypeptide comprising the KCTD17 IDR having the amino acid sequence as shown in SEQ ID NO:3. The polypeptide of the present invention can be used as a phase transition protein element to form a fusion protein with a target protein (e.g., an antigen protein, especially an autoimmune antigen protein).

[0010] On the other hand, the present invention provides a nucleic acid molecule comprising a polynucleotide encoding the above polypeptide.

[0011] In some embodiments, the nucleic acid molecule comprises the polynucleotide as shown in SEQ ID NO:4.

[0012] On the other hand, the present invention provides a vector comprising the above nucleic acid molecule.

[0013] In some embodiments, the vector can be an expression vector. Further, in some embodiments, the vector can be a prokaryotic expression vector or a eukaryotic expression vector.

[0014] In some embodiments, the vector can be a plasmid.

[0015] In some embodiments, the expression vector can further comprise a polynucleotide encoding a light-induced polymerization protein domain. The light-induced polymerization protein domain can refer to the domain of a protein that can dimerize or even polymerize in response to light stimulation. Examples thereof include, but are not limited to, Cry2 PHR, the N-terminal extension (NTE) domain of phytochrome B, and the LOV2 domain of phototropin 1.

[0016] In some embodiments, the expression vector can further comprise a polynucleotide encoding a fluorescent protein tag. The fluorescent protein tag can refer to a protein that can spontaneously generate fluorescence and has the functions of fluorescence labeling and tracing. Examples thereof include, but are not limited to, mCherry, GFP, RFP, YFP, dsRed, etc.

[0017] In some embodiments, the expression vector can further comprise a multiple cloning site (MCS) to facilitate the insertion of various target protein genes into the expression vector.

[0018] In some embodiments, the expression vector can sequentially comprise a multiple cloning site (MCS), a polynucleotide encoding KCTD17 IDR, a polynucleotide encoding a fluorescent protein tag, and a polynucleotide encoding a light-induced polymerization protein domain (especially Cry2 PHR) from upstream to downstream, and optionally, each element can be connected by a linker.

[0019] In some embodiments, the nucleotide sequence of the expression vector optoDroplet-KCTD17 IDR is as shown in SEQ ID NO: 26.

[0020] In some embodiments, the expression vector may further comprise a target protein gene. The target protein gene may be a gene that expresses any protein, including but not limited to, an antigen protein gene, particularly an autoimmunity antigen protein gene, and examples thereof include but are not limited to, the Titin gene, the GAD65 gene, the GFAP gene, the MBP gene, the Hu gene, the Yo gene, the Ri gene, the CV2 gene, the Amphiphysin gene, the Ma1 gene, the Ma2 gene, the SOX1 gene, the Zic4 gene, the Recoverin gene, and the PKCγ gene.

[0021] In the present invention, the positions of elements such as KCTD17 IDR, the light-induced polymerization protein domain, and the fluorescent protein tag are not particularly limited as long as they can form 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.

[0022] In some embodiments, the expression vector can be obtained by a method comprising the following steps: coupling KCTD17 IDR with a fluorescent protein tag (e.g., mCherry) and a light-induced polymerization protein domain (e.g., Cry2 PHR), constructing them into the pEGFP-N1 plasmid, and retaining a multiple cloning site before KCTD17 IDR and the fluorescent tag mCherry for inserting the target protein gene.

[0023] In addition, in the present invention, for other elements that must be present in the vector, particularly the expression vector, elements well-known in the art can be used and will not be elaborated herein.

[0024] On the other hand, the present invention provides a cell comprising the above vector. In some embodiments, the cell may be a HEK293T cell, a Hela cell, a Hep2 cell, and examples thereof include but are not limited to, HEK293T cells, Hela cells, Hep2 cells, and the like.

[0025] The present invention provides a new phase-transition IDR protein and develops a smaller optoDroplet tool: optoDroplet-KCTD17 IDR. For the first time, the present invention combines the expression of this tool with that of a target protein (such as an antigen protein, particularly a neuroautoimmune antigen protein), places the target protein gene into this tool to form an expression plasmid, which can significantly promote the aggregation of the target protein and form a droplet-like structure, facilitating the amplification of the target protein signal. For example, in some embodiments, the signal intensity of the target protein measured on a confocal fluorescence microscope can be increased by more than ten times.

[0026] The optoDroplet-KCTD17 IDR of the present invention has its own advantages compared with the previous optoDroplet tool: First, the KCTD17 IDR protein becomes smaller, with only 54 amino acids; in addition, its ability to undergo phase transition is very strong. Even without the help of Cry2, it can cause the small GFP protein to undergo protein phase transition; Second, the original optoDroplet tool uses the pHR-FusN-mCherry-Cry2 plasmid, with a plasmid size of 11954bp, which contains some sequence characteristics of viral plasmids, such as non-essential elements like LTR, RRE, and cPPT. Based on the pEGFP-N1 vector, the present invention introduces the target protein gene and two other essential elements for phase transition: KCTD17 IDR and Cry2 PHR, and finally obtains a simple expression plasmid optoDroplet-KCTD17 IDR, with a plasmid size of approximately 6500bp. Brief Description of the Drawings

[0027] Figure 1 A figure showing the phase transition of the fusion protein of KCTD17 and GFP (GFP-KCTD17, upper figure) and the fusion protein of KCTD17 IDR (209-262aa) and GFP (GFP-KCTD17 IDR, lower figure) in the cytoplasm to form a droplet-like structure.

[0028] Figure 2 A figure showing the protein phase transition of KCTD17 in vitro. Among them: A. Prokaryotic protein expression plasmid map of KCTD17: The MBP tag promotes protein expression and helps protein folding. Immediately after MBP is an HRV3C protease cleavage site, which can remove the MBP tag later, followed by a GFP tag to facilitate observing whether the KCTD17 protein aggregates and forms a droplet-like structure in the test tube and under the microscope; B. Protein electrophoresis map (Coomassie brilliant blue staining) of the prokaryotic expressed protein of KCTD17 and the protein after digestion with HRV3C enzyme; C. Observation of the protein phase transition of KCTD17 at different protein concentrations under a fluorescence microscope after removing the MBP tag.

[0029] Figure 3 Figure showing that full-length KCTD17 promotes the phase transition of the Titin / GAD65 fusion protein.

[0030] Figure 4 Schematic diagram of the optoDroplet-KCTD17 IDR expression plasmid of the present invention (B) and the schematic diagram of the original optoDroplets tool using the pHR-FusN-mCherry-Cry2 plasmid (A).

[0031] Figure 5 Figure showing the protein phase transition of optoDroplet-KCTD17 IDR-Titin in vitro.

[0032] Figure 6 Figure showing the protein phase transition of optoDroplet-KCTD17 IDR-GAD65 in vitro.

[0033] Figure 7 Figure showing that the optoDroplet-KCTD17 IDR system can tolerate the effects of cell fixation. Detailed implementation manners

[0034] Hereinafter, the present invention will be described in detail through examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0036] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0037] Example 1 Discovery of the phase transition protein KCTD17

[0038] The forces between proteins and proteins or RNA molecules can cause them to separate or aggregate from each other. When the molecules reach a certain concentration, "phase separation" occurs, where similar components aggregate together and unrelated molecules are isolated outside. Phase transition proteins usually have an important feature, that is, they have an intrinsically disordered region (IDR). IDR is a common domain in phase separation proteins, which is characterized by a low-complexity sequence region, such as a repeat sequence of a single amino acid, such as being rich in arginine, etc.

