Bimolecular fluorescence complementation system based on nano antibody NbS and epitope SYN and application of bimolecular fluorescence complementation system

Abstract

The invention relates to the technical field of protein engineering, and discloses a bimolecular fluorescence complementation system based on a nano antibody NbS and an epitope SYN and application of the bimolecular fluorescence complementation system, the system comprises a first carrier and a second carrier, the first carrier comprises the epitope SYN and one of fluorescent protein fragments G1 and G2; the second carrier comprises a nano antibody NbS and one of fluorescent protein fragments G1 and G2; the fluorescent protein fragments in the first carrier and the second carrier are different; the sequence of the epitope SYN is as shown in SEQ ID NO: 1, the sequence of the nano antibody NbS is as shown in SEQ ID NO: 2, the fluorescent protein fragment G1 is a protein fragment containing amino acids at the first to 214th sites of the super-folded GFP protein, and the fluorescent protein fragment G2 is a protein fragment containing amino acids at the 215th to 238th sites of the super-folded GFP protein. According to the system, the sensitivity of the BiFC can be improved under the condition of reducing background signals, and on the basis of not depending on the existing protein-protein interaction in vivo, the BiFC is completed and can be used as a tool in vivo.

Description

Technical Field

[0001] The invention relates to the technical field of protein engineering, and in particular to a bimolecular fluorescence complementation system based on nano antibody NbS and epitope SYN and applications thereof. Background Art

[0002] Bimolecular fluorescence complementation (BiFC) is a non-invasive fluorescence-based technique that can directly visualize the subcellular localization of protein-protein interactions in living cells using confocal laser scanning microscopy. In theory, BiFC is based on the interaction between two proteins connected by two non-fluorescent split protein domains, and the co-folding of the two non-fluorescent split protein domains into a β-barrel structure under the interaction, resulting in the reconstitution of the fluorophore. Given the irreversibility of the split fluorescent protein assembly, this stability of BiFC can be used to capture transient interactions and low-affinity complexes.

[0003] Compared with the peak-shifted GFP (sGFP or GFP5) and enhanced GFP (EGFP) widely used in plant science, superfolded GFP (sfGFP) exhibits stronger folding ability and maturation efficiency, as well as resistance to oxidative environments. In transient expression experiments in Arabidopsis protoplasts, it was found that sfGFP was twice as bright as sGFP and 25% brighter than EGFP. Further comparative analysis also showed that the fluorescence intensity of the sfGFP-based BiFC (split-sfGFP) was the strongest among the five BiFC systems based on GFP variants. The split-sfGFP system usually divides the sfGFP sequence between the tenth and eleventh β-strands into two non-fluorescent parts: sfGFP1-10 and sfGFP11, of which sfGFP11 is a short amino acid peptide chain containing the conserved residue E222, and the sfGFP1-10 fragment contains the remaining three residues that constitute the sfGFP chromophore.

[0004] Although the split-sfGFP system has been used for protein quantification, visualization of protein subcellular localization, single-molecule imaging, detection of cell-cell contact sites, and in vitro protein complex assembly, there are still some defects or limitations: for example, BiFC is usually used to detect whether target proteins interact with each other in vivo, and its usage is relatively simple; for example, when the expression level of split fluorescent protein is high, non-specific interactions will occur, resulting in spontaneous self-assembly and accumulation of background signals.

[0005] At present, the BiFC system, especially the split-GFP system, is mainly used to detect the interaction between target proteins in cells, such as membrane proteins with hydrophobic properties that cannot be studied in vivo using biochemical methods, and the contact points between organelles and possible downstream cellular responses. However, these BiFCs based on the detection of interactions between target proteins in vivo are limited in the interaction reaction area due to the self-localization restrictions of the target proteins, so the specific details of judging and analyzing downstream cell pathways or in vivo transport are very limited. When studying unknown proteins in vivo, it is impossible to use BiFC based on general protein-protein interactions to study and reveal their sorting and transport pathways at the organelle level. On the other hand, in addition to exploring the interactions between target proteins, BiFC has not played a greater tool attribute, especially how to use it as a sensor to achieve subcellular level tracing in vivo. All of these are of great significance to the subsequent sorting and transportation of target molecules, especially the targeted delivery of drugs. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and to provide a bimolecular fluorescence complementation system based on nanoantibody NbS and epitope SYN and its application. The bimolecular fluorescence complementation system can expand the application scope of BiFC technology and improve the sensitivity of BiFC under the condition of reducing background signals. It can complete BiFC and use it as a tool in vivo without relying on existing protein-protein interactions in the body. At the same time, BiFC can be used as a sensor at the cellular level in the body to trace the target molecules at the organelle level.

