A bimolecular fluorescence complementation system based on nanobody NbS and epitope SYN and application thereof

Abstract

The application relates to the technical field of protein engineering, and discloses a bimolecular fluorescence complementation system based on a nanobody NbS and an epitope SYN and application thereof. The system comprises a first carrier and a second carrier. The first carrier comprises an epitope SYN and one of fluorescent protein fragments G1 and G2. The second carrier comprises a nanobody 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 shown in SEQ ID NO: 1. The sequence of the nanobody NbS is shown in SEQ ID NO: 2. The fluorescent protein fragment G1 is a protein fragment comprising the 1-214th amino acid of a super-folded GFP protein. The fluorescent protein fragment G2 is a protein fragment comprising the 215-238th amino acid of the super-folded GFP protein. The system can improve the sensitivity of BiFC under the condition of reducing background signals. On the basis of not depending on existing protein-protein interaction in the body, the BiFC can be completed and used as a tool in the body.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein engineering, and particularly relates to a BiFC system based on nanobody NbS and epitope SYN and application thereof. BACKGROUND

[0002] BiFC is a non-invasive fluorescence-based technique that allows direct visualization of 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 split protein domains without fluorescence, and the two split protein domains without fluorescence are folded into a beta barrel structure under the interaction, thereby causing the reconstruction of the fluorophore. Due to the irreversibility of split fluorescent protein assembly, the stability of BiFC can be used to capture transient interactions and low-affinity complexes.

[0003] Compared with the peak-shift GFP (sGFP or GFP5) and enhanced GFP (EGFP) widely used in plant science, super-fold GFP (sfGFP) shows stronger folding ability and maturation efficiency, and resistance to oxidative environment. In the transient expression experiment of Arabidopsis protoplast, it is found that the brightness of sfGFP is twice that of sGFP, and also 25% brighter than EGFP. Further comparative analysis also shows that the fluorescence intensity of split-sfGFP based on sfGFP is 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 beta strands into two parts that do not emit fluorescence: sfGFP1-10 and sfGFP11, wherein sfGFP11 is a short amino acid peptide chain containing the conserved residue E222, and the sfGFP1-10 fragment contains the remaining three residues that make up the sfGFP chromophore.

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

[0005] Currently, BiFC system, especially split-GFP system, is mainly used to detect the interaction between target proteins in cells, such as membrane proteins with hydrophobic characteristics which cannot be studied in vivo by biochemical methods, and the contact points between organelles and the possible downstream cellular responses. However, these BiFCs based on the interaction between target proteins in vivo have limited interaction reaction regions due to the localization of target proteins, and thus the specific details of downstream cellular pathways or in vivo transport are very limited. When studying unknown proteins in vivo, BiFC based on protein-protein interaction under normal conditions cannot be used to study and reveal the sorting and transport pathways at the organelle level. On the other hand, BiFC has not been used as a sensor for subcellular level tracking in vivo, which has important significance for the subsequent sorting and transport of target molecules, especially the targeted delivery of drugs. SUMMARY

[0006] The purpose of the present application is to overcome the problems existing in the prior art, provide a BiFC system based on nanobody NbS and epitope SYN and application thereof, which can expand the application range of BiFC technology and improve the sensitivity of BiFC under the condition of reducing background signal, complete BiFC without relying on existing protein-protein interaction in vivo and use BiFC as a tool in vivo; at the same time, BiFC can be used as a sensor at the cellular level in vivo to track target molecules at the organelle level.

[0007] To achieve the above purpose, the present application provides a BiFC system based on nanobody NbS-epitope SYN, which comprises a first carrier and a second carrier,

[0008] The first carrier comprises an epitope SYN and one of a fluorescent protein fragment G1 and a fluorescent protein fragment G2;

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

[0010] The sequence of the epitope SYN is shown in SEQ ID NO: 1, the sequence of the nanobody NbS is shown in SEQ ID NO: 2, the fluorescent protein fragment G1 is a protein fragment comprising the 1st-214th amino acid of a super-folded GFP protein, and the fluorescent protein fragment G2 is a protein fragment comprising the 215th-238th amino acid of a super-folded 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 sequence of the signal peptide SP is as shown in SEQ ID NO: 6, and the sequence of the protein tag HA is as 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 sequence of the signal peptide SP is as shown in SEQ ID NO: 6, the sequence of the red fluorescent protein RFP is as shown in SEQ ID NO: 8, and the sequence of the endoplasmic reticulum retention signal HDEL is as shown in SEQ ID NO: 9.