[0039] The present invention discovered through screening a protein KCTD17 with an IDR located at the C-terminus. Its IDR sequence is 209-262aa, and its amino acid sequence is shown in SEQ ID NO:3. In order to verify that the KCTD17 protein can undergo protein phase transition, the present invention conducted intracellular verification experiments and in vitro verification experiments.

[0040] Intracellular experiment:

[0041] The GFP fusion expression plasmid of KCTD17 was constructed, and the expression of the GFP fusion protein KCTD17 was observed in HEK293T cells, forming droplet-like structures.

[0042] I. Main reagents, instruments and sources:

[0043] PCR enzyme: Max DNA Polymerase (Takara, catalog number: R045A);

[0044] NEB restriction enzymes: EcoRI-HF and BamHI-HF;

[0045] Cell line: HEK293T cells;

[0046] Gel extraction kit: Omega Gel Extraction Kit;

[0047] Plasmid: pEGFP-C1 was purchased from Clontech; the plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Weizhen Biology Co., Ltd. (CH873972);

[0048] PEI linear transfection reagent PEI MAX (24765-1) was purchased from Polysciences;

[0049] Plasmid extraction kit: Omega plasmid mini kit;

[0050] Pure water instrument: Millipore 5UV Water Purification System;

[0051] Pipette: Eppendorf;

[0052] PCR instrument: Bio-Rad T100.

[0053] Using the above reagents and consumables, the KCTD17 gene can be subcloned into the pEGFP-C1 plasmid according to the following steps. In this example, the KCTD17 gene was amplified by PCR and inserted into pEGFP-C1 to obtain a eukaryotic expression plasmid with correct expression.

[0054] II. Experimental method

[0055] 1. Primer design:

[0056] The KCTD17 gene was constructed into the plasmid pEGFP-C1 with a GFP tag. PCR amplification primers: the upstream primer of the KCTD17 gene, KCTD17-F (SEQ ID NO:5), and the downstream primer of the KCTD17 gene, KCTD17-R (SEQ ID NO:6). The primer sequences are as follows. The upstream primer includes the homologous arm sequence before the insertion position on the target vector pEGFP-C1 and the specific primer at the 5' end of the KCTD17 gene. The downstream primer includes the reverse complementary sequence of the homologous arm after the insertion position on pEGFP-C1 and the specific primer at the 3' end of the KCTD17 gene:

[0057] Primer Sequence KCTD17-F 5’-CTCGAGCTCAAGCTTCGAATTCTATGCAGACGCCGCGGCCG-3’ KCTD17-R 5’-CAGTTATCTAGATCCGGTGGATCCGATGGGAACCCCAAGTCCCTGGAGGTG-3’

[0058] The primers were synthesized by Genewiz.

[0059] 2. PCR reaction:

[0060] The PCR system is shown as follows:

[0061]

[0062] Mix the PCR reaction system evenly, and start PCR amplification after a short transient centrifugation;

[0063] The PCR reaction program is shown as follows:

[0064] Pre-denaturation: 94℃, 2min. Denaturation: 98℃, 10sec. Annealing: 63℃, 10sec. Extension: 72℃, 20s(1Kb / 10s) Number of cycles: 35 cycles Extension: 72℃, 2min Storage condition: 10℃

[0065] 3. After PCR is completed, perform gel electrophoresis and cut and recover the gene product.

[0066] 4. Linearization of the pEGFP-C1 vector: Digest pEGFP-C1 with EcoRI and BamHI to obtain a linearized vector fragment. The reaction system is as follows:

[0067]

[0068]

[0069] Incubate at 37°C for 2 hours for enzymatic digestion, perform agarose gel electrophoresis, and cut and recover the large vector fragment.

[0070] 5. Seamless ligation

[0071] The reaction system is shown as follows:

[0072] Reaction component Volume Large fragment of pEGFP-C1 plasmid 3μl KCTD17 gene fragment 2μl NEBuilder HiFi DNA Assembly Master Mix 10μl <![CDATA[ddH 2 O]]> 5μl Total volume 20μl

[0073] After mixing the reaction system, place it at 50°C for 5 - 15 minutes.

[0074] 6. Transformation: Take 10 μl of the homologous recombination product and transform it into competent Escherichia coli cells. After recovery, spread it on an LB solid culture plate resistant to kanamycin (Kan) for culture.

[0075] 7. Pick a single colony and add it to an LB medium containing Kan antibiotic. Incubate the bacteria at 37 °C overnight with shaking.

[0076] 8. Extract the recombinant plasmid pEGFP-C1-KCTD17. After preliminary screening by electrophoresis (since the recombinant plasmid with successful recombination has a larger molecular weight and slower migration speed compared to the template empty plasmid), sequence it and screen for recombinants with correct sequencing results.

[0077] 9. Take the correct pEGFP-C1-KCTD17 plasmid and transfect the cultured HEK293 cells according to the transfection method of the PEI transfection reagent to obtain HEK293 cells expressing the pEGFP-C1-KCTD17 gene.

[0078] 10. Fix and prepare slides of the cells transfected for 24 h using a fixative to obtain cell smears. The fixative is a fixative such as acetone, formaldehyde, paraformaldehyde, methanol, ethanol, etc. Observe the results directly under a microscope and take pictures of the fixed cell smears.

[0079] The experimental results are as Figure 1 shown in the upper figure above. After the KCTD17 gene was fused with GFP, it led to a significant change in the morphology of the GFP protein. The green fluorescent protein in almost all cells aggregated into clusters and showed a droplet-like structure.

[0080] In vitro experiment:

[0081] In this invention, a prokaryotic expression plasmid of KCTD17 was constructed, and the KCTD17 protein was obtained by expression in Escherichia coli, and it was observed that the fusion protein KCTD17 could form a droplet-like structure in a test tube.

[0082] I. Construction of the prokaryotic expression plasmid of KCTD17

[0083] 1. Main reagents, instruments and sources:

[0084] PCR enzyme: Max DNA Polymerase (Takara, catalog number: R045A);

[0085] NEB restriction endonucleases: EcoRI-HF and BamHI-HF;

[0086] Cell line: HEK293T cells;

[0087] Gel extraction kit: Omega Gel Extraction Kit;

[0088] Plasmids: pET-22b was purchased from sigma, 69744; pMal-c2x was purchased from Addgene, 75286; pEGFP-C1 was purchased from Clontech, and the plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Shandong Weizhen Biotechnology Co., Ltd. (CH873972);

[0089] Plasmid extraction kit: Omega plasmid mini kit;

[0090] Pure water instrument: Millipore 5UV Water Purification System;

[0091] Pipettor: Eppendorf;

[0092] Ni-NTA agarose: QIAGEN 30210

[0093] PCR instrument: Bio-Rad T100.

[0094] Using the above reagents and consumables, the KCTD17 gene can be subcloned into the pET22b plasmid according to the following steps.

[0095] 2. Experimental method

[0096] In this example, the KCTD17 gene was amplified by PCR, other elements such as MBP and GFP tags were added, and inserted into pET22b to obtain the prokaryotic expression plasmid of KCTD17 as Figure 2 shown in A below, and transformed into BL21(DE3) competent cells to obtain a prokaryotic expression strain with high expression of KCTD17.

