Nanometer antibody NbA and epitope AYD interaction system for plant cell research and biosensor

Abstract

The invention relates to the technical field of protein engineering, in particular to a nano antibody NbA and epitope AYD interaction system for plant cell research, a biosensor and a nano antibody and epitope interaction system, which comprise a first carrier and a second carrier, the first carrier contains a nano antibody NbA; the second carrier contains an epitope AYD; the gene sequence of the nano antibody NbA is as shown in SEQ ID NO: 1, and the gene sequence of the epitope AYD is as shown in SEQ ID NO: 2. The nano antibody NbA and the epitope AYD are applied to plant cells for the first time, and a novel protein-protein interaction tool and a high-specificity protein tag are provided for future research in the plant field on the basis that original molecular solubility and biophysical characteristics are not affected. Specific binding of the nano antibody NbA and the epitope AYD of the gene sequence in plant cells is realized, and a research basis is provided for researching other frontier problems in the plant field in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, and particularly relates to a nanobody NbA and epitope AYD interaction system applicable to plant cell research and a biosensor. BACKGROUND

[0002] Single-chain camelid antibody (V HH ) fragments are the smallest antigen-binding units, consisting of a peptide linker and a whole IgG antibody fragment formed by connecting the variable domains of heavy and light chains, and are called nanobodies, with a molecular weight of about 15 kD. The unpaired variable domains have high potential in various medical applications. Compared with classic antibodies, nanobodies exhibit similar functional characteristics in specificity and affinity. At the same time, nanobodies also have the advantages of small size, high solubility and strong stability, which are beneficial to biophysical research.

[0003] Beta-amyloid is a main peptide component of senile plaques and is formed by proteolysis of amyloid precursor protein. It has been found that the epitope of beta-amyloid recognized by the variable domain of single-chain camelid anti-beta-amyloid antibody (referred to as beta-amyloid nanobody, NbA) is called AYD. These antibodies are found in the blood of camelids, and the interaction between nanobody NbA and epitope AYD is further characterized by in vitro enzyme-linked immunosorbent assay and biological affinity analysis, but there is no prior art to apply it to plant cells.

[0004] Therefore, it is of great significance and value to improve and prove that nanobody NbA and its epitope AYD can be successfully applied in plant cells. SUMMARY

[0005] In view of the above deficiencies of the prior art, the purpose of the present application is to provide a nanobody NbA and epitope AYD interaction system and a biosensor for plant cell research, aiming to solve the prior art that there is no precedent for applying the interaction between nanobody NbA and epitope AYD to plant cells.

[0006] The technical scheme of the present application is as follows:

[0007] A nanobody NbA and epitope AYD interaction system for plant cell research comprises a first carrier and a second carrier;

[0008] The first carrier contains nanobody NbA; and the second carrier contains epitope AYD.

[0009] The genetic sequence of the nanobody NbA is shown as SEQ ID NO: 1, and the genetic sequence of the epitope AYD is shown as SEQ ID NO: 2.

[0010] The nanobody NbA and epitope AYD interaction system for plant cell research, wherein the protein sequence of the nanobody NbA is shown as SEQ ID NO: 3, and the protein sequence of the epitope AYD is shown as SEQ ID NO: 4.

[0011] The nanobody NbA and epitope AYD interaction system for plant cell research, wherein the first vector comprises the nanobody NbA and a red fluorescent protein RFP connected to the nanobody NbA, and the protein sequence of the red fluorescent protein RFP is shown as SEQ ID NO: 5.

[0012] The nanobody NbA and epitope AYD interaction system for plant cell research, wherein the first vector comprises the nanobody NbA, a red fluorescent protein RFP and a sequence-specific vacuole sorting signal Aleu connected in sequence, the protein sequence of the red fluorescent protein RFP is shown as SEQ ID NO: 5, and the protein sequence of the sequence-specific vacuole sorting signal Aleu is shown as SEQ ID NO: 6.

[0013] The nanobody NbA and epitope AYD interaction system for plant cell research, wherein the second vector comprises the epitope AYD and a green fluorescent protein GFP connected to the epitope AYD, and the protein sequence of the green fluorescent protein GFP is shown as SEQ ID NO: 7.

[0014] The nanobody NbA and epitope AYD interaction system for plant cell research, wherein the second vector comprises the epitope AYD, a green fluorescent protein GFP and an endoplasmic reticulum membrane protein CNX connected