Bimolecular fluorescence complementation system triggered based on interaction of two groups of nano antibodies and epitopes and application of bimolecular fluorescence complementation system
By using a bimolecular fluorescent complementary system based on the interaction of two sets of nanobodies and epitopes, combined with endocytosis absorption experiments, non-toxic visual tracking of the Golgi apparatus and TGN/EE was achieved, solving the problem of difficulty in tracking upstream cell compartments of vacuoles in existing technologies and avoiding interference from fluorescent proteins.
Patent Information
- Application Number
- CN202511024203.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-24
- Publication Date
- 2025-12-05
AI Technical Summary
Existing bimolecular fluorescence complementary systems are difficult to effectively track upstream cell compartments of vacuoles such as the Golgi apparatus and TGN/EE in living cells, and co-expression of common fluorescent proteins can interfere with the transport process.
A bimolecular fluorescence complementary system based on the interaction between two sets of nanobodies and epitopes was adopted. By combining the complementarity of two split green fluorescent protein fragments through endocytosis absorption experiments, specific compartmental tracing of the target protein was achieved, avoiding drug treatment. The non-toxic visualization tracking of Golgi apparatus and TGN/EE was realized by utilizing nanobodies-epitaxy interactions.
It enables non-toxic visual tracking of the Golgi apparatus and TGN/EE, avoids interference from fluorescent proteins during co-expression, and has high specificity without affecting the normal transport process of proteins.
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Figure CN121065221A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of protein engineering, in particular to a two-molecular fluorescence complementation system based on two groups of nanobodies and epitope interaction triggered and application thereof, more particularly to a strategy for non-toxic visualization and tracking of Golgi and trans-Golgi network / early endosome (TGN / EE) in living cells. BACKGROUND
[0002] Golgi and trans-Golgi network / early endosome (TGN / EE) as the central compartment of intracellular membrane transport system has always been a hotspot and focus of research, not only involved in intracellular secretion, endocytosis and vacuole transport process, but also the multi-compartment spanning transport routes around this part and compartment, especially their merging or branching point problems, still remain to be solved. Therefore, it has very important research and practical significance to realize the tracking of transport in Golgi and TGN / EE in living cells.
[0003] However, there is a major limitation in evaluating Golgi and TGN / EE transport. If the transport system is not subjected to pharmacological treatment, proteins are rarely detected in the transport compartment, which makes the interpretation of the observation results more complicated. On the other hand, although endocytosis absorption experiment can achieve specific targeting of target compartment based on nanobody-epitope interaction, the fluorescent protein that has to be connected for exploring the transport of the protein to be tested may appear in every cell compartment in the transport process without distinction according to the transport of the protein to be tested in the co-expression process, which interferes with the transport of the target compartment. The two-molecular fluorescence complementation (BiFC) technology based on nanobody NbS and epitope SYN can only track limited cell compartments in living cells, especially most of them only stay in the tracking of endoplasmic reticulum (ER). If two parts of BiFC fusion protein are expressed at the same time, the output of BiFC fluorescence signal will be completed at the early stage of protein synthesis (ER), causing signal interference and making it impossible to track the subsequent vacuole upstream cell compartments, especially Golgi and TGN / EE.
[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY
[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a two-molecular fluorescence complementation system based on two groups of nanobodies and epitope interaction triggered and application thereof, aiming to solve the problem of limited cell compartments that can be tracked in living cells by the existing two-molecular fluorescence complementation system, especially the vacuole upstream cell compartments in the middle part of the intracellular membrane transport system that are difficult to achieve natural tracking, Golgi and TGN / EE.
[0006] The technical scheme of the present application is as follows:
[0007] A two-group nanobody and epitope interaction triggered bimolecular fluorescence complementation system based on the interaction, comprising a first carrier, a second carrier and a third carrier;
[0008] The first carrier comprises: an epitope SYN, an epitope AYD, and one protein fragment G2 in a red fluorescent protein and split green fluorescent protein system;
[0009] The second carrier comprises: a nanobody NbA and an anchor protein;
[0010] The third carrier comprises: a nanobody NbS, and the other protein fragment G1 in the split green fluorescent protein system and a to-be-tested protein;
[0011] The sequence of the epitope SYN is shown as SEQ ID NO: 1, the sequence of the epitope AYD is shown as SEQ ID NO: 2, the sequence of the nanobody NbA is shown as SEQ ID NO: 3, and the sequence of the nanobody NbS is shown as SEQ ID NO: 4.