[0097] 1) Primer design:

[0098] To construct the KCTD17 gene, MBP gene (from pMal-c2x), and GFP tag (from pEGFP-C1 plasmid) onto the prokaryotic expression plasmid pET22b, a set of primers for gene assembly was designed. The primers included the upstream primer of the MBP gene, MBP-Nde1-F (SEQ ID NO:7), the downstream primer of the MBP gene, MBP-R (SEQ ID NO:8), the upstream primer of the EGFP gene, EGFP-F (SEQ ID NO:9), the downstream primer of the EGFP gene, EGFP-R (SEQ ID NO:10), the upstream primer of the KCTD17 gene, pET22b-KCTD17-F (SEQ ID NO:11), and the downstream primer of the KCTD17 gene, pET22b-KCTD17-R (SEQ ID NO:12). The upstream primers included the fragment sequences overlapping with the upstream vector or gene and the specific primer sequences of the 5' end of the gene and elements, and the downstream primers included the fragment sequences overlapping with the downstream vector or gene and the reverse complementary sequences of the specific primer sequences of the 3' end of the gene and elements. The primers were synthesized by Genewiz.

[0099]

[0100]

[0101] 2) PCR amplification and recovery:

[0102] Select the above set of primers and the corresponding DNA templates, and use the PrimeSTAR (Takara, R045A) system to amplify the MBP, EGFP, and KCTD17 genes. After PCR is completed, electrophoresis is performed and the gene products are recovered by cutting the gel.

[0103] 3) Linearization of the pET22b vector:

[0104] Digest pET22b with NdeI and XhoI to obtain a linearized vector fragment. The reaction system is as follows:

[0105] Reaction component Volume pET22b plasmid 1μg rCutSmart buffer 5μl NEB restriction endonuclease NdeI 1μl NEB restriction endonuclease XhoI 1μl <![CDATA[ddH 2 O]]> Make up to 50μl

[0106] After mixing the reaction system, incubate it in a 37°C incubator for 2 h, perform electrophoresis, and recover the large vector fragment by cutting the gel.

[0107] 4) Seamless ligation:

[0108] Use the NEBuilder HiFi DNA Assembly Master Mix to perform seamless ligation of multiple fragments with the recovered MBP, EGFP tag, KCTD17 antigen gene, and linearized pET22b. The reaction system is as follows:

[0109] Reaction component Volume Large fragment of pET22b plasmid 3μl MBP fragment 1μl KCTD17 gene fragment 1μl EGFP fragment 1μl NEBuilder HiFi DNA Assembly Master Mix 10μl <![CDATA[ddH 2 O]]> 4μl Total volume 20μl

[0110] After the reaction system is mixed evenly, place it at 50 °C for reaction for 5 - 15 minutes.

[0111] 5) Transformation:

[0112] Take 10 μl of the homologous recombination product to transform Escherichia coli competent cells. After recovery, coat it on an LB solid culture plate resistant to ampicillin (Amp) for culture.

[0113] 6) Pick a single colony and add it to an LB medium containing Amp antibiotic, and culture it with shaking overnight at 37 °C.

[0114] 7) Extract the recombinant plasmid pET22b - MBP - EGFP - KCTD17. After preliminary screening by electrophoresis (the recombinant plasmid with successful recombination will have a slower migration speed than the template empty plasmid due to the increase in molecular weight), sequence it and screen the recombinants with correct sequencing results.

[0115] II. Prokaryotic expression and purification of KCTD17

[0116] After preliminary small - scale expression exploration, express the KCTD17 protein in large quantities (0.5 mM IPTG, 19 °C, 220 rpm, overnight induction), resuspend it with buffer (25 mM Tris, 0.5 M NaCl, 5% glycerol, 0.05% DDM, 20 mM Imidazole, pH 8.0), disrupt it under high pressure for 5 min, and centrifuge at high speed (15000 rpm, 40 min, 4 °C). The following steps are used for separate purification.

[0117] (1) His - column affinity purification

[0118] Washing Buffer: 25 mM Tris, 150 mM NaCl, 5% glycerol, 0.05% DDM, pH 8.0;

[0119] Imidazole gradient eluent: 25 mM Tris, 150 mM NaCl, 5% glycerol, 0.05% DDM, pH 8.0 containing 20 mM, 50 mM, 100 mM, 250 mM, 500 mM imidazole at different concentrations (5 - 10 column volumes).

[0120] Steps:

[0121] 1) Equilibrate with Buffer 1 for 2 - 5 column bed volumes at a flow rate of 2 mL / min;

[0122] (2) Filter the cell lysate (50 mM PBS, pH 7.4, 0.5 M NaCl) through a 0.45 μm filter membrane, load the sample, and the flow rate is 1 mL / min;

[0123] (3) Wash with Washing Buffer for 2 - 5 column volumes at a flow rate of 2 mL / min;

[0124] (4) Perform stepwise elution with gradient eluents containing 20, 50, 100, 250, and 500 mM Imidazole at a flow rate of 2 mL / min. Collect the elution peaks at each stage and detect the molecular weight and purity of the fusion protein by SDS - PAGE;

[0125] (2) Purification by molecular sieve

[0126] Molecular sieve Buffer: 25 mM Tris, 150 M NaCl, pH 8.0;

[0127] Column: SD200 increase.

[0128] Steps:

[0129] (1) Before loading, the sample should be filtered through a 0.2 μm pore size filter membrane or centrifuged at 10000 g for 5 min to remove residues;

[0130] (2) Concentrate the protein sample to 0.5 - 1.0 ml and inject the sample into the protein purifier using a pipette;

[0131] (3) Run the molecular sieve program;

[0132] (4) According to the elution peak diagram of the molecular sieve, collect different fractions and concentrate to obtain the target protein.

[0133] After the above purification steps, the recombinantly expressed KCTD17 protein was obtained. As shown in Figure 2 B below, using HRV3C

[0134] protease can cleave the MBP tag.

[0135] III. In vitro phase transition experiment of KCTD17 protein

[0136] Test protein phase separation in the presence and absence of a crowding agent (10% dextran). The protein dilution buffer is 25 mM Tris - HCl 7.4, 150 mM KCl, 2.5% glycerol, and 0.5 mM DTT. The protein is diluted to 1 - 20 μM respectively, and the total solution volume is 20 μL. Add HRV3C protease to the diluted protein at a ratio of 1:50. Add the sample to a 96 - well microplate and take images after all droplets have settled to the bottom of the plate. As shown inFigure 2 As shown in B, after digestion with HRV3C protease, the KCTD17 fusion protein dissociates and releases the MBP tag (inhibiting protein phase transition), as Figure 2 shown in C, as the protein concentration increases, the KCTD17 protein is more likely to form green droplet-like structures in solution. The droplets formed by the KCTD17 protein can fuse in vitro, indicating that the protein is in a fluid state and undergoes protein phase transition. Therefore, in vitro experiments show that KCTD17 can undergo protein phase transition.

[0137] Based on the performance of the KCTD17 protein in in vivo and in vitro experiments, it is confirmed that the present invention has found a new phase transition protein.

[0138] Example 2 KCTD17 can promote the phase transition of autoimmunity antigen proteins

[0139] Currently, the CBA method for detecting nerve autoantibodies mainly uses expression plasmids constructed with natural full-length genes, which are transfected into 293 and hep2 cells to prepare detection materials. For example, the Titin antigen (MGT30) or the GAD65 antigen gene is constructed into a eukaryotic expression vector for the detection of autoantibodies. The expression of Titin / GAD65 antigen in the cytoplasm is diffusely distributed without a specific morphology. In the present invention, the antigens Titin / GAD65 are added to the N-terminus of the KCTD17 gene of the pEGFP-C1-KCTD17 recombinant plasmid to construct a new recombinant expression plasmid, which expresses the fusion protein of Titin / GAD65 and KCTD17. After transfection of cells, it is found that introducing the phase transition protein KCTD17 can change the original diffuse distribution of the Titin / GAD65 protein, but form a certain aggregation state. It shows that KCTD17 can promote the protein phase transition of the Titin / GAD65 fusion protein and form a denser aggregate.