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

[0016] Preferably, the second vector comprises a VSS, a cyan fluorescent protein CFP, a fluorescent protein fragment G1 and a nanobody NbS connected in sequence, the sequence of the 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.

[0017] The second aspect of the present application provides a biosensor comprising the dual-molecular fluorescence complementation system as described above.

[0018] The third aspect of the present application provides an application of the dual-molecular fluorescence complementation system as described above or the biosensor as described above in tracking of organelle level in vivo.

[0019] The fourth aspect of the present application provides an application of the dual-molecular fluorescence complementation system as described above or the biosensor as described above in detection of drug targeted delivery.

[0020] Compared with the prior art, the present application has the following advantages and beneficial effects:

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

[0022] 2, A further core element of the present application is that one of the split GFP fragments can be targeted to a desired organelle or a certain in vivo site as needed, and the second split GFP fragment can be linked to a protein or signal molecule to be detected without affecting its physiological process; when the split GFP fragment protein linked to the protein or signal to be detected reaches the target organelle or target site, the nanobody NbS and the epitope SYN linked on the split GFP fragment can output a detectable green fluorescent protein signal at the action site or organelle of the triggered BiFC through the highly specific interaction therebetween. This BiFC triggered by the nanobody NbS-epitope SYN interaction can serve as a subcellular level sensor to reveal the sorting and transport path of the protein or signal molecule in vivo, and provide a new idea for the targeted delivery of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a working principle schematic diagram of the BiFC system in Example 1 of the present application;

[0024] Figure 2 is a test result diagram of the BiFC experiment triggered by the nanobody NbS-epitope SYN interaction in the BiFC system in Example 1 of the present application;

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

[0026] Figure 4 is a co-immunoprecipitation test result diagram of the BiFC system in Example 1 of the present application;

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

[0028] Figure 6 is a test result diagram of the BiFC system in Example 2 of the present application in a tracing experiment at the organelle level of living cells in vivo;

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

[0030] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0031] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not to be understood as being crucial to the invention. Any numeric range recited is intended to include all derivatives of and combinations of the recited range limits. Numeric ranges include all values and subranges between the upper and lower ends of the recited range.

[0032] The application provides a bimolecular fluorescence complementation system based on nanobody NbS-epitope SYN, which comprises a first carrier and a second carrier, the first carrier comprises: epitope SYN and one of fluorescent protein fragment G1 and fluorescent protein fragment G2; the second carrier comprises: nanobody NbS and one of fluorescent protein fragment G1 and fluorescent protein fragment G2; and the fluorescent protein fragments in the first carrier and the second carrier are different.

[0033] In the bimolecular fluorescence complementation system, the epitope is SYN (alpha-synuclein epitope), which is from the NCBI database (NP_001362219.1), and the specific sequence is shown as SEQ ID NO: 1.

[0034] In the bimolecular fluorescence complementation system, the nanobody is NbS (nanobody of alpha-synuclein), and the specific sequence is shown as SEQ ID NO: 2.

[0035] In the bimolecular fluorescence complementation system, the split GFP system is realized by super-fold GFP (sfGFP), and the protein sequence is from the NCBI database. Specifically, the sequence of the super-fold GFP is shown as SEQ ID NO: 3.

[0036] In the application, the split GFP system adopts a two-segment method, wherein the fluorescent protein G1 fragment comprises 1-10 beta chains, specifically, the fluorescent protein fragment G1 is a protein fragment comprising amino acids 1-214 of the super-fold GFP protein; the fluorescent protein G2 fragment comprises the 11th beta chain, specifically, the fluorescent protein fragment G2 is a protein fragment comprising amino acids 215-238 of the super-fold GFP protein.