[0007] In order to achieve the above object, the present invention provides a bimolecular fluorescence complementation system based on nanobody NbS-epitope SYN, which comprises a first carrier and a second carrier.

[0008] The first vector comprises: epitope SYN and one of fluorescent protein fragment G1 and fluorescent protein fragment G2;

[0009] The second vector comprises: nanobody NbS and one of fluorescent protein fragment G1 and fluorescent protein fragment G2; and the fluorescent protein fragments in the first vector and the second vector are different;

[0010] The sequence of the epitope SYN is shown as SEQ ID NO: 1, the sequence of the nanobody NbS is shown as SEQ ID NO: 2, the fluorescent protein fragment G1 is a protein fragment comprising amino acids 1 to 214 of the superfolded GFP protein, and the fluorescent protein fragment G2 is a protein fragment comprising amino acids 215 to 238 of the superfolded GFP protein.

[0011] Preferably, the sequence of the fluorescent protein fragment G1 is as shown in SEQ ID NO:4.

[0012] Preferably, the sequence of the fluorescent protein fragment G2 is as shown in SEQ ID NO:5.

[0013] Preferably, the first vector comprises a signal peptide SP, a fluorescent protein fragment G1, a protein tag HA and an epitope SYN connected in sequence, the signal peptide SP sequence is shown in SEQ ID NO: 6, and the protein tag HA sequence is shown in SEQ ID NO: 7;

[0014] Preferably, the second vector comprises a signal peptide SP, a nanobody NbS, a red fluorescent protein RFP, a fluorescent protein fragment G2 and an endoplasmic reticulum retention signal HDEL connected in sequence, the signal peptide SP sequence is shown in SEQ ID NO:6, the red fluorescent protein RFP sequence is shown in SEQ ID NO:8, and the endoplasmic reticulum retention signal HDEL sequence is shown in SEQ ID NO:9.

[0015] Preferably, the first vector comprises the epitope SYN, the red fluorescent protein RFP, the fluorescent protein fragment G2 and the endoplasmic reticulum retention signal HDEL connected in sequence;

[0016] Preferably, the second vector comprises VSS, cyan fluorescent protein CFP, fluorescent protein fragment G1 and nanobody NbS connected in sequence, wherein the sequence of VSS is as shown in SEQ ID NO:10, and the sequence of cyan fluorescent protein CFP is as shown in SEQ ID NO:11.

[0017] A second aspect of the present invention provides a biosensor, which comprises the bimolecular fluorescence complementation system as described above.

[0018] The third aspect of the present invention provides the use of the bimolecular fluorescence complementation system or the biosensor described above in tracing at the organelle level in vivo.

[0019] A fourth aspect of the present invention provides use of the bimolecular fluorescence complementation system as described above or the biosensor as described above in detecting targeted drug delivery.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0021] 1. The nanoantibody NbS-epitope SYN interaction used in the present invention can not only be used as a tool to replace the existing protein-protein interaction in the body to complete BiFC, but also reduce the background signal to a certain extent without large-scale expression to cause spontaneous fluorescence complementation, so that BiFC has higher specificity.

[0022] 2. A further core element of the present invention is that one of the split GFP fragments can be positioned at the target organelle or a certain site in the body as needed, and the second split GFP fragment can be connected to the protein or signal molecule to be detected without affecting its physiological process; when the split GFP fragment protein connected to the protein or signal to be detected reaches the target organelle or target site, the nano antibody NbS and epitope SYN connected to the split GFP fragment can output a detectable green fluorescent protein signal at the site of action or organelle that triggers BiFC through highly specific interactions between them. This BiFC triggered by the nano antibody NbS-epitope SYN interaction can be used as a sensor at the subcellular level in the body to reveal the sorting and transportation path of the detection protein or signal molecule in the body, providing new ideas for the study of targeted drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of the working principle of the bimolecular fluorescence complementation system in Example 1 of the present invention;