[0140] I. Main reagents, instruments and sources:

[0141] PCR enzyme: Max DNA Polymerase (Takara, catalog number: R045A);

[0142] NEB restriction endonucleases: EcoRI-HF and BamHI-HF

[0143] Cell line: HEK293T cells;

[0144] Gel extraction kit: Omega Gel Extraction Kit;

[0145] Plasmids: pEGFP-C1-KCTD17 (obtained in Example 1); the plasmid pEGFP-N1-Titin / GAD65 containing the Titin gene and the GAD65 gene was synthesized by General Biosystems (the Titin gene and the GAD65 gene were subcloned into pEGFP-N1 respectively, and the insertion sites were Nhe1 and Xho1).

[0146] Plasmid extraction kit: Omega plasmid mini kit;

[0147] Pure water instrument: Millipore 5UV Water Purification System;

[0148] Pipette: eppendorf;

[0149] PCR instrument: Bio-Rad T100;

[0150] Using the above reagents and consumables, the Titin / GAD65 gene can be subcloned into the pEGFP-C1-KCTD17 plasmid according to the following steps.

[0151] II. Experimental methods

[0152] 1. Construction of pEGFP-C1-Titin / GAD65-KCTD17 plasmid:

[0153] The Titin gene and the GAD65 gene were inserted into pEGFP-C1-KCTD17 by a two-step method.

[0154] 1) Primer design:

[0155] The Titin / GAD65 gene was constructed into pEGFP-C1-KCTD17.

[0156] The first-round PCR amplification primers Titin-F (SEQ ID NO: 13), Titin-R (SEQ ID NO: 14), GAD65-F (SEQ ID NO: 15), and GAD65-R (SEQ ID NO: 14) were designed.

[0157] The primer sequences are as follows in the table. The upstream primers include the homologous arm sequence before the insertion position on the target vector pEGFP-C1-KCTD17 and the Titin / GAD65 gene-specific upstream primer, and the downstream primers include the reverse complementary sequence of the homologous arm after the insertion position on pEGFP-C1-KCTD17 and the Titin / GAD65 gene vector-specific downstream primer.

[0158]

[0159] The primers were synthesized by Genewiz, Inc.

[0160] 2) First-round PCR reaction

[0161] First, dilute the primers for the first-round PCR amplification to a final concentration of 10 μM according to the concentration;

[0162] The PCR system is as follows:

[0163]

[0164] Mix the PCR reaction system evenly, and start PCR amplification after brief transient centrifugation;

[0165] The PCR reaction program is as follows:

[0166]

[0167]

[0168] After PCR is completed, perform gel electrophoresis and cut and recover the gene product.

[0169] Recover the PCR product according to the instructions of the gel recovery kit. The concentration of the recovered product from the first-round PCR is approximately 50 - 100 ng / μl.

[0170] 3) Second-round PCR reaction

[0171] The reaction system is as follows:

[0172] Reagent Volume (μl) KOD buffer 5 2mM dNTPs 5 <![CDATA[25mM MgSO 4 > 2 First-round PCR recovery product 16 50ng / μl pEGFP-C1-KCTD17 plasmid 1 KOD DNA Polymerase 1 <![CDATA[ddH 2 O]]> 20 Total volume 50

[0173] Mix the PCR reaction system evenly, and start PCR amplification after brief transient centrifugation; The PCR reaction program is as follows:

[0174] Pre-denaturation: 94℃, 2min. Denaturation: 98℃, 10sec. Annealing: 55℃, 20sec. Extension: 68℃, 7min 30s Number of cycles 13 cycles Extension: 68℃, 10min Storage condition: 10℃

[0175] 4) Dpn1 digestion:

[0176] After the second-round PCR reaction is completed, add 1 μl of Dpn1 enzyme to the reaction tube and react at 37 °C for 1 - 2 h to completely remove the template plasmid in the PCR reaction system.

[0177] 5) Transformation:

[0178] Take 10 μl of the PCR product after Dpn1 digestion and transform Escherichia coli competent cells. After recovery, spread it on a solid LB culture plate with Kan resistance for culture.

[0179] 6) Pick a single colony, add it to an LB medium containing Kan antibiotic, and culture it with shaking at 37 °C overnight.

[0180] 7) Recombinant plasmids were extracted. After preliminary screening by electrophoresis and confirmation, sequencing was performed, and the correctly sequenced recombinant plasmid pEGFP-C1-Titin / GAD65-KCTD17 was selected.

[0181] 2. Experiment on observing protein phase transition in cells

[0182] The correct pEGFP-C1-Titin / GAD65-KCTD17 plasmid was taken and transfected into cultured HEK293 cells according to the transfection method of PEI transfection reagent, and HEK293 cells expressing the pEGFP-C1-Titin / GAD65-KCTD17 gene were obtained.

[0183] The cells transfected for 24 h were fixed and made into slides using a fixative. The prepared cell slides were obtained. The fixative was a fixative such as acetone, formaldehyde, paraformaldehyde, methanol, ethanol, etc. The fixed cell slides were directly observed under a microscope and photographed.

[0184] The experimental results are as Figure 3 shown. After the KCTD17 gene was fused with the Titin / GAD65 antigen gene, a significant change occurred in the morphology of the Titin / GAD65 protein, and the Titin / GAD65 protein in almost all cells aggregated into clusters.

[0185] Example 3 Determination of the KCTD17 IDR sequence

[0186] Phase transition proteins usually have an important intrinsically disordered region (IDR), and this intrinsically disordered region usually determines the phase transition of the protein.

[0187] In the present invention, a series of plasmids of the C-terminal protein of KCTD17 were constructed, and the most core IDR region (209 - 262 aa) in KCTD17 was screened through intracellular experiments and named KCTD17 IDR.

[0188] It was determined through the following experiment. As shown in the lower figure below, the separate KCTD17 IDR (209 - 262 aa) was able to make the coupled GFP protein exhibit a droplet-like structure. Figure 1 I. Main reagents, instruments and sources:

[0189] PCR enzyme:

[0190] Max DNA Polymerase (Takara, catalog number: R045A);

[0191] NEB restriction enzymes: EcoRI-HF and BamHI-HF;

[0192] Cell line: HEK293T cells;

[0193] Gel extraction kit: Omega Gel Extraction Kit;

[0194] Plasmid: pEGFP-C1 (purchased from Clontech); The plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Weizhen Biological Co., Ltd. in Shandong (CH873972);

[0195] Plasmid extraction kit: Omega plasmid mini kit;

[0196] Pure water instrument: Millipore 5UV Water Purification System;

[0197] Pipette: Eppendorf;

[0198] PCR instrument: Bio-Rad T100.

[0199] Using the above reagents and consumables, the KCTD17 gene can be subcloned into the pEGFP-C1 plasmid according to the following steps. In this example, the KCTD17 IDR gene was amplified by PCR and inserted into pEGFP-C1 to obtain a eukaryotic expression plasmid with correct expression.

[0200] II. Construction of pEGFP-C1-KCTD17 IDR plasmid

[0201] 1. Primer design:

[0202] The KCTD17 IDR gene was constructed onto the plasmid pEGFP-C1 with a GFP tag.