[0012] The two-group nanobody and epitope interaction triggered bimolecular fluorescence complementation system based on the interaction, wherein the protein fragment G1 comprises a protein fragment of amino acids 1-214 of a super-folded GFP protein; and the protein fragment G2 comprises a protein fragment of amino acids 215-233 of the super-folded GFP protein.
[0013] The two-group nanobody and epitope interaction triggered bimolecular fluorescence complementation system based on the interaction, wherein the sequence of the protein fragment G1 is shown as SEQ ID NO: 5.
[0014] The two-group nanobody and epitope interaction triggered bimolecular fluorescence complementation system based on the interaction, wherein the sequence of the protein fragment G2 is shown as SEQ ID NO: 6.
[0015] The two-group nanobody and epitope interaction triggered bimolecular fluorescence complementation system based on the interaction, wherein the first carrier comprises, in sequence, an epitope SYN, a red fluorescent protein RFP, a protein fragment G2 and an epitope AYD; and the sequence of the red fluorescent protein RFP is shown as SEQ ID NO: 7.
[0016] The two-group nanobody and epitope interaction triggered bimolecular fluorescence complementation system based on the interaction, wherein the anchor protein is a trans-Golgi network / early endosome marker compartment membrane protein SYP61, a vacuolar sorting receptor VSR4s; and the to-be-tested protein is a trans-Golgi marker compartment membrane protein ST and a cis-Golgi marker compartment membrane protein Man1.
[0017] The two groups of nanobodies and epitope interaction triggered double-molecular fluorescence complementation system, wherein the sequence of the trans-Golgi network / early endosome marker compartment membrane protein SYP61 is shown as SEQ ID NO: 8; the sequence of the vacuolar sorting receptor VSR4s is shown as SEQ ID NO: 9; the sequence of the trans-Golgi marker compartment membrane protein ST is shown as SEQ ID NO: 10; and the sequence of the cis-Golgi marker compartment membrane protein Man1 is shown as SEQ ID NO: 11.
[0018] A biosensor comprising a two groups of nanobodies and epitope interaction triggered double-molecular fluorescence complementation system.
[0019] A two groups of nanobodies and epitope interaction triggered double-molecular fluorescence complementation system or biosensor is used for tracking protein transport in different cell compartments.
[0020] A two groups of nanobodies and epitope interaction triggered double-molecular fluorescence complementation system or biosensor is used for tracking protein transport in different cell compartments.
[0021] Beneficial effects: the present application provides a two groups of nanobodies and epitope interaction triggered double-molecular fluorescence complementation system and its application, the double-molecular fluorescence complementation system comprises a first carrier, a second carrier and a third carrier; the first carrier comprises: an epitope SYN, an epitope AYD, and one protein fragment G2 in a red fluorescent protein and a split green fluorescent protein system; the second carrier comprises: a nanobody NbA and an anchor protein; and the third carrier comprises: a nanobody NbS, and another protein fragment G1 in a split green fluorescent protein system and a protein to be detected. The present application is based on a nanobody-epitope interaction triggered double-molecular fluorescence complementation (BiFC) technology, two groups of different nanobody-epitope interactions are adopted, and endocytosis experiments are combined to realize tracking of a specific compartment of a protein to be detected through complementation of two split green fluorescent protein fragments; and the system does not utilize any drug treatment, realizes non-toxic visualization tracking of a transport process in a cell compartment upstream of a vacuole, mainly in a Golgi body and a trans-Golgi network / early endosome (TGN / EE), in a living cell; meanwhile, the two groups of nanobody-epitopes adopted by the system not only have high specificity, but also do not affect physiological processes such as transport of the proteins combined. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 A schematic diagram for tracking a cell compartment upstream of a vacuole based on two groups of nanobody-epitope interaction triggered BiFC;
[0023] Figure 2 Schematic diagram for live cell visualization tracking of Golgi and TGN / EE using TGN / EE marker compartment membrane protein SYP61 and vacuolar sorting receptor VSR4;
[0024] Figure 3 Resulting image for live cell visualization tracking of target protein transport in Golgi or TGN / EE based on two sets of nanobody-epitope interaction triggered bimolecular fluorescence complementation for example 1. DETAILED DESCRIPTION
[0025] The present application provides a bimolecular fluorescence complementation system triggered by two sets of nanobody and epitope interaction and application thereof, in order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application is further described in detail below. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.