[0024] Figure 2 This is a test result diagram of a bimolecular fluorescence complementation experiment triggered by the nanobody NbS-epitope SYN interaction in the bimolecular fluorescence complementation system in Example 1 of the present invention;

[0025] Figure 3 is a test result diagram when the NbS-SYN nanobody-epitope interaction in Example 1 of the present invention is missing;

[0026] Figure 4 This is a graph showing the immunoprecipitation test results of the bimolecular fluorescence complementation system in Example 1 of the present invention;

[0027] Figure 5 is a schematic diagram of the working principle of the bimolecular fluorescence complementation system as a biosensor at the subcellular level in Example 2 of the present invention;

[0028] Figure 6 This is a test result diagram of the bimolecular fluorescence complementation system in Example 2 of the present invention used in a tracing experiment at the organelle level in living cells;

[0029] Figure 7 and Figure 8 This is a test result diagram when the NbS-SYN nanobody-epitope interaction in Example 2 of the present invention is missing. DETAILED DESCRIPTION

[0030] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0031] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0032] The present invention provides a bimolecular fluorescence complementation system based on nano antibody NbS-epitope SYN, the system comprising a first vector and a second vector, the first vector comprising: epitope SYN and one of a fluorescent protein fragment G1 and a fluorescent protein fragment G2; the second vector comprising: nano antibody NbS and one of a fluorescent protein fragment G1 and a fluorescent protein fragment G2; and the fluorescent protein fragments in the first vector and the second vector are different.

[0033] In the bimolecular fluorescence complementation system of the present invention, the epitope used is SYN (α-synuclein epitope), which comes from the NCBI database (NP_001362219.1), and the specific sequence is shown in SEQ ID NO:1.

[0034] In the bimolecular fluorescence complementation system described in the present invention, the nanobody adopts NbS (nanobody of α-synuclein), and the specific sequence is shown in SEQ ID NO:2.

[0035] In the bimolecular fluorescence complementation system of the present invention, the split GFP system is implemented using superfolded GFP (sfGFP), and the protein sequence is derived from the NCBI database. Specifically, the sequence of the superfolded GFP is shown in SEQ ID NO:3.

[0036] In the present invention, the split GFP system adopts a two-segment method, wherein the fluorescent protein G1 fragment contains 1-10β chains, specifically, the fluorescent protein fragment G1 is a protein fragment containing the 1st to 214th amino acids of the superfolded GFP protein; the fluorescent protein G2 fragment contains the 11th β chain, specifically, the fluorescent protein fragment G2 is a protein fragment containing the 215th to 238th amino acids of the superfolded GFP protein.

[0037] For the convenience of description, herein, the fluorescent protein fragment G1 may be abbreviated as GFP G1 fragment, and the fluorescent protein fragment G2 may be abbreviated as GFP G2 fragment.

[0038] In a specific embodiment, the sequence of the fluorescent protein fragment G1 is shown in SEQ ID NO:4.

[0039] In a specific embodiment, the sequence of the fluorescent protein fragment G2 is shown in SEQ ID NO:5.

[0040] In a specific embodiment, the first vector comprises the epitope SYN and the fluorescent protein G1, and the second vector comprises the nanobody NbS and the fluorescent protein G2.

[0041] Preferably, the first vector comprises a signal peptide SP, a fluorescent protein fragment G1, a protein tag HA and an epitope SYN connected in sequence to constitute an SP-G1-HA-SYN vector, and the G1-HA-SYN fusion protein is expressed and synthesized in vivo, and the signal peptide SP sequence is shown in SEQ ID NO:6, and the protein tag HA sequence is shown in SEQ ID NO:7; the second vector comprises a signal peptide SP, a nanobody NbS, a red fluorescent protein RFP, a fluorescent protein fragment G2 and an endoplasmic reticulum retention signal HDEL connected in sequence to constitute an SP-NbS-RFP-G2-HDEL vector, and the NbS-RFP-G2-HDEL fusion protein is expressed and synthesized in vivo, and the signal peptide SP sequence is shown in SEQ ID NO:6, the red fluorescent protein RFP sequence is shown in SEQ ID NO:8, and the endoplasmic reticulum retention signal HDEL sequence is shown in SEQ ID NO:9.