[0203] PCR amplification primers: The upstream primer of the KCTD17 IDR gene, KCTD17-F1 (SEQ ID NO:16), and the downstream primer of the KCTD17 IDR gene, KCTD17-R1 (SEQ ID NO:17). The primer sequences are shown in the following table. The upstream primer includes the homologous arm sequence before the insertion position on the target vector pEGFP-C1 and the specific primer at the 5' end of the KCTD17 IDR gene. The downstream primer includes the reverse complementary sequence of the homologous arm after the insertion position on pEGFP-C1 and the specific primer at the 3' end of the KCTD17 IDR gene:

[0204]

[0205] The primers were synthesized by Genewiz.

[0206] 2. PCR Reaction:

[0207] The PCR system is as follows:

[0208]

[0209] Mix the PCR reaction system evenly, and start PCR amplification after brief transient centrifugation;

[0210] The PCR reaction program is as follows:

[0211] Pre-denaturation: 94℃, 2min. Denaturation: 98℃, 10sec. Annealing: 63℃, 10sec. Extension: 72℃, 20s(1Kb / 10s) Number of cycles: 35 cycles Extension: 72℃, 2min Storage condition: 10℃

[0212] 3. After PCR is completed, perform gel electrophoresis and cut the gel to recover the gene product.

[0213] 4. Linearization of pEGFP-C1 vector: Digest pEGFP-C1 with EcoRI and BamHI double enzymes to obtain a linearized vector fragment. The reaction system is as follows:

[0214] Reaction component Volume pEGFP-C1 plasmid Total amount 2μg rcutsmart buffer 5μl NEB restriction endonuclease EcoRI-HF 1μl NEB restriction endonuclease BamHI-HF 1μl <![CDATA[ddH 2 O]]> Up to 50μl Total volume 50μl

[0215] Incubate with enzymes at 37°C for 2 hours, perform agarose gel electrophoresis, and cut the gel to recover the large vector fragment.

[0216] 5. Seamless Ligation

[0217] The reaction system is as follows:

[0218] Reaction component Volume Large fragment of pEGFP-C1 plasmid 3μl KCTD17IDR gene fragment 2μl NEBuilder HiFi DNA Assembly Master Mix 10μl ddH2O 5μl Total volume 20μl

[0219] After mixing the reaction system, place it at 50°C for 5 - 15 minutes.

[0220] 6. Transformation: Take 10 μl of the homologous recombination product to transform Escherichia coli competent cells, and spread them on a solid LB culture plate with Kan resistance after recovery for culture.

[0221] 7. Pick single colonies, add them to an LB medium containing Kan antibiotic, and culture them with shaking overnight at 37°C.

[0222] 8. Extract the recombinant plasmid pEGFP-C1-KCTD17 IDR, send it for sequencing, and screen the recombinants with correct sequencing results.

[0223] III. Intracellular Experiments

[0224] Take the correct pEGFP-C1-KCTD17 IDR plasmid and transfect the cultured HEK293 cells according to the transfection method of the PEI transfection reagent to obtain HEK293 cells expressing the pEGFP-C1-KCTD17 IDR gene.

[0225] The cells transfected for 24 h were fixed and made into cell smears using a fixative. The fixative is a fixative such as acetone, formaldehyde, paraformaldehyde, methanol, ethanol, etc. The fixed cell smears were directly observed under a microscope and photographed.

[0226] The experimental results are as Figure 1 shown in the lower middle figure below. After the separate KCTD17 IDR gene was fused with GFP, it led to a significant change in the morphology of the GFP protein. The green fluorescent protein in almost all cells aggregated into clusters, indicating that the KCTD17 IDR sequence indeed has a certain phase transition ability.

[0227] Example 4 New optoDroplet tool: Design and construction of optoDroplet-KCTD17 IDR expression plasmid

[0228] Although KCTD17 IDR can promote phase transition of GFP in vivo, KCTD17 IDR cannot cause a similar obvious phase transition phenomenon of the fusion protein brought by full-length KCTD17 to Titin / GAD65-GFP, indicating that the phase transition ability of KCTD17IDR is weakened compared with full-length KCTD17. Therefore, in the present invention, KCTD17 IDR plus the Cry2 element was designed to make its phase transition ability similar to that of full-length KCTD17, and the phase transition process is light-regulated. Only when irradiated with blue light, Cry2 can function to make the fusion protein undergo an obvious protein phase transition and aggregation. Therefore, the present invention designed an optoDroplet-KCTD17 IDR expression system.

[0229] The original optoDroplets tool uses the pHR-FusN-mCherry-Cry2 plasmid, and the plasmid size is 11954 bp, which contains some sequence characteristics of viral plasmids, such as non-essential elements such as LTR, RRE, and cPPT (as Figure 4 shown in A in

[0230] order to simplify the optoDroplets plasmid, the inventors introduced an antigen gene and two other essential phase transition elements: KCTD17 IDR and Cry2 PHR on the basis of the pEGFP-N1 vector. mCherry and Cry2 were obtained by PCR amplification from pHR-FusN-mCherry-Cry2. The two phase transition elements KCTD17 IDR and Cry2 PHR were in series, and an antigen gene was introduced at the N-terminus. A linker was added between the antigen gene and the phase transition element, and finally a new and simple optoDroplets expression plasmid: optoDroplet-KCTD17 IDR was obtained (as Figure 4As shown in B (SEQ ID NO: 26), the plasmid size is only 6504 bp. In addition, the present invention retains multiple cloning sites, which facilitates the insertion of various antigen genes into the expression plasmid.

[0231] I. Main reagents, instruments and sources:

[0232] PCR enzyme: Max DNA Polymerase (Takara, catalog number: R045A);

[0233] NEB restriction endonucleases: NotI-HF and BamHI-HF;

[0234] Cell line: HEK293T cells;

[0235] Gel extraction kit: Omega Gel Extraction Kit;

[0236] Plasmids: pEGFP-N1 was purchased from Clontech; the plasmid pEnter KCTD17 containing the KCTD17 gene was purchased from Shandong Weizhen Biotechnology Co., Ltd. (CH873972); pHR-FusN-mCherry-Cry2 was purchased from Addgene 101221;

[0237] Plasmid extraction kit: Omega plasmid mini kit;

[0238] Water purification instrument: Millipore 5UV Water Purification System;

[0239] Pipettor: Eppendorf;

[0240] PCR instrument: Bio-Rad T100.

[0241] Using the above reagents and consumables, the KCTD17 IDR, Cry2 and antigen genes can be subcloned into the pEGFP-N1 plasmid according to the following steps.

[0242] Experimental method

[0243] 1. Primer design:

[0244] The KCTD17 IDR, mCherry (from pHR-FusN-mCherry-Cry2), and Cry2 (pHR-FusN-mCherry-Cry2) were subcloned into the pEGFP-N1 plasmid. PCR amplification primers: the upstream primer for the KCTD17 IDR gene, KCTD17IDR-F (SEQ ID NO:18); the downstream primer for the EGFP gene, KCTD17 IDR-R (SEQ ID NO:19); mCherry and Cry2 genes were amplified from pHR-FusN-mCherry-Cry2, with the upstream primer mCherry-Cry2-F (SEQ ID NO:20) and the downstream primer mCherry-Cry2-R (SEQ ID NO:21). The primer sequences are as follows. The upstream primers include the homologous arm sequence before the insertion position on the destination vector pEGFP-N1 and the specific primer at the 5' end of the KCTD17 gene, and the downstream primers include the reverse complementary sequence of the homologous arm after the insertion position on pEGFP-N1 and the specific primer at the 3' end of the KCTD17 gene:

[0245]

[0246] The primers were synthesized by GenScript Corporation.

[0247] 2. PCR amplification and recovery: Select a set of primers and the corresponding DNA templates, and use the PrimeSTAR (Takara, R045A) system to amplify the MBP, EGFP, and KCTD17 genes.