[0037] For the convenience of description, in the present application, the fluorescent protein fragment G1 can be abbreviated as GFP G1 fragment, and the fluorescent protein fragment G2 can be abbreviated as GFP G2 fragment.

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

[0039] In a specific embodiment, the sequence of the fluorescent protein fragment G2 is shown as 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, constituting a SP-G1-HA-SYN vector, for in vivo expression of a G1-HA-SYN fusion protein, wherein the sequence of the signal peptide SP is shown as SEQ ID NO: 6, and the sequence of the protein tag HA is shown as SEQ ID NO: 7; and 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, constituting a SP-NbS-RFP-G2-HDEL vector, for in vivo expression of a NbS-RFP-G2-HDEL fusion protein, wherein the sequence of the signal peptide SP is shown as SEQ ID NO: 6, the sequence of the red fluorescent protein RFP is shown as SEQ ID NO: 8, and the sequence of the endoplasmic reticulum retention signal HDEL is shown as 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 an epitope SYN, a red fluorescent protein RFP, a fluorescent protein fragment G2 and an endoplasmic reticulum retention signal HDEL connected in sequence; and the second vector comprises a VSS, a cyan fluorescent protein CFP, a fluorescent protein fragment G1 and a nanobody NbS connected in sequence, wherein the sequence of the VSS is shown as SEQ ID NO: 10, and the sequence of the cyan fluorescent protein CFP is shown as SEQ ID NO: 11.

[0044] In the present application, when detecting the BiFC experiment triggered by the nanobody NbS-epitope SYN interaction, the nanobody NbS and the epitope SYN are connected to two split GFP fragments respectively, and the endoplasmic reticulum retention signal HDEL is used for auxiliary verification, which increases the contact opportunity of the two fragments and expands the probability of BiFC for convenient detection; when verifying by Co-IP experiment, the HA protein tag is introduced to connect the G1 fragment for auxiliary detection.

[0045] The application also provides a biosensor comprising the above-mentioned bimolecular fluorescence complementation system. The specific structure of the bimolecular fluorescence complementation system is described in the above-mentioned embodiments. Since the biosensor of the application adopts all the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described here one by one.

[0046] The application also provides the use of the above-mentioned bimolecular fluorescence complementation system or the above-mentioned biosensor in in vivo organelle level tracing.

[0047] The application also provides the use of the above-mentioned bimolecular fluorescence complementation system or the above-mentioned biosensor in detecting drug targeted delivery.

[0048] In the application, in the detection of bimolecular fluorescence complementation based on nanobody NbS-epitope SYN interaction triggering for in vivo organelle level tracing experiment of living cells, the method of connecting vacuole sorting signal to split GFP fragment is used to reveal that the patent can be tracked at the organelle level. The vacuole sorting signal is Aleurain (Aleu) of sequence-specific vacuole sorting signal (ssVSS), and its protein sequence is shown as SEQ ID NO: 10; and the cyan fluorescence protein CFP is connected, and its protein sequence is shown as SEQ ID NO: 11, which is used to show the sorting transport pathway of the split GFP fragment connected with the vacuole sorting signal.

[0049] The application will be described in detail through the following examples, but the protection scope of the application 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-fold GFP full-length protein sequence (SEQ ID NO: 3):

[0056] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.

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

[0058] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEK.

[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] The sequence of the red fluorescent protein RFP (SEQ ID NO:8):

[0066] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQ DGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGARSM.

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

[0068] HDEL.

[0069] The protein sequence of sequence-specific vacuole 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 embodiment illustrates the bimolecular fluorescence complementary system based on the NbS-epitope SYN interaction triggered by nanobody provided by the present invention and its working principle.

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

[0075] NbS, a-synuclein nanobody.

[0076] SYN, a-synuclein epitope.

[0077] G1, 1-10 beta chain of GFP.