[0042] In another specific embodiment, the first vector comprises the epitope SYN and the fluorescent protein G2, and the second vector comprises the nanobody NbS and the fluorescent protein G1.

[0043] Preferably, the first vector comprises the epitope SYN, the red fluorescent protein RFP, the fluorescent protein fragment G2 and the endoplasmic reticulum retention signal HDEL connected in sequence; the second vector comprises VSS, the cyan fluorescent protein CFP, the fluorescent protein fragment G1 and the nanobody NbS connected in sequence, wherein the sequence of VSS is as shown in SEQ ID NO:10, and the sequence of the cyan fluorescent protein CFP is as shown in SEQ ID NO:11.

[0044] In the present invention, when detecting the bimolecular fluorescence complementation experiment triggered by the nanoantibody NbS-epitope SYN interaction, the nanoantibody NbS and the epitope SYN are respectively connected to two split GFP fragments, and the endoplasmic reticulum retention signal HDEL is used for auxiliary verification to increase the contact opportunity of the two fragments and expand the probability of BiFC for easy detection; when performing co-immunoprecipitation (Co-IP) experimental verification, the HA protein tag is introduced to connect the G1 fragment for auxiliary detection.

[0045] The present invention also provides a biosensor, which includes the bimolecular fluorescence complementation system as described above. The specific structure of the bimolecular fluorescence complementation system refers to the above embodiments. Since the biosensor of the present invention adopts all the technical solutions of all the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0046] The present invention also proposes the use of the bimolecular fluorescence complementation system or the biosensor described above in tracing at the organelle level in vivo.

[0047] The present invention also proposes the use of the bimolecular fluorescence complementation system as described above or the biosensor as described above in detecting targeted drug delivery.

[0048] In the present invention, when detecting bimolecular fluorescence complementation triggered by nano-antibody NbS-epitope SYN interaction for tracing experiments at the organelle level in living cells, a method of connecting the vacuolar sorting signal to the split GFP fragment is used to reveal that the present patent can be traced at the organelle level. Among them, the vacuolar sorting signal uses a sequence-specific vacuolar sorting signal (ssVSS) Aleurain (Aleu), whose protein sequence is shown in SEQ ID NO: 10; and is connected to a cyan fluorescent protein CFP, whose protein sequence is shown in SEQ ID NO: 11, to show the sorting and transport pathway of the split GFP fragment connected to the vacuolar sorting signal.

[0049] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0050] In the following examples, the protein sequences involved include:

[0051] SYN protein sequence (SEQ ID NO: 1):

[0052] MPIRRKQKGTAYLRKLIRNSASRSGENWRNGHETLRTSAFDVPFDLL CSSSCTQNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA.

[0053] NbS protein sequence (SEQ ID NO:2):

[0054] MQVQLQESGGGSVQTGGSLRLSCVASGYSGYMAWFRQAPGKEREG IAAIYRGDKITYYAHSVQGRFTISQANAKNTVYLLMNSLKPEDTAIYYCA ARRVVADSPLLSKTYAYWGQGTQVTVSS.

[0055] Super folded GFP full-length protein sequence (SEQ ID NO: 3):

[0056] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.

[0057] GFP G1 fragment protein sequence (SEQ ID NO: 4):

[0058] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYK TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEK.

[0059] GFP G2 fragment protein sequence (SEQ ID NO: 5):

[0060] RDHMVLLEFVTAAGITHGMDELYK.

[0061] Signal peptide SP protein sequence (SEQ ID NO: 6):

[0062] MRLCKFTALSSLLFSLLLLSASAM.

[0063] Protein tag HA protein sequence (SEQ ID NO: 7):

[0064] YPYDVPDYA.

[0065] Red fluorescent protein RFP protein sequence (SEQ ID NO: 8):

[0066] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQ DGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGARSM.

[0067] Endoplasmic reticulum retention signal HDEL protein sequence (SEQ ID NO: 9):

[0068] HDEL.

[0069] Protein sequence of sequence-specific vacuolar sorting signal (ssVSS)-Aleurain (Aleu) (SEQ ID NO: 10):

[0070] MSRLSLLLVLVAGLFAVAFARTANFADENPIRQVVSDSFHELESASA. Cyan fluorescent protein CFP protein sequence (SEQ ID NO: 11):

[0071] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0072] Example 1

[0073] This example is used to illustrate the bimolecular fluorescence complementation system triggered by the nanobody NbS-epitope SYN interaction provided by the present invention and its working principle.