[0248] 3. After PCR is completed, electrophorese on a gel and cut the gel to recover the gene products KCTD17 IDR and mCherry-Cry2.

[0249] 4. Vector linearization: Digest pEGFP-N1 with NheI and NotI to obtain a linearized vector fragment. The reaction system is as follows:

[0250] Reaction component Volume pEGFP-N1 plasmid Total amount 2ug rcutsmart buffer 5μl BamHI-HF 1μl NotI-HF 1 μl ddH2O Up to 50 μl Total volume 50 μl

[0251] Incubate at 37°C for 2 hours for enzymatic digestion, perform agarose gel electrophoresis, and cut the gel to recover the large vector fragment.

[0252] 5. Seamless ligation

[0253] The reaction system is as follows:

[0254]

[0255]

[0256] After the reaction system was mixed evenly, it was placed at 50 °C for reaction for 30 minutes.

[0257] 6. Transformation: Take 10 μl of the homologous recombination product and transform Escherichia coli competent cells. After recovery, spread it on an LB solid culture plate with Kan resistance for culture.

[0258] 7. Pick a single colony and add it to an LB medium containing Kan antibiotic, and culture it with shaking at 37 °C overnight.

[0259] 8. Extract the recombinant plasmid optoDroplet-KCTD17 IDR, sequence it, and screen the recombinant with correct sequencing results.

[0260] Example 5 Construction of optoDroplet-KCTD17 IDR-Titin Vector and Expression of Titin Protein

[0261] Anti-striated muscle antibody is a characteristic of systemic myasthenia gravis, and Titin is the main autoantigen recognized by anti-striated muscle antibody. Although the commonly used detection method for Titin is ELISA, the CBA method is also widely used at present. The key material for CBA is to obtain a plasmid that can highly express the antigen. Therefore, the present invention designed a photoinduced phase transition expression plasmid for Titin.

[0262] I. Main Reagents, Instruments and Sources:

[0263] PCR Enzyme: Max DNA Polymerase (Takara, catalog number: R045A);

[0264] NEB Restriction Endonucleases: NheI-HF and XhoI-HF;

[0265] Cell Line: HEK293T cells;

[0266] Gel Extraction Kit: Omega Gel Extraction Kit;

[0267] Plasmids: optoDroplet-KCTD17 IDR (from Example 4); the plasmid pEGFP-N1-Titin containing the Titin gene was synthesized by General Biosystems (subcloning the Titin gene into pEGFP-N1);

[0268] Plasmid Extraction Kit: Omega plasmid mini kit;

[0269] Water Purifier: Millipore 5UV Water Purification System;

[0270] Pipette: Eppendorf;

[0271] PCR instrument: Bio-Rad T100.

[0272] Using the above reagents and consumables, the Titin and antigen genes can be subcloned into the optoDroplet-KCTD17IDR plasmid according to the following steps.

[0273] II. Construction of the optoDroplet-KCTD17 IDR-Titin vector

[0274] 1) Primer design:

[0275] The primers include the upstream primer Titin-NheI-F (SEQ ID NO: 22) of the Titin gene and the downstream primer Titin-XhoI-R (SEQ ID NO: 23) of the Titin gene.

[0276] Primer Sequence Titin-NheI-F 5’-GTGAACCGTCAGATCCGCTAGCATGAGGTGCGAGGAGGGCAAAGATAATT-3’ Titin-XhoI-R 5’-CAGAATTCGAAGCTTGAGCTCGAGAGCCGCCGTCATTCTTGGGAG-3’

[0277] 2) PCR amplification and recovery:

[0278] Select a set of primers and the corresponding DNA template (pEGFP-N1-Titin), use the PrimeSTAR (Takara, R045A) system to amplify the Titin gene. After PCR is completed, electrophorese and cut the gel to recover the gene product.

[0279] 3) Vector linearization:

[0280] Digest optoDroplet-KCTD17 IDR with NheI and XhoI to obtain a linearized vector fragment. The reaction system is as follows:

[0281] Reaction components Volume optoDroplet-KCTD17 IDR plasmid Total 2 μg rcutsmart buffer 5 μl NheI-HF 1 μl XhoI-HF 1 μl <![CDATA[ddH 2 O]]> Up to 50 μl Total volume 50 μl

[0282] Incubate at 37°C for 2 hours for enzymatic digestion, perform Agarose gel electrophoresis, cut the gel to recover the large vector fragment, and recover the DNA according to the instructions of the gel recovery kit.

[0283] 4) Seamless ligation:

[0284] Use NEBuilder HiFiDNA Assembly Master Mix to perform seamless ligation of the recovered Titin gene and the linearized optoDroplet-KCTD17 IDR. The reaction system is as follows:

[0285] Reaction components Volume optoDroplet-KCTD17 IDR plasmid backbone 3 μl Titin gene 1 μl NEBuilder HiFi DNA Assembly Master Mix 10 μl <![CDATA[ddH 2 O]]> 4 μl Total volume 20 μl

[0286] 5) Recombinant clone screening:

[0287] Take 5 μl of the ligation product to transform Escherichia coli competent cells. After recovery, spread them on an LB solid culture plate with Kan resistance for culture. Pick single colonies, add them to an LB medium containing Kan antibiotic, shake culture overnight at 37 °C, extract plasmids, and sequence to screen the correct recombinant optoDroplet-KCTD17 IDR-Titin.

[0288] III. Expression of optoDroplet-KCTD17 IDR-Titin

[0289] 1. Cell transfection:

[0290] Digest the cultured Hep2 cells with trypsin, terminate digestion with DMEM complete medium containing 10% serum. Aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, discard the supernatant, add DMEM complete medium containing 10% serum, and gently pipette to mix evenly to prepare a cell suspension.

[0291] Place the sterilized glass slides in a cell culture plate, treat them with polylysine (PDL), rinse 3 times and then air-dry. Add the prepared cell suspension to the cell culture plate, gently mix, and place it in an incubator at 37 °C, 5% CO 2 overnight culture. Observe the cells the next day, and transfection can be carried out when the density reaches 40 - 50%.

[0292] Mix the prepared optoDroplet-KCTD17 IDR-Titin plasmid with the transfection reagent PEI at a mass-volume ratio of 1:3, vortex, and let stand for 10 min, then transfect into the prepared cells respectively, and culture at 37 °C, 5% CO 2 for 48 h.

[0293] Blue light excitation of optoDroplet-KCTD17 IDR-Titin transfected cells: Place the well plate or culture dish on the confocol, and excite with 405 nm blue light for 5 - 120 S;

[0294] After the cells were excited by blue light, the fluorescence of Titin in optoDroplet-KCTD17 IDR-Titin and the conventional plasmid pEGFP-N1-Titin was observed under the red channel. Before the blue light excitation, the Titin fusion protein expressed by optoDroplet-KCTD17 IDR-Titin was consistent with that of pEGFP-N1-Titin, showing a diffuse cytoplasmic distribution. However, after 30 s of blue light excitation, obvious aggregated red fluorescence signals appeared in the cells transfected with optoDroplet-KCTD17 IDR-Titin, and larger droplet-like forms could be seen after 120 s of excitation. Compared with before the blue light excitation, the Titin fusion protein aggregated due to phase transition after excitation, and its fluorescence intensity increased by 9 times.