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

[0079] HDEL, endoplasmic reticulum (ER) 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 electroporation competence were isolated according to established procedures; 6-8 weeks 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 (cellulase R-10, CAS NO. MX7352, YAKULT HONSHA CO., Ltd., Japan) and placed in the dark at 25°C overnight. Cells were filtered through a 100 pm nylon filter and resuspended by centrifugation in 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) at 15 minutes, 80 g, and this process was repeated five times. For each electroporation, approximately 2-5 x 10 6Five hundred μΐ of protoplast suspension 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). 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 images were acquired using a Leica TCS-SP8 confocal laser scanning microscope with a x63 (1.2 numerical aperture) water immersion objective. Fluorophores were excited (ex) and emission (em) detected by line switching using HyD detectors in sequential mode: 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); 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).

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

[0088] Protoplast cells as described above were diluted five times with 250 mM NaCl and cells were pelleted by centrifugation at 80 g for 10 min. After resuspension of the cell pellet, 2x binding buffer (40 mM HEPES, 300 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.1) was added 1 : 1 and mixed at 4 °C for 30 min, after which the supernatant was collected by centrifugation at 12,000 g for 15 min. Antibody-containing magnetic beads were fused with the collected supernatant at 4 °C for 60-120 min. The magnetic beads were then collected with a magnetic plate and rinsed with binding buffer, after which loading buffer was added to the magnetic beads and mixed, denatured at 95 °C for 10 min and ready for loading. The antibodies used for subsequent incubation 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 a secondary antibody, and rat monoclonal anti-HA-peroxidase (Roche 12013819001, 1 :3,000).

[0089] 2. Bimolecular fluorescence complementation system.

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

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

[0092] 3. Working principle and verification results of the BiFC system.

[0093] (1) Working principle of the BiFC system

[0094] The BiFC system described in the present application is a BiFC triggered by the interaction between the nanobody NbS and the epitope SYN, and its principle is shown in Figure 1 .

[0095] In the BiFC system, the key element is the highly specific interaction between the NbS-labeled split GFP G2 fragment (11th beta chain, i.e., a protein fragment composed of amino acids 215-238 of the super-folded GFP protein) and the corresponding SYN-labeled second split GFP G1 fragment (1-10 beta chain, i.e., a protein fragment composed of amino acids 1-214 of the super-folded GFP protein), which triggers the close proximity of G1 and G2 BiFC fragments, thereby producing a detectable BiFC-based fluorescent output signal.

[0096] (2) The NbS-labeled G2 fragment is connected to the red fluorescent protein RFP, and under the action of the endoplasmic reticulum ER resident signal HDEL, the fusion protein NbS-RFP-G2-HDEL is retained 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 in the endoplasmic reticulum ER causes G1 and G2 to fuse, the fluorescent chromophore is reconstituted, and a detectable GFP green fluorescent protein signal Figure 2 ) is output.

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

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

[0099] The above results show that the nanobody NbS-epitope SYN interaction in the double fluorescent molecule complementary system provided by the application can not only replace protein-protein interaction to complete BiFC as a tool, but also reduce the background signal to a certain extent without causing spontaneous fluorescence complementation caused by large-scale expression, so that BiFC has high specificity.

[0100] Example 2

[0101] This example is used to illustrate the double-molecule fluorescent system based on the nanobody NbS-epitope SYN interaction trigger and its use for tracking at the organelle level in living cells in vivo.

[0102] 1. Double-molecule fluorescent complementary system.

[0103] The first vector comprises, in sequence, the epitope SYN, the red fluorescent protein RFP, the fluorescent protein fragment G2, and the endoplasmic reticulum resident signal HDEL.

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

[0105] 2. Working principle and test results of the double-molecule fluorescent complementary system for tracking at the organelle level in living cells in vivo, wherein the test method refers to Example 1.

[0106] (1) The working principle of the double-molecule fluorescent complementary system based on the nanobody NbS-epitope SYN interaction trigger as a biological sensor for tracking at the subcellular level is shown in Figure 5 .

