Novel living cell tubulin probe as well as construction method and application thereof

Abstract

The invention discloses a novel living cell microtubulin probe and a construction method and application thereof.The probe is composed of EGFP and SPASTIN protein 1-325 amino acid, and the SPASTIN protein 1-325 amino acid is shown as SEQ ID NO.10. By means of a truncated body version, the SPASTIN protein loses the microtubule shearing function, interference to other physiological processes of cells is reduced, and the activity of the SPASTIN protein is improved. The combination with other non-specific structures can be reduced, the positioning accuracy of the probe is improved, the specific region or substructure of the microtube can be more accurately positioned, and the background interference is reduced; the problems of cell function interference, photobleaching, toxicity and the like of existing markers are solved, and a tool is provided for deep understanding of dynamic changes of the microtubules and roles of the microtubules in various biological processes of the microtubules.

Description

Technical Field

[0001] The invention relates to the field of molecular markers, and in particular to a novel living cell tubulin probe, and also to a construction method and application of the probe. Background Art

[0002] Microtubules are an important component of the cytoskeleton and are mainly composed of α and β tubulin dimers. They form slender tubular structures in cells with a diameter of about 25nm and are extremely stable and dynamic. Microtubules not only provide mechanical support for cells and maintain cell shape, but also participate in a variety of key biological processes such as the transport of intracellular substances, cell division, and cell signaling.

[0003] The function of microtubules is closely related to their dynamic properties. Microtubules can quickly respond to the needs of cells by accelerating or slowing down the polymerization and dissociation of dimers, and this dynamic change is essential for the normal function of cells. In addition, microtubules are also regarded as important targets for drug development. Many anticancer drugs (such as paclitaxel and vincristine) inhibit the proliferation of cancer cells by affecting the dynamic stability of microtubules. Marking the state and functional changes of microtubules helps to evaluate the effects of these drugs and provide a basis for personalized treatment. Therefore, marking tubulin in living cells is crucial to understanding its dynamic behavior and function.

[0004] Existing labeling technologies mainly include the use of fluorescent protein fusion expression, chemical fluorescent probes, and small molecule dyes. Among them, the fusion of fluorescent protein (such as GFP, mCherry, etc.) genes to the coding sequence of tubulin (such as α and β tubulin) allows researchers to observe the dynamic changes of microtubules in real time through a fluorescence microscope. However, the fusion of fluorescent proteins may affect the normal function of tubulin, especially under high expression conditions, which may cause changes in the assembly and disassembly rates of microtubules, thereby affecting the physiological state of cells and experimental results; chemical fluorescent probes need to optimize staining conditions for different cells, and excessive or insufficient use of probes may cause signal distortion; small molecule dyes usually have a strong photobleaching phenomenon. Over time, the fluorescent signal may weaken rapidly, limiting the ability to observe for a long time, and the cell permeability and toxicity of the dye may affect the physiological state of the cell.

[0005] The SPAST gene is located at 2p22.3 and contains 17 exons. The protein SPASTIN encoded by it contains 616 amino acids and is expressed in many eukaryotic organisms. It is a key microtubule-associated protein. The SPASTIN protein mainly contains four domains: AAA-ATPase region, microtubule-binding domain (MBD), microtubule interacting and trafficking domain (MIT), and hydrophobic domain (HD). SPASTIN promotes the depolymerization of microtubules by binding to microtubules, thereby regulating the dynamic stability of microtubules and affecting the dynamic balance of microtubules. This role is particularly important during cell division, as it helps the assembly of the spindle and the correct distribution of chromosomes. In addition, SPASTIN plays a key role in the formation of neuronal processes, ensuring the normal function and development of nerve cells.

[0006] Therefore, there is an urgent need for a new method for labeling tubulin in living cells that can overcome the shortcomings of existing technologies and provide lower cell interference and longer dynamic observation capabilities, which will have important scientific significance and application potential. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a novel living cell tubulin probe; a second object of the present invention is to provide the use of the novel living cell tubulin probe in labeling living cell microtubules; a third object of the present invention is to provide a method for preparing the novel living cell tubulin probe.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] 1. A novel living cell microtubule protein probe, the probe is composed of EGFP and amino acids 1-325 of SPASTIN protein, and the amino acids 1-325 of SPASTIN protein are shown in SEQ ID NO.10.

[0010] Preferably, the nucleotide sequence of EGFP is shown in SEQ ID NO.7.

[0011] Preferably, the nucleotide sequence of positions 1-325 of the SPASTIN protein is as shown in SEQ ID NO.9.

[0012] 2. Application of the novel living cell tubulin probe in labeling living cell microtubules.

[0013] 3. The preparation method of the novel living cell microtubule protein probe is as follows: pEGFP-puro is used as a template, and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are used as primers to amplify EGFP; SEQ ID NO.3 and SEQ ID NO.4 are used as primers, and the cDNA of HEK-293T cells is used as a template to amplify the nucleotides encoded at positions 1-325 of the SPASTIN protein, and then the nucleotides encoded at positions 1-325 of the EGFP and SPASTIN protein are connected to the XbaI and NotI restriction sites of the vector PcDNA3.1-DsRED through homologous recombination.

[0014] The beneficial effects of the present invention are: a new type of living cell microtubule protein probe, SPASTIN protein has a strong binding affinity with microtubules, and through the truncated version, SPASTIN protein loses the function of microtubule shearing, reduces interference with other physiological processes of cells, and can reduce binding to other non-specific structures, and improves the positioning accuracy of the probe. This means that it can more accurately locate specific areas or substructures of microtubules and reduce background interference.

[0015] It overcomes the problems of existing labeling, such as interference with cell function, photobleaching, and toxicity, and provides a tool for a deeper understanding of the dynamic changes of microtubules and their roles in various biological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1 EGFP-SPAST 1-161 cellular localization;

[0018] Figure 2 EGFP-SPAST 1-325 cellular localization. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0020] Example 1

[0021] A method for constructing a novel protein probe for specifically labeling microtubules comprises the following steps:

[0022] A. Total RNA from HEK-293T cells was extracted using the Tiangen RNAsimple Total RNA Extraction Kit (DP419), and 1 μg of RNA was reverse transcribed using the ABClonal Reverse Transcription Kit (RK20433) to obtain 20 μL of cDNA.

[0023] B. Amplification of target fragment and linearization of vector: Using pEGFP-puro as template, amplify the EGFP sequence (SEQ ID NO.7) by PCR; the primers for amplifying EGFP are as follows:

[0024] EGFP upstream primer: 5′-TATAGGGAGACCCAAGCTGGCCACCatggtgagcaagggcgag-3′ (SEQ ID NO. 1);

[0025] EGFP downstream primer: 5′-tccacccggagaattcatggtcgccaccggcttgtacagctcgtccat-3′ (SEQ ID NO. 2).

[0026] Using cDNA as a template, the SPAST gene sequence corresponding to amino acids 1-325 of the SPASTIN protein (SEQ ID NO.8) was obtained by PCR. The amplification primer sequences are as follows:

[0027] SPAST 1-325 Upstream primer: 5′-atggacgagctgtacaagccggtggcgaccatgaattctccgggtgga-3′ (SEQ ID NO. 3);

[0028] SPAST 1-325 Downstream primer: 5′-AAGACTTCCTCTGCCCTCTTAagcaaggttgctgtccac-3′ (SEQ ID NO. 4).

[0029] Using cDNA as a template, the SPAST gene sequence corresponding to amino acids 1-616 of the SPASTIN protein (SEQ ID NO.9) was obtained by PCR; the amplification primer sequences are as follows:

[0030] SPAST 1-616 Upstream primer: 5'-atggacgagctgtacaagccggtggcgaccatgaattctccgggt-3' (SEQ ID NO. 5);

[0031] SPAST 1-616Downstream primer: 5′-AAGACTTCCTCTGCCCTCTTAaacagtggtatctccaaa-3′ (SEQ ID NO. 6).

[0032] The Taq enzyme premix in this step was purchased from Novozymes Nanjing Novozymes Biotech Co., Ltd. The PCR amplification system was ddH 2 O 19μL, 2×Taq Plus Master Mix (P212) 25μL, upstream primer (10μM) 2μL, downstream primer (10μM) 2μL, template cDNA 2μL; the primers and template should match the target fragment to be amplified.

[0033] The linearization treatment of the vector PcDNA3.1-DsRED was carried out by digestion with XbaI and NotI. The restriction endonucleases in this step were purchased from Jiangsu Best Biotechnology Co., Ltd. The digestion reaction system was ddH 2 O 13 μL, 10× CutOne TM ColorBuffer 2μL, LightNing TM XbaI 1 μl, LightNing TM NotI 1 μl, vector plasmid 3 μl; reaction conditions: 37°C for 1 h.

[0034] C. Agarose gel electrophoresis and gel recovery of the target fragment: The amplified product and the enzyme-digested product obtained in step B were separated by electrophoresis in a 1.2% agarose gel, and gel recovery was performed using the Yisheng Gel Extraction Kit (19101ES70) to obtain the corresponding target fragment EGFP fragment, the SPAST gene sequence fragment corresponding to the amino acids 1-325 of the SPASTIN protein, and the SPAST gene sequence fragment corresponding to the amino acids 1-616 of the SPASTIN protein.

[0035] D. Homologous recombination: The EGFP fragment obtained in step C, the SPAST gene sequence fragment corresponding to the amino acids 1-325 of the SPASTIN protein, and the linearized PcDNA3.1 vector fragment were subjected to homologous recombination reaction to obtain a recombinant product, namely EGFP-SPAST 1-325 Expression vector; the EGFP fragment obtained in step C, the SPAST gene sequence fragment corresponding to the amino acids 1-616 of the SPASTIN protein, and the linearized PcDNA3.1 vector fragment are subjected to homologous recombination reaction to obtain a recombinant product, namely EGFP-SPAST 1-616 Expression vector.