[0026] Those skilled in the art can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs. It should also be understood that terms such as those defined in general dictionaries should be understood as having meanings consistent with those in the context of the prior art, and unless specifically defined as such, should not be interpreted in an idealized or overly formal sense.
[0027] The present application provides a bimolecular fluorescence complementation system triggered by two sets of nanobody and epitope interaction, which comprises a first carrier, a second carrier and a third carrier;
[0028] The first carrier comprises: an epitope SYN, an epitope AYD, and one of a protein fragment G2 in a red fluorescent protein and split green fluorescent protein system;
[0029] The second carrier comprises: a nanobody NbA and an anchor protein;
[0030] The third carrier comprises: a nanobody NbS, and the other protein fragment G1 in a split green fluorescent protein system and a target protein;
[0031] The sequence of the epitope SYN is shown as SEQ ID NO: 1, the sequence of the epitope AYD is shown as SEQ ID NO: 2, the sequence of the nanobody NbA is shown as SEQ ID NO: 3, and the sequence of the nanobody NbS is shown as SEQ ID NO: 4.
[0032] In the embodiment, based on the BiFC (Bimolecular Fluorescence Complementation) technology triggered by nanobody-epitope interaction, two different nanobody-epitope interactions are adopted, and the specific compartment of the protein to be tested is traced by the complementation of two split green fluorescent protein fragments in combination with endocytosis experiment; and the system does not use any drug treatment, realizes the non-toxic visualization tracking of the transport process of the cell compartment upstream of the vacuole, mainly Golgi and TGN / EE in living cells; at the same time, the two groups of nanobody-epitope adopted by the system not only have high specificity, but also do not affect the transport and other physiological processes of the proteins combined respectively. Specifically, the bimolecular fluorescence complementation system triggered by the interaction of the two groups of nanobody and epitope can explore the Golgi and TGN / EE which are difficult to achieve natural tracing in the middle of the intracellular membrane transport system; at the same time, the endocytosis experiment is combined with the bimolecular fluorescence complementation technology triggered by the nanobody-epitope interaction, which more accurately tracks and displays the target cell compartment to be tested, avoiding the excessive interfering fluorescent signals of non-target compartments to be tested generated by ordinary fluorescent proteins in endocytosis experiments.
[0033] In some embodiments, the nanobody NbS can specifically bind to the epitope SYN; the nanobody NbS is an α-synuclein nanobody, and its sequence is shown as SEQ ID NO: 4; the epitope SYN adopts an α-synuclein epitope from the NCBI database (NP_001362219.1), and its sequence is shown as SEQ ID NO: 1.
[0034] In some embodiments, the nanobody NbA can specifically bind to the epitope AYD; the nanobody NbA is a β-amyloid nanobody, and its sequence is shown as SEQ ID NO: 3; the epitope AYD adopts a β-amyloid epitope from the NCBI database (PDB: 2LMQ_R), and its sequence is shown as SEQ ID NO: 2.
[0035] In some embodiments, the split GFP system is implemented with a soluble optimized superfolder GFP (sfGFP OPT), the protein sequence of which is shown as SEQ ID NO: 12; the split sfGFP OPT system employs a two-segment approach, i.e., the protein fragment G1 comprises a protein fragment of amino acids 1-214 of the superfolder GFP protein (1-10 beta strands, denoted as GFP G1); and the protein fragment G2 comprises a protein fragment of amino acids 215-233 of the superfolder GFP protein (11th beta strand, denoted as GFP G2).