[0295] Example 6 Vector construction and protein expression of optoDroplet-K10C-GAD65

[0296] Autoimmune encephalitis (AE) is an immune-mediated neurological disease mediated by autoantibodies against neurons, which can present symptoms such as cognitive dysfunction, behavioral abnormalities, epileptic seizures, mental disorders, involuntary movements, and auto-neurological dysfunction. Detection of GAD65 antibody is of great significance for the diagnosis of autoimmune encephalitis. Currently, the commonly used detection methods for GAD65 are ELISA and CBA methods. The key material for CBA is a plasmid that can highly express the antigen. Therefore, the present invention designed an optoDroplet-KCTD17 IDR-GAD65 expression plasmid.

[0297] I. Vector construction of optoDroplet-KCTD17 IDR-GAD65

[0298] 1) Primer design:

[0299] The primers include the upstream primer GAD65-Nhe1-F (SEQ ID NO:24) of the GAD65 gene and the downstream primer GAD65-Xho1-R (SEQ ID NO:25) of the GAD65 gene.

[0300]

[0301] 2) PCR amplification and recovery:

[0302] Select the GAD65-Nhe1-F and GAD65-Xho1-R primers and the corresponding DNA template (the plasmid pEGFP-N1-GAD65 containing the GAD65 gene was synthesized by General Biosystems (the GAD65 gene was subcloned into pEGFP-N1)), and use the PrimeSTAR (Takara, R045A) system to amplify the GAD65 gene. After PCR is completed, electrophorese and cut the gel to recover the gene product.

[0303] 3) Double digestion of the vector:

[0304] Digest optoDroplet-KCTD17 IDR with NheI and XhoI to obtain the large linearized vector fragment, as in Example 5; incubate with enzymes at 37 °C for 2 hours, perform Agarose gel electrophoresis, cut the gel to recover the large vector fragment, and recover the DNA according to the instructions of the gel recovery kit.

[0305] 4) Seamless ligation:

[0306] Use NEBuilder to perform seamless ligation of multiple fragments on the recovered GAD65 gene and the large linearized optoDroplet-KCTD17 IDR fragment. The reaction system is as follows:

[0307] Reaction components Volume optoDroplet-KCTD17 IDR plasmid backbone 3 μl GAD65 gene 1 μl NEBuilder HiFi DNA Assembly Master Mix 10 μl <![CDATA[ddH 2 O]]> 6 μl Total volume 20 μl

[0308] 5) Screening of recombinant clones:

[0309] Take 5 μl of the ligation product to transform competent Escherichia coli cells. After recovery, spread them on a Kan-resistant LB solid culture plate for culture. Pick a single colony, add it to an LB medium containing Kan antibiotic, and culture it with shaking at 37 °C overnight. Extract the plasmid and sequence it to screen the correct recombinant optoDroplet-KCTD17 IDR-GAD65.

[0310] II. Expression of optoDroplet-KCTD17 IDR-GAD65

[0311] 1. Cell transfection:

[0312] Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, pour out the supernatant, add DMEM complete medium containing 10% serum, and gently pipette and mix to make a cell suspension.

[0313] Place the sterilized glass slides in a cell culture plate, treat them with polylysine (PDL), rinse three times and then air dry. Add the prepared cell suspension to the cell culture plate, gently mix, and place it in an incubator at 37°C with 5% CO 2 Incubate overnight. Observe the cells the next day. Transfection can be carried out when the density reaches 40 - 50%.

[0314] Mix the prepared optoDroplet-K10C-GAD65 plasmid with the transfection reagent PEI at a mass-to-volume ratio of 1:3, vortex, and let stand for 10 min. Transfect the prepared cells respectively and incubate at 37°C with 5% CO 2 Incubate for 48 h.

[0315] Blue light excitation of optoDroplet-K10C-GAD65 transfected cells: Place the well plate on a Confocol and excite it for 5 - 120 s in the 405 nm blue light channel;

[0316] After the cells are excited by blue light, observe the fluorescence of GAD65 in optoDroplet-KCTD17 IDR-GAD65 in the red channel. Before the blue light excitation, the GAD65 fusion protein expressed by optoDroplet-KCTD17 IDR-GAD65 is the same as that of pEGFP-N1-GAD65, showing a diffuse cytoplasmic distribution. However, after the blue light excitation for 30 s, obvious aggregated red fluorescence signals appear in the cells transfected with optoDroplet-KCTD17IDR-GAD65, and larger droplet-like shapes can be seen after the excitation for 120 s. Compared with before the blue light excitation, the GAD65 fusion protein aggregates due to phase transition after excitation, and its fluorescence intensity increases by 11 times ( Figure 6 ).

[0317] Example 7 The optoDroplet-KCTD17 IDR system antigen aggregation can tolerate methanol fixation

[0318] Previously, the phase transition phenomenon of optoDroplet was observed in living cells, but whether the phase transition of this protein can tolerate the influence of cell fixation operation is unknown. In the present invention, after the cells transfected with optoDroplet-KCTD17 IDR are excited by blue light, the cells are quickly fixed, and then the situation of the phase transition protein is observed. Both methanol and paraformaldehyde (PFA) can retain the protein phase boundary structure induced by blue light in optoDroplet-KCTD17 IDR.

[0319] I. Main reagents, instruments and sources:

[0320] Cell line: HEK293T cells;

[0321] Plasmids: optoDroplet-KCTD17 IDR-Titin / GDA65 (from Examples 5 and 6);

[0322] Plasmid extraction kit: Omega plasmid mini kit;

[0323] Water purification instrument: Millipore 5UV Water Purification System;

[0324] Pipette: Eppendorf;

[0325] Blue light viewer: Beijing Liuyi Biotechnology Co., Ltd., WD-9403X;

[0326] Using the above reagents and consumables, the following steps can be used to observe whether the Titin and GDA65 antigens expressed by the optoDroplet-KCTD17 IDR system can tolerate cell fixation.

[0327] II. Cell transfection:

[0328] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, discard the supernatant, add DMEM complete medium containing 10% serum, and gently pipette and mix to make a cell suspension.

[0329] 2. Place the sterilized glass slides in a cell culture plate, treat with polylysine (PDL), rinse 3 times and air dry. Add the prepared cell suspension to the cell culture plate, gently mix, and place in an incubator at 37°C, 5% CO 2 overnight. Observe the cells the next day. Transfection can be performed when the density reaches 40 - 50%.

[0330] 3. Mix the prepared optoDroplet-KCTD17 IDR-Titin and optoDroplet-KCTD17 IDR-GAD65 plasmids with the transfection reagent PEI at a mass-volume ratio of 1:3, vortex, and let stand for 10 min. Transfect into the prepared cells respectively and incubate at 37°C, 5% CO 2 for 48 h.

[0331] 4. Fixation of optoDroplet-KCTD17 IDR-Titin and optoDroplet-KCTD17 IDR-GAD65 transfected cells:

[0332] (1) Place the well plate on the blue light viewer and excite with blue light for 1 - 5 minutes;

[0333] (2) Fix the cells with acetone / cold methanol for 10 minutes and wash twice with PBS;

[0334] After cell fixation, observe the fluorescence of Titin and GAD65 proteins in the optoDroplet-KCTD17 IDR system under the red channel. In the transfected cells, the Titin and GAD65 fusion proteins expressed by the optoDroplet-KCTD17 IDR system showed obvious aggregation and presented a droplet-like morphology, indicating that the proteins expressed by optoDroplet-KCTD17 IDR can tolerate the effects of fixation reagents such as methanol and PFA after phase transition ( Figure 7 ).