[0074] In this example, immunoprecipitation (anti-RFP antibody coated magnetic beads; IP, α-RFP) was performed and immunoblotting (IB) was probed with antibodies to detect anchors (α-RFP and α-HA).

[0075] NbS, α-synuclein nanobody.

[0076] SYN, α-synuclein epitope.

[0077] G1, 1-10 β-strand of GFP.

[0078] G2, the 11th beta chain of GFP.

[0079] HDEL, endoplasmic reticulum retention signal.

[0080] VSS, vacuolar sorting signal.

[0081] Aleu, Aleurain, one of the vacuolar sorting signals.

[0082] 1. Experimental methods:

[0083] (1) Protoplast isolation and gene expression

[0084] Protoplasts with electrotransformation ability were isolated according to established procedures; 6-8 week old tobacco leaves were perforated and 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 Co., Ltd.) and placed in 25°C dark and cultured overnight. The cells were filtered through a 100 μm nylon filter and resuspended in EPB electrofection buffer (137 g / L sucrose, 2.4 g / L HEPES, 6 g / L KCl, 600 mg / L CaCl2·2H2O, pH adjusted to 7.2 with KOH) by centrifugation at 80 g for 15 min. This process was repeated five times. 6500 μl of protoplast suspension of each protoplast was mixed with 10-50 μg of plasmid DNA; transfection was performed by applying a 160V single square wave pulse for 10 ms (Gene Pulser XcellTM, Bio-Rad Laboratories Co., Ltd., Shanghai). After transfection, each sample was supplemented with 2 ml of TEX buffer and incubated at 25°C in the dark for 18-24 hours.

[0085] (2) Confocal microscopy imaging

[0086] Confocal image acquisition was performed using a Leica TCS-SP8 confocal laser scanning microscope with a ×63 (1.2 numerical aperture) water immersion objective. Fluorophores were excited (ex) and emitted (em) detected by line switching in sequential mode using a HyD detector: GFP (ex / em, 488nm / 500-530nm), RFP (ex / em, 552nm / 580-640nm) and CFP (ex / em, 448nm / 460-490nm); the pinhole was set to 1 Airy unit; after acquisition, images were post-processed using ImageJ (v.1.51, https: / / imagej.nih.gov / ij / index.html).

[0087] (3) Protein extraction and co-immunoprecipitation (Co-IP)

[0088] The protoplast cells described above were diluted five times with 250mM NaCl and centrifuged at 80g for 10 minutes to precipitate the cells. After resuspending the cell pellet, 2× binding buffer (40mM HEPES, 300mM NaCl, 2mMCaCl2, 2mM MgCl2, pH 7.1) was added in a 1:1 ratio, mixed at 4°C for 30 minutes, and then centrifuged at 12,000g for 15 minutes to collect the supernatant. The magnetic beads containing the antibody were fused with the collected supernatant at 4°C for 60-120 minutes. The magnetic beads were then collected with a magnetic plate and rinsed with binding buffer, and then the loading buffer was added to mix with the magnetic beads, denatured at 95°C for 10 minutes, and then loaded for use. The antibodies used in subsequent incubations were as follows: mouse monoclonal anti-RFP (ChromoTek, CAS NO. 6g6, 1:2,000) and goat anti-mouse IgG (H+L) labeled with horseradish peroxidase (HRP) (Immunoway, CAS NO. RS0001, 1:20,000) as secondary antibodies, and rat monoclonal anti-HA-peroxidase (Roche 12013819001, 1:3,000).

[0089] 2. Bimolecular fluorescence complementation system.

[0090] The first vector is composed of a signal peptide SP, a fluorescent protein fragment G1, a protein tag HA and an epitope SYN connected in sequence to form an SP-G1-HA-SYN vector, which expresses and synthesizes a G1-HA-SYN fusion protein in vivo.

[0091] The second vector is composed of a signal peptide SP, a nanobody NbS, a red fluorescent protein RFP, a fluorescent protein fragment G2 and an endoplasmic reticulum retention signal HDEL connected in sequence, forming an SP-NbS-RFP-G2-HDEL vector, which expresses and synthesizes the NbS-RFP-G2-HDEL fusion protein in vivo.