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

[0108] (2) The G2 fragment labeled with SYN is connected with red fluorescent protein RFP under the action of endoplasmic reticulum ER resident signal HDEL, so that SYN-RFP-G2-HDEL is positioned in the endoplasmic reticulum ER lumen; the second split G1 fragment is connected with one of the to-be-detected vacuole sorting signals (VSS) Aleu to form Aleu-CFP-G1-NbS fusion protein through the blue fluorescent protein CFP labeled with NbS; when co-expressed, the GFP green fluorescent protein signal is formed in the endoplasmic reticulum lumen through the NbS-SYN nanobody-epitope interaction, and the blue fluorescent protein CFP signal also appears in the endoplasmic reticulum (ER) Figure 6 ), indicating that the vacuole sorting signal Aleu is transported to the vacuole through the endoplasmic reticulum sorting.

[0109] (3) The results without the NbS-SYN nanobody-epitope interaction are shown in Figure 7 and Figure 8 , and the results show that, without triggering the BiFC, the blue fluorescent protein CFP signal fills the vacuole, indicating that the vacuole sorting signal Aleu fusion protein cannot be left in the endoplasmic reticulum lumen to show its sorting transport pathway through the endoplasmic reticulum through the nanobody NbS-epitope SYN interaction.

[0110] The above results show that a further core element of the double fluorescent molecule complementary system described in the present application is that one of the split GFP fragments needs to be positioned in a target organelle or a certain in-vivo site, and the second split GFP fragment can be connected with a to-be-detected protein or signal molecule. When the split GFP fragment protein connected with the to-be-detected protein or signal reaches the target organelle or target site, the nanobody NbS and the epitope SYN connected on the split GFP fragment can output a detectable green fluorescent protein signal at the triggering BiFC action site or organelle through the highly specific interaction therebetween, so as to show the sorting transport path of the to-be-detected protein or signal molecule in the body.

[0111] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept range of the present application, various simple modifications can be made to the technical solutions of the present application, including that various technical features are combined in any other suitable manner, and these simple modifications and combinations should also be considered as the disclosed content of the present application, and all fall within the protection scope of the present application.

Claims

1. A bimolecular fluorescence complementary 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 carrier comprises: nanobody NbS and one of fluorescent protein fragments G1 and G2; and the fluorescent protein fragments in the first carrier are different from those in the second carrier. The sequence of the epitope SYN is shown in SEQ ID NO:1, the sequence of the nanobody NbS is shown in SEQ ID NO:2, the fluorescent protein fragment G1 is a protein fragment of amino acids 1 to 214 of the superfolded GFP protein, and the fluorescent protein fragment G2 is a protein fragment of amino acids 215 to 238 of the superfolded GFP protein.

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

5.

4. The bimolecular fluorescence complementary 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. The sequence of the signal peptide SP is shown in SEQ ID NO:6, and the sequence of the protein tag HA is shown in SEQ ID NO:

7.

5. The bimolecular fluorescence complementary system according to claim 1 or 2, characterized in that, 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 resident signal HDEL connected in sequence. The sequence of the signal peptide SP is shown in SEQ ID NO:6, the sequence of the red fluorescent protein RFP is shown in SEQ ID NO:8, and the sequence of the endoplasmic reticulum resident signal HDEL is shown in SEQ ID NO:

9.

6. The bimolecular fluorescence complementary system according to claim 1, characterized in that, The first vector contains, in sequence, epitope SYN, red fluorescent protein RFP, fluorescent protein fragment G2, and endoplasmic reticulum resident signal HDEL.

7. The bimolecular fluorescence complementary system according to claim 1, characterized in that, The second carrier comprises VSS, cyan fluorescent protein CFP, fluorescent protein fragment G1 and nanobody NbS connected in sequence, wherein the sequence of VSS is shown in SEQ ID NO:10 and the sequence of cyan fluorescent protein CFP is shown in SEQ ID NO:

11.

8. A biosensor, characterized in that, The biosensor includes the bimolecular fluorescence complementary system as described in any one of claims 1-7.

9. The application of the bimolecular fluorescence complementary system according to any one of claims 1-7 or the biosensor according to claim 8 in in vivo organelle-level tracing.

10. The application of the bimolecular fluorescence complementary system according to any one of claims 1-7 or the biosensor according to claim 8 in the detection of targeted drug delivery.

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