[0335] Sequence:

[0336] Amino acid sequence of KCTD17 (SEQ ID NO:1):

[0337] MQTPRPAMRMEAGEAAPPAGAGGRAAGGWGKWVRLNVGGTVFLTTRQTLCREQKSFLSRLCQG

[0338] EELQSDRDETGAYLIDRDPTYFGPILNFLRHGKLVLDKDMAEEGVLEEAEFYNIGPLIRIIKDRMEE

[0339] KDYTVTQVPPKHVYRVLQCQEEELTQMVSTMSDGWRFEQLVNIGSSYNYGSEDQAEFLCVVSKE

[0340] LHSTPNGLSSESSRKTKSTEEQLEEQQQQEEEVEEVEVEQVQVEADAQEKGSRPHPLRPEAELAVR

[0341] ASPRPLARPQSCHPCCYKPEAPGCEAPDHLQGLGVPI

[0342] Nucleotide sequence of KCTD17 (SEQ ID NO:2):

[0343] ATGCAGACGCCGCGGCCGGCGATGAGGATGGAGGCCGGGGAGGCAGCGCCGCCGGCGGGGGCGGGCGGCCGCGCCGCAGGCGGCTGGGGCAAGTGGGTGCGGCTCAACGTGGGGGGCACGGTGTTCCTGACCACCCGGCAGACGCTGTGCCGCGAGCAGAAGTCCTTCCTCAGCCGCCTGTGCCAGGGGGAAGAGCTGCAGTCGGACCGGGATGAGACCGGGGCCTACCTCATTGACCGTGACCCCACCTACTTCGGGCCCATCCTGAACTTCCTCCGGCATGGCAAGCTGGTGCTGGACAAGGACATGGCTGAGGAGGGGGTCCTGGAGGAAGCCGAGTTCTACAACATCGGCCCGCTGATCCGCATCATCAAAGACCGGATGGAAGAGAAGGACTACACGGTCACCCAGGTCCCACCCAAGCACGTGTACCGCGTGCTGCAGTGCCAGGAGGAGGAGCTCACGCAAATGGTCTCCACCATGTCTGATGGCTGGCGCTTCGAGCAGCTGGTGAACATCGGCTCCTCCTACAACTACGGCAGCGAGGACCAGGCAGAGTTCCTGTGTGTGGTGTCCAAGGAGCTCCACAGCACCCCAAACGGGCTGAGCTCAGAGTCCAGCCGCAAAACCAAGAGCACGGAGGAGCAGCTGGAGGAGCAGCAGCAGCAGGAGGAGGAGGTGGAGGAGGTGGAGGTGGAACAGGTGCAGGTGGAGGCAGATGCACAGGAGAAAGGTTCCCGTCCGCACCCTCTCAGACCTGAGGCTGAGCTTGCAGTGAGGGCTTCTCCTCGGCCCCTCGCCCGCCCCCAGAGCTGCCATCCCTGCTGTTACAAGCCAGAGGCACCCGGATGTGAGGCCCCAGATCACCTCCAGGGACTTGGGGTTCCCATC

[0344] KCTD17 IDR amino acid sequence (SEQ ID NO:3):

[0345] KTKSTEEQLEEQQQQEEEVEEVEVEQVQVEADAQEKGSRPHPLRPEAELAVRAS

[0346] Nucleotide sequence of KCTD17 IDR (SEQ ID NO:4):

[0347] AAAACCAAGAGCACGGAGGAGCAGCTGGAGGAGCAGCAGCAGCAGGAGGAGGAGGTGGAGGAGGTGGAGGTGGAACAGGTGCAGGTGGAGGCAGATGCACAGGAGAAAGGTTCCCGTCCGCACCCTCTCAGACCTGAGGCTGAGCTTGCAGTGAGGGCTTCT

[0348] Full sequence of optoDroplet-KCTD17 IDR vector (SEQ ID NO:26):

[0349]

[0350] Titin nucleotide sequence (SEQ ID NO:27)

[0351] ATGAGGTGCGAGGAGGGCAAAGATAATTGGATTAGATGCAATATGAAGCTGGTGCCTGAGCTGACCTACAAAGTGACCGGCCTGGAGAAGGGCAACAAGTATCTGTATAGAGTGAGCGCCGAGAACAAAGCCGGCGTGAGCGATCCTAGCGAGATCCTGGGCCCCCTGACCGCCGATGACGCCTTCGTGGAGCCTACCATGGACCTGAGCGCCTTCAAAGACGGCCTGGAGGTCATTGTGCCTAATCCTATCACCATTCTGGTGCCTAGCACCGGCTACCCCAGACCCACCGCCACCTGGTGTTTTGGCGACAAAGTGCTGGAGACCGGCGATAGAGTGAAGATGAAAACCCTGTCCGCCTACGCCGAGCTGGTCATTTCCCCCTCCGAGAGATCCGATAAGGGCATCTATACCCTGAAGCTGGAGAATAGAGTGAAGACCATTTCCGGCGAGATTGACGTGAACGTGATCGCCAGACCCTCCGCCCCCAAGGAGCTGAAGTTTGGCGATATTACCAAGGACTCCGTGCACCTGACCTGGGAGCCCCCCGATGATGATGGCGGCAGCCCCCTGACCGGCTATGTGGTGGAGAAAAGGGAGGTGAGCAGGAAAACCTGGACCAAGGTCATGGATTTTGTGACCGATCTGGAGTTTACCGTGCCTGATCTGGTGCAGGGCAAAGAGTACCTGTTTAAAGTGTGTGCCAGGAATAAGTGCGGCCCTGGCGAGCCCGCCTACGTGGATGAGCCCGTGAATATGAGCACCCCCGCCACCGTGCCCGACCCACCTGAAAATGTGAAATGGAGGGACAGAACCGCCAATTCCATCTTTCTGACCTGGGATCCTCCCAAGAATGACGGCGGC

[0352] GAD65 nucleotide sequence (SEQ ID NO:28)

[0353]

Claims

1. A polypeptide, which is the KCTD17 IDR with the amino acid sequence shown in SEQ ID NO:

3.

2. A nucleic acid molecule, which encodes the polypeptide according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein the nucleic acid molecule is the polynucleotide shown in SEQ ID NO:

4.

4. A vector for expressing the polypeptide according to claim 1, which contains the nucleic acid molecule according to claim 2 or 3.

5. An expression vector, which contains a part composed of, from upstream to downstream in sequence, a multiple cloning site where each element is optionally connected by a linker, a polynucleotide encoding the KCTD17 IDR according to claim 1, a polynucleotide encoding a fluorescent protein tag, and a polynucleotide encoding a light-induced polymerization protein domain Cry2 PHR, wherein, the fluorescent protein tag is selected from mCherry and GFP.

6. The expression vector according to claim 5, wherein, the expression vector is optoDroplet-KCTD17 IDR shown in SEQ ID NO:

26.

7. An expression vector, which contains a target protein gene, and the target protein gene is inserted into the expression vector according to claim 5 through a multiple cloning site, wherein, the target protein gene is selected from the Titin gene or the GAD65 gene.

8. The expression vector according to claim 7, wherein, the target protein is located at the N-terminus of KCTD17 IDR.

9. The expression vector according to claim 5, wherein, the expression vector is obtained by a method including the following steps: coupling KCTD17 IDR with a fluorescent protein tag and a light-induced polymerization protein domain Cry2 PHR, constructing them into the pEGFP-N1 plasmid, and retaining a multiple cloning site before KCTD17 IDR and the fluorescent protein tag to insert the target protein gene.

10. A cell, which contains the vector according to any one of claims 4-9.

11. The cell according to claim 10, wherein the cell is selected from HEK293T cells, Hela cells, and Hep2 cells.

Images