[0092] 3. The working principle and verification results of the bimolecular fluorescence complementation system.

[0093] (1) Working principle of the bimolecular fluorescence complementation system

[0094] The bimolecular fluorescence complementation system of the present invention is based on bimolecular fluorescence complementation (BiFC) triggered by the interaction between the nanoantibody NbS and the epitope SYN. Figure 1 shown.

[0095] In the bimolecular fluorescence complementation system, the core element is the highly specific interaction between the split GFP G2 fragment (the 11th β-chain, i.e., the protein fragment consisting of amino acids 215 to 238 of the superfolded GFP protein) labeled with the nanobody NbS and the second split GFP G1 fragment (1-10 β-chain, i.e., the protein fragment consisting of amino acids 1 to 214 of the superfolded GFP protein) labeled with the corresponding epitope SYN, which triggers the close proximity of the G1 and G2 BiFC fragments, thereby generating a detectable BiFC-based fluorescence output signal.

[0096] (2) The NbS-labeled G2 fragment is linked to the red fluorescent protein RFP. Under the action of the endoplasmic reticulum ER retention signal HDEL, the fusion protein NbS-RFP-G2-HDEL stays in the endoplasmic reticulum. When the SYN-labeled G1 fragment, i.e., the fusion protein G1-HA-SYN, is expressed at the same time, the NbS-SYN nanobody-epitope interaction causes G1 and G2 to fuse in the endoplasmic reticulum ER, the fluorescent chromophore is reconstructed, and a detectable GFP green fluorescent protein signal is output ( Figure 2 ).

[0097] (3) When the NbS-SYN nanobody-epitope interaction is missing, G1 and G2 cannot fuse and reassemble the fluorescent chromophore to complete BiFC, so there is no GFP signal ( Figure 3 ).

[0098] (4) Co-immunoprecipitation (Co-IP) results Figure 4As shown, the results demonstrate the specificity of the NbS-SYN nanobody-epitope interaction.

[0099] The above results show that in the dual fluorescent molecular complementation system provided by the present invention, the nanoantibody NbS-epitope SYN interaction can not only be used as a tool to replace protein-protein interaction to complete BiFC, but also reduce the background signal to a certain extent without large-scale expression to cause spontaneous fluorescence complementation, so that BiFC has higher specificity.

[0100] Example 2

[0101] This example is used to illustrate the bimolecular fluorescence system triggered by the nanobody NbS-epitope SYN interaction provided by the present invention and its use for tracing at the organelle level in living cells.

[0102] 1. Bimolecular fluorescence complementation system.

[0103] The first vector contains the epitope SYN, the red fluorescent protein RFP, the fluorescent protein fragment G2 and the endoplasmic reticulum retention signal HDEL which are linked in sequence.

[0104] The second vector contains VSS, cyan fluorescent protein CFP, fluorescent protein fragment G1 and nanobody NbS connected in sequence.

[0105] 2. The working principle and test results of the bimolecular fluorescence complementation system for tracing organelle levels in living cells, wherein the test method refers to Example 1.

[0106] (1) The working principle of the bimolecular fluorescence complementation system based on the nanoantibody NbS-epitope SYN interaction as a biosensor for subcellular tracking is as follows Figure 5 shown.

[0107] The nanobody NbS marks the split GFP G1 fragment and connects to the vacuolar sorting signal (VSS) to be detected. The corresponding epitope SYN marks the second split GFP G2 fragment and uses the endoplasmic reticulum ER retention signal HDEL to locate the G2 fusion protein in the endoplasmic reticulum lumen. When BiFC occurs, it indicates that the vacuolar sorting signal fusion protein is retained in the endoplasmic reticulum lumen through the nanobody NbS-epitope SYN interaction, revealing that the vacuolar sorting signal reaches the vacuole through the endoplasmic reticulum, completing the tracing of the vacuolar sorting signal.

[0108] (2) The SYN-labeled G2 fragment is linked to the red fluorescent protein RFP. Under the action of the ER retention signal HDEL, SYN-RFP-G2-HDEL is localized in the ER lumen. The second split G1 fragment is linked to one of the vacuolar sorting signals (VSS) to be tested, Aleu, through the NbS-labeled cyan fluorescent protein CFP to form Aleu-CFP-G1-NbS fusion protein. When co-expressed, the GFP green fluorescent protein signal is formed in the ER lumen through the NbS-SYN nanobody-epitope interaction, and the cyan fluorescent protein CFP signal also appears in the ER ( Figure 6 ), indicating that the vacuolar sorting signal Aleu is sorted and transported to the vacuole through the endoplasmic reticulum.

[0109] (3) The results of no NbS-SYN nanobody-epitope interaction are as follows Figure 7 and Figure 8 As shown, the results showed that without triggered BiFC, the cyan fluorescent protein CFP signal filled the vacuole, indicating that the vacuolar sorting signal Aleu fusion protein could not be retained in the ER lumen through the nanobody NbS-epitope SYN interaction, indicating that its sorting transport pathway passed through the ER.

[0110] The above results show that the further core element of the dual fluorescent molecular complementation system described in the present invention is that one of the split GFP fragments needs to be located at the target organelle or a certain site in the body, and the second split GFP fragment can be connected to the protein or signal molecule to be detected. When the split GFP fragment protein connected to the protein or signal to be detected reaches the target organelle or target site, the nano antibody NbS and the epitope SYN connected to the split GFP fragment can output a detectable green fluorescent protein signal at the site of action or organelle that triggers BiFC through a highly specific interaction between them, thereby showing the sorting and transportation path of the protein or signal molecule to be detected in the body.

[0111] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A bimolecular fluorescence complementation system based on nanobody NbS and epitope SYN, characterized in that: The system includes a first carrier and a second carrier, The first vector comprises: epitope SYN and one of fluorescent protein fragment G1 and fluorescent protein fragment G2; The second vector comprises: nanobody NbS and one of fluorescent protein fragment G1 and fluorescent protein fragment G2; and the fluorescent protein fragments in the first vector and the second vector are different; The sequence of the epitope SYN is shown as SEQ ID NO: 1, the sequence of the nanobody NbS is shown as SEQ ID NO: 2, the fluorescent protein fragment G1 is a protein fragment comprising amino acids 1 to 214 of the superfolded GFP protein, and the fluorescent protein fragment G2 is a protein fragment comprising amino acids 215 to 238 of the superfolded GFP protein.

2. The bimolecular fluorescence complementation system according to claim 1, characterized in that: The sequence of the fluorescent protein fragment G1 is shown in SEQ ID NO:

4.

3. The bimolecular fluorescence complementation system according to claim 1, characterized in that: The sequence of the fluorescent protein G2 is shown in SEQ ID NO:

5.

4. The bimolecular fluorescence complementation system according to claim 1 or 2, characterized in that: The first vector comprises a signal peptide SP, a fluorescent protein fragment G1, a protein tag HA and an epitope SYN connected in sequence, wherein the signal peptide SP sequence is shown in SEQ ID NO: 6, and the protein tag HA sequence is shown in SEQ ID NO: 7; Preferably, the second vector comprises a signal peptide SP, a nanobody NbS, a red fluorescent protein RFP, a fluorescent protein fragment G2 and an endoplasmic reticulum retention signal HDEL connected in sequence, the signal peptide SP sequence is shown in SEQ ID NO:6, the red fluorescent protein RFP sequence is shown in SEQ ID NO:8, and the endoplasmic reticulum retention signal HDEL sequence is shown in SEQ ID NO:

9.

5. The bimolecular fluorescence complementation system according to claim 1, characterized in that: The first vector comprises the epitope SYN, the red fluorescent protein RFP, the fluorescent protein fragment G2 and the endoplasmic reticulum retention signal HDEL connected in sequence; Preferably, the second vector comprises VSS, cyan fluorescent protein CFP, fluorescent protein fragment G1 and nanobody NbS connected in sequence, wherein the sequence of VSS is as shown in SEQ ID NO:10, and the sequence of cyan fluorescent protein CFP is as shown in SEQ ID NO:

11.

6. A biosensor, characterized in that: The biosensor comprises the bimolecular fluorescence complementation system according to any one of claims 1-5.

7. Use of the bimolecular fluorescence complementation system according to any one of claims 1 to 5 or the biosensor according to claim 6 in tracing at the organelle level in vivo.

8. Use of the bimolecular fluorescence complementation system according to any one of claims 1 to 5 or the biosensor according to claim 6 in detecting targeted drug delivery.

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