[0036] The homologous recombinase used in this step was purchased from Jiangsu Best Biotechnology Co., Ltd. 1 -325 The homologous recombination reaction system of the expression vector is as follows: 120ng of linearized vector, 14ng of EGFP, 20ng of the SPAST gene sequence fragment corresponding to amino acids 1-325 of SPASTIN protein, and 10ng of LightNing TM DNA Assembly Mix Plus 5 μL, add ddHO 2 0 to 10 μL;

[0037] EGFP-SPAST 1-616 The homologous recombination reaction system of the expression vector is as follows: 120ng of linearized vector, 14ng of EGFP, 37ng of the SPAST gene sequence fragment corresponding to amino acids 1-616 of SPASTIN protein, and 10ng of LightNing TM DNA Assembly Mix Plus 5 μL, add ddHO 2 0 to 10 μL;

[0038] The homologous recombination reaction conditions were 50°C for 15 min.

[0039] E. Transformation of recombination products and plasmid extraction: Take 5 μL of the homologous recombination product obtained in step D and add it to 50 μL Stb13 competent cells, mix well by slowly pipetting, place on ice for 30 minutes, heat shock at 42°C for 45 seconds, add 500 μL of antibiotic-free LB medium after ice bath for 2 minutes, and shake and culture at 37°C for 60 minutes; centrifuge at 5000rpm for 1 minute, remove 400 μL of supernatant, use the remaining medium to resuspend the bacterial precipitate and evenly spread it on a plate containing ampicillin antibiotics, and invert it at 37°C for overnight culture. Pick a single colony of the positive clone and inoculate it into ampicillin-resistant LB medium for overnight culture, then use the endotoxin-free plasmid mini-extraction kit (DP118) purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the plasmid to obtain EGFP-SPAST. 1-325 Expression plasmids.

[0040] Example 2

[0041] The application of a novel protein probe for specifically labeling microtubules in eukaryotic cells comprises the following steps:

[0042] A. Cell preparation: HEK-293T cells digested with 0.25% Trypsin-EDTA (Gibco, 25200056) were plated into a 6-well cell culture plate, with about 800,000 cells per well, until the cell density reached 60-70% for cell transfection.

[0043] B. Prepare PEI-nucleic acid complex: Take 1 μg EGFP-SPAST 1-325 Plasmid or 1 μg EGFP-SPAST 1-616 The plasmid was added with 1.5 μL PEI Transfection Reagen (MCE, HY-K2014), mixed by pipetting, incubated at room temperature for 5 min, diluted with 400 μL serum-free basal medium, gently mixed and incubated at room temperature for 30 min, and then further diluted by adding 600 μL serum-free basal medium.

[0044] C. Cell transfection: Aspirate the original culture medium, add the diluted PEI-nucleic acid complex to different cells, culture at 37°C for 12 hours, and then add an equal amount of fresh growth medium.

[0045] D. After 36 hours of culture, EGFP-SPAST can be observed under a fluorescence microscope. 1-325 Plasmids and EGFP-SPAST 1-616 Plasmid expression and EGFP-SPAST 1-325 EGFP-SPAST 1-616 The morphology of microtubules was observed by protein labeling.

[0046] The results are as follows Figure 1 and Figure 2 shown. Figure 1 The results showed that EGFP-SPAST 1-616 The protein appears as a dot-like marker distributed on the microtubule network in the cell. SPASTIN protein can be observed to be distributed in the cytoplasm in a dot-like manner interacting with microtubules. These dot-like positions usually appear at the ends of microtubules or in the depolymerization area of ​​microtubules. EGFP-SPAST 1-325 The protein showed persistent and abnormal tight binding to the microtubule structure within the cell, and a highly overlapping spatial distribution with tubulin protein was observed.

[0047] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A novel living cell microtubule protein probe, characterized in that: The probe consists of EGFP and amino acids 1-325 of SPASTIN protein, and the amino acids 1-325 of SPASTIN protein are shown in SEQ ID NO.

10.

2. The novel living cell microtubule protein probe according to claim 1, characterized in that: The nucleotide sequence of EGFP is shown in SEQ ID NO.

7.

3. The novel living cell microtubule protein probe according to claim 1, characterized in that: The nucleotide sequence of the SPASTIN protein at positions 1-325 is shown in SEQ ID NO.

9.

4. Use of the novel living cell tubulin probe according to any one of claims 1 to 3 in labeling living cell microtubules.

5. The method for preparing the novel living cell microtubule protein probe according to any one of claims 1 to 3, characterized in that: Using pEGFP-puro as a template and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers, EGFP was amplified; then, using SEQ ID NO.3 and SEQ ID NO.4 as primers and the cDNA of HEK-293T cells as a template, the nucleotides encoding positions 1-325 of the SPASTIN protein were amplified, and then, through homologous recombination, the nucleotides encoding positions 1-325 of the EGFP and SPASTIN protein were connected to the XbaI and NotI restriction sites of the vector PcDNA3.1-DsRED.

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