[0036] Specifically, a schematic diagram for tracking the cell compartments upstream of the vacuole (Golgi and TGN / EE) based on two sets of nanobody-epitope interactions triggering BiFC is shown in FIG. 1, the core elements of which are as follows: a split GFP G2 fragment (11th beta strand 215-233) is targeted into the target cell compartment upstream of the vacuole via endocytosis using a set of nanobody-epitope interactions, and when a test fusion protein carrying a second split GFP G1 fragment (1-10 beta strands 1-214) is transported through the target cell compartment, the close proximity of the G1 and G2 fragments is triggered by another set of nanobody-epitope interactions to produce a detectable BiFC-based fluorescence output signal while targeting the test fusion protein in the corresponding target cell compartment, thus completing the non-toxic in vivo visualization tracking of the test fusion protein. This strategy can be simplified into three stages: 1) assembling a "bait", the bi-epitope-linked G2 fragment fusion protein is absorbed into the cell via endocytosis to avoid the BiFC fluorescence signal generated at the initial stage due to co-expression; 2) setting up a "cage", the compartment-specific membrane protein with nanobodies targets the endocytosed bi-epitope G2 fusion fragment to the specific compartment via nanobody-epitope interactions, preventing it from continuing to sort into the vacuole via the endocytosis pathway; and 3) capturing the "prey", the test protein carrying another nanobody and G1 fragment will be detected in the compartment based on the BiFC green fluorescence signal once it is sorted and transported through the corresponding compartment where the "cage" is set up. Figure 1
[0037] In some embodiments, the sequence of the protein fragment G1 is shown as SEQ ID NO: 5.
[0038] In some embodiments, the sequence of the protein fragment G2 is shown as SEQ ID NO: 6. Specifically, the split green fluorescent protein fragment G2 is absorbed into the cell via endocytosis in a bi-epitope-linked manner.
[0039] In some embodiments, the first vector comprises epitope SYN, red fluorescent protein RFP, protein fragment G2 and epitope AYD connected in sequence; the sequence of the red fluorescent protein RFP is shown as SEQ ID NO: 7. The first vector is used to provide split green fluorescent protein fragment G2 labeled with double epitopes SYN and AYD.
[0040] Specifically, in order to obtain split green fluorescent protein fragment G2 labeled with double epitopes expressed alone, the cell culture solution is collected by filtration, and the secreted split green fluorescent protein fragment G2 labeled with double epitopes is obtained, which is then added into a new cell culture solution to be endocytosed into new cells.
[0041] In some embodiments, the second vector comprises anchor protein and nanobody NbA connected in sequence. The second vector is used to provide anchor protein specific to different compartments labeled with NbA.
[0042] In some embodiments, the third vector comprises target protein to be tested, split green fluorescent protein fragment G1 and nanobody NbS connected in sequence. The third vector is used to provide target protein to be tested connected with split green fluorescent protein fragment G1 labeled with NbS.
[0043] It should be noted that the split green fluorescent protein fragments in the first vector and the third vector are different, but the fluorescent protein fragment in the first vector can be split green fluorescent protein fragment G1, and the fluorescent protein fragment in the third vector can be split green fluorescent protein fragment G2, which can all have the same effect.
[0044] In some embodiments, the anchor protein is selected from the group consisting of SYP61, a marker compartment membrane protein of TGN / EE, and VSR4s, a vacuole sorting receptor; the protein to be tested is selected from the group consisting of ST, a trans-Golgi marker compartment membrane protein, and Man1, a cis-Golgi marker compartment membrane protein; ST, a trans-Golgi marker compartment membrane protein, and Man1, a cis-Golgi marker compartment membrane protein, are selected as an illustrative example of the application.
[0045] In some embodiments, the sequence of SYP61, a marker compartment membrane protein of trans-Golgi network / early endosome (TGN / EE), is shown as SEQ ID NO: 8; the sequence of VSR4s, a vacuole sorting receptor, is shown as SEQ ID NO: 9; the sequence of ST, a trans-Golgi marker compartment membrane protein, is shown as SEQ ID NO: 10; and the sequence of Man1, a cis-Golgi marker compartment membrane protein, is shown as SEQ ID NO: 11.
[0046] Specifically, when detecting the visual tracking of the model experiment of the target protein in the Golgi or trans-Golgi network / early endosome (TGN / EE) transport, the nanobody NbA connected to the TGN / EE marker compartment membrane protein SYP61 is used to target the endocytosed double epitope labeled split green fluorescent protein fragment G2 at the TGN / EE, and when the NbS and split green fluorescent protein fragment G1 labeled target protein is transported through the TGN / EE, the BiFC signal can be generated by the NbS-SYN interaction and the target protein can be targeted at the TGN / EE, realizing the tracking of the target protein in the TGN / EE.
[0047] In order to further track the transport in the Golgi, the vacuolar sorting receptor VSR4 is used, which can circulate between the TGN / EE and the Golgi, and when connected to the nanobody NbA, it can first bind to the endocytosed double epitope labeled split green fluorescent protein fragment G2 at the TGN / EE, and then circulate between the TGN / EE and the Golgi. When the target fusion protein is transported through the Golgi, the BiFC signal can be generated by the NbS-SYN interaction.
[0048] The schematic diagram of in vivo visualization of Golgi and TGN / EE using TGN / EE marker compartment membrane protein SYP61 and vacuolar sorting receptor VSR4 is as follows Figure 2As shown, the red G2 fragment fusion protein labeled with dual epitopes SYN and AYD, SYN-RFP-G2-AYD, is taken into the cell by endocytosis. ① Under the action of the TGN / EE compartment membrane protein SYP61 labeled with NbA, SYP61-NbA targets the endocytosed SYN-RFP-G2-AYD to the TGN / EE through the NbA-AYD interaction. When the test protein labeled with NbS and G1 fragments is sorted through the TGN / EE, the test fusion protein is targeted to the TGN / EE through the NbS-SYN interaction and generates a green fluorescence output signal based on BiFC, showing that it is sorted through the TGN / EE. Since the endocytosis pathway reaches the vacuole via the TGN / EE, multivesicular bodies / late endosomes (MVB / LE), therefore, the vacuole sorting receptor VSR4 labeled with NbA, NbA-VSR4, is used to make the endocytosed SYN-RFP-G2-AYD circulate between the TGN / EE and the Golgi apparatus under the NbA-AYD interaction. When the test protein labeled with NbS and G1 fragments only passes through the Golgi apparatus, the bound test fusion protein continues to circulate between the TGN / EE and the Golgi apparatus under the NbS-SYN interaction and is finally stabilized in the TGN / EE, thus generating a green fluorescence output signal that is located in the TGN / EE but indicates that it is transported through the Golgi apparatus; when the test protein labeled with NbS and G1 fragments is finally targeted to the Golgi apparatus, the bound test fusion protein will be located in the Golgi apparatus under the NbS-SYN interaction and generate a green fluorescence signal based on BiFC. If the sorting path of the test protein is unknown, both strategies can be used to track the Golgi apparatus and the TGN / EE at the same time, and the results can be analyzed and judged.
[0049] In some embodiments, the marker compartment membrane protein ST of the trans-Golgi and the marker compartment membrane protein Man1 of the cis-Golgi are used to simulate the transport of the test protein through the Golgi apparatus, thereby revealing that the BiFC system based on two groups of nanobodies and epitope interaction triggered by the present application can track the transport in the Golgi apparatus. This provides a new solution strategy for future drug targeted delivery, especially drug reverse delivery in a natural physiological state, and also provides the possibility of realizing the in vivo reverse real-time tracking of upstream organelles, especially the Golgi apparatus and the TGN, by biosensors.
[0050] In addition, the present application also provides a biosensor comprising a BiFC system triggered by two groups of nanobodies and epitope interaction.
[0051] In addition, the application also provides a two-molecular fluorescence complementation system triggered by the interaction of two groups of nanobodies and epitopes or the biosensor of claim 8 for tracking the transport of proteins in different cell compartments; and the application of the two-molecular fluorescence complementation system triggered by the interaction of two groups of nanobodies and epitopes or the biosensor of claim 8 in detecting drug targeted delivery.
[0052] The following further illustrates the application by examples. It should also be understood that the following examples are only used to further illustrate the application and cannot be understood as limiting the protection scope of the application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the application are within the protection scope of the application.
[0053] Example 1
[0054] This example uses a two-molecular fluorescence complementation system triggered by the interaction of two groups of nanobodies and epitopes to verify the transport of target proteins in the Golgi or TGN / EE, as follows:
[0055] 1) Protoplast isolation and gene expression
[0056] Protoplasts with electroporation ability were isolated according to the established procedure. After the 6-8 week tobacco leaves were punctured, they were incubated in TEX buffer (3.05 g / L Gamborg B5 medium, 500 mg / L MES, 750 mg / L CaCl2·2H2O, 250 mg / L NH4NO3, adjusted to pH 5.7 with KOH) containing 0.2% w / v macerozyme (macerozyme R-10, CAS NO. 9032-75-1, YAKULT HONSHA CO., Ltd., Japan) and 0.4% w / v cellulose (cellulose R-10, CAS NO. MX7352, YAKULT HONSHA CO., Ltd., Japan) and placed in the dark at 25°C overnight. The tobacco leaf protoplasts were filtered through a 100 μm nylon filter and rinsed with EPB electroporation buffer (137 g / L sucrose, 2.4 g / L HEPES, 6 g / L KCl, 600 mg / L CaCl2·2H2O, adjusted to pH 7.2 with KOH), centrifuged at 80 g for 15 minutes, resuspended, and the precipitate and excess EPB solution were removed using a peristaltic pump. For each electroporation, 2-5 x 10 6500 μl of protoplast suspension of one protoplast was mixed with 10-50 μg of plasmid DNA. Transfection was performed by applying a 160 V single square wave pulse for 10 ms (Gene Pulser Xcell™, Bio-Rad Laboratories Co., Ltd., Shanghai). The transfection system was divided into two groups: one group was used for endocytosis absorption experiment, and the transfection vector SYN-RFP-G2-AYD was used; the other group was used for subsequent confocal imaging experiment, and sample 1 (SYP61-CFP and SYP61-NbA), sample 2 (SYP61-CFP), sample 3 (SYP61-G1-NbS), sample 4 (NbA-VSR4 and ST-G1-NbS), sample 5 (SYP61-NbA and ST-G1-NbS), sample 6 (NbA-VSR4 and Man1-G1-NbS), and sample 7 (SYP61-NbA and Man1-G1-NbS) were used. After transfection, each sample was supplemented with 2 ml of TEX buffer and incubated at 25°C in the dark for 18-24 hours. CFP is a cyan fluorescent protein, and its sequence is shown as SEQ ID NO: 13.
[0057] 2) Endocytosis absorption experiment
[0058] The cell sample expressing only SYN-RFP-G2-AYD protein incubated at 25°C in the dark for 18-24 hours was taken out, and the solution containing the target fusion protein was collected by filtering with a 100 μm nylon filter membrane. In the subsequent confocal imaging experiment, the sample was replaced with TEX buffer as a culture solution and co-cultured with other expression samples 1-7, and the subsequent experiment was completed by absorption through the endocytosis pathway. After incubation at 25°C in the dark for 18-24 hours, the confocal microscope imaging experiment was performed.
[0059] 3) Confocal microscope imaging
[0060] Leica TCS-SP8 confocal laser scanning microscope with a x63 (1.2 numerical aperture) water immersion objective was used for confocal image acquisition of samples 1-7. Fluorophores were excited (ex) and emission (em) were detected by using HyD detectors in sequential mode with line switching: GFP (ex / em, 488 nm / 500-530 nm), RFP (ex / em, 552 nm / 580-640 nm) and CFP (ex / em, 448 nm / 460-490 nm). The pinhole was set to 1 Airy unit. Post-acquisition images were post-processed using ImageJ (v.1.51, https: / / imagej.nih.gov / ij / index.html), and the results are shown in FIGS. 1-7. Figure 3
[0061] wherein,Figure 3 The analysis and conclusions of a-j in the above are as follows:
[0062] a, endocytosed red bi-moiety G2 fragment fusion protein SYN-RFP-G2-AYD is targeted to TGN / EE by endocytosis pathway under the action of TGN / EE compartment membrane protein SYP61-NbA, and will be sorted into vacuole without the anchor protein.
[0063] b, endocytosed SYN-RFP-G2-AYD is prevented from being sorted into vacuole by NbA-AYD interaction under the action of NbA-labeled TGN / EE compartment membrane protein SYP61-NbA, as shown by red dots in the figure, and is co-localized with cyan TGN / EE compartment membrane protein-labeled SYP61-CFP, showing that it is targeted to TGN / EE, as indicated by the arrow in the figure.
[0064] c, without SYP61-NbA anchor protein, endocytosed SYN-RFP-G2-AYD is sorted into vacuole by endocytosis pathway, as shown by the central large vacuole in red in the figure, and is not co-localized with cyan TGN / EE compartment membrane protein-labeled SYP61-CFP. This result shows that G2 fragment can be targeted to target specific compartment under a group of nanobody-epitope interactions by endocytosis absorption, laying a foundation for subsequent visualization tracking in living cells.
[0065] d, an example schematic diagram simulating tracking of a target protein to be tested in TGN / EE transport. TGN / EE compartment membrane protein SYP61 is connected with NbS and G1 fragment, and endocytosed SYN-RFP-G2-AYD is targeted to TGN / EE by SYP61-G1-NbS under NbS-SYN interaction and produces green fluorescence output signal based on BiFC, showing that it is sorted through TGN / EE. The process of future target protein to be tested sorted through TGN / EE and visualized is simulated, the core of which is to achieve compartment-specific targeting of G2 fragment and BiFC produced by transport of target protein to be tested to specific compartment. In order to avoid BiFC completed at the initial stage of protein synthesis caused by co-expression, causing signal interference and unable to perform subsequent tracking of different cell compartments, G2 fragment fusion protein can be absorbed into cells by endocytosis pathway to achieve compartment-specific targeting. Therefore, two groups of nanobody-epitope interactions are needed to achieve: one group of nanobody-epitope interactions completes compartment-specific targeting of endocytosed G2 fragment, and the other group of nanobody-epitope interactions completes BiFC.
[0066] e、Results show that the target protein of interest transported through TGN / EE will produce BiFC green fluorescence output signal locating at TGN / EE under nanobody-epitope interaction, as indicated by the arrow in the figure, like NbS and G1 fragment labeled TGN / EE compartment membrane protein SYP61-G1-NbS.
[0067] f、Schematic diagram of the example of tracking the target protein of interest in Golgi transport using vacuolar sorting receptor VSR4. According to the characteristics of vacuolar sorting receptor VSR4 that can circulate between TGN / EE and Golgi, NbA-labeled VSR4 binds endocytosed SYN-RFP-G2-AYD in TGN / EE under NbA-AYD interaction and realizes its circulation between TGN / EE and Golgi. If the final destination of the target protein is Golgi, as shown in the figure, use Golgi membrane protein to connect G1 fragment and NbS, when NbA-VSR4-bound SYN-RFP-G2-AYD circulates to Golgi, it will bind the target fusion protein located in Golgi under NbS-SYN interaction and produce BiFC-based green fluorescence signal.
[0068] g and i use anti-Golgi marker compartment membrane protein ST or cis-Golgi marker compartment membrane protein Man1 to connect G1 fragment and NbS, respectively, and the results show that under NbS-SYN interaction, NbA-VSR4-bound SYN-RFP-G2-AYD is targeted to anti-Golgi or cis-Golgi and produces BiFC green fluorescence signal at the corresponding location, as indicated by the arrow in the figure.
[0069] Control groups h and j, if VSR4 is not used, but SYP61-NbA is used, G2 can only be targeted to TGN / EE and cannot reach Golgi, and G1 fragment and NbS-labeled anti-Golgi marker compartment membrane protein ST or cis-Golgi marker compartment membrane protein Man1 can only be located in Golgi and cannot be transported to TGN / EE, so no BiFC green fluorescence signal is produced, as shown in the BiFC channel of the figure.
[0070] wherein, NbS is an alpha-synuclein nanobody. SYN is an alpha-synuclein epitope. NbA is a beta-amyloid nanobody. AYD is a beta-amyloid epitope. G1 is a 1-10 beta chain of GFP. G2 is a 11 beta chain of GFP. SYP61 is a marker compartment membrane protein of TGN / EE. VSR4 is a vacuolar sorting receptor. ST is a marker compartment membrane protein of trans-Golgi. Man1 is a marker compartment membrane protein of cis-Golgi. RFP is a red fluorescent protein. CFP is a cyan fluorescent protein.
[0071] In summary, the present application provides a two-molecular fluorescence complementation system based on two groups of nanobody-epitope interaction triggering and its application, the two-molecular fluorescence complementation system comprises a first vector, a second vector and a third vector; the first vector comprises: an epitope SYN, an epitope AYD, and one protein fragment G2 in a split green fluorescent protein system; the second vector comprises: a nanobody NbA and an anchor protein; the third vector comprises: a nanobody NbS, and the other protein fragment G1 in the split green fluorescent protein system and a protein to be detected. The present application is based on a two-molecular fluorescence complementation (BiFC) technology triggered by nanobody-epitope interaction, two groups of different nanobody-epitope interactions are adopted, and endocytosis experiment is combined to realize specific compartment tracing of the protein to be detected by complementation of two split green fluorescent protein fragments; and the system does not utilize any drug treatment, realizes non-toxic visualization tracing of the transport process in the cell compartments upstream of the vacuole, mainly the Golgi and trans-Golgi network / early endosome (TGN / EE) in living cells; meanwhile, the two groups of nanobody-epitope adopted by the system not only have high specificity, but also do not affect the transport and other physiological processes of the proteins combined. It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can improve or change according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.
Claims
1. A bimolecular fluorescence complementation system based on the interaction of two sets of nanobodies and epitopes triggered, characterized in that, The bimolecular fluorescence complementation system comprises a first vector, a second vector and a third vector; The first vector comprises an epitope SYN, an epitope AYD, and one protein fragment G2 in a red fluorescent protein and split green fluorescent protein system; The second vector comprises a nanobody NbA and an anchor protein; The third vector comprises a nanobody NbS, and another protein fragment G1 in the split green fluorescent protein system and a protein to be detected; The sequence of the epitope SYN is shown as SEQ ID NO: 1, the sequence of the epitope AYD is shown as SEQ ID NO: 2, the sequence of the nanobody NbA is shown as SEQ ID NO: 3, and the sequence of the nanobody NbS is shown as SEQ ID NO:
4.
2. The two set of Nanobodies and epitope interaction triggered based bimolecular fluorescence complementation system according to claim 1, characterized in that, The protein fragment G1 comprises a protein fragment of amino acids 1-214 of a super-folded GFP protein; and the protein fragment G2 comprises a protein fragment of amino acids 215-233 of the super-folded GFP protein.
3. The two set of Nanobodies and epitope interaction triggered bimolecular fluorescence complementation system according to claim 2, characterized in that, The sequence of the protein fragment G1 is shown as SEQ ID NO:
5.
4. The two set of Nanobodies and epitope interaction triggered bimolecular fluorescence complementation system according to claim 2, characterized in that, The sequence of the protein fragment G2 is shown as SEQ ID NO:
6.
5. The dual molecular fluorescence complementation system based on two sets of nanobodies and epitope interaction-triggered according to claim 1, characterized in that, The first vector comprises, in sequence, an epitope SYN, a red fluorescent protein RFP, a protein fragment G2, and an epitope AYD; and the sequence of the red fluorescent protein RFP is shown as SEQ ID NO:
7.
6. The two set of Nanobodies and epitope interaction triggered bimolecular fluorescence complementation system according to claim 1, characterized in that, The anchor protein is a trans-Golgi network / early endosome marker compartment membrane protein SYP61, a vacuolar sorting receptor VSR4s; and the protein to be detected is a trans-Golgi marker compartment membrane protein ST and a cis-Golgi marker compartment membrane protein Man1.
7. The two set of Nanobodies and epitope interaction triggered bimolecular fluorescence complementation system according to claim 6, characterized in that, The sequence of the trans-Golgi network / early endosome marker compartment membrane protein SYP61 is shown as SEQ ID NO: 8; the sequence of the vacuolar sorting receptor VSR4s is shown as SEQ ID NO: 9; the sequence of the trans-Golgi marker compartment membrane protein ST is shown as SEQ ID NO: 10; and the sequence of the cis-Golgi marker compartment membrane protein Man1 is shown as SEQ ID NO:
11.
8. A biosensor characterized by The biosensor comprises two groups of nanobodies and epitope interaction triggered bimolecular fluorescence complementation systems according to any one of claims 1-7.
9. Use of the two groups of nanobodies and epitope interaction triggered bimolecular fluorescence complementation systems according to any one of claims 1-7 or the biosensor according to claim 8 for tracking protein transport in different cellular compartments.
10. Use of the two groups of nanobodies and epitope interaction triggered bimolecular fluorescence complementation systems according to any one of claims 1-7 or the biosensor according to claim 8 in detecting drug targeted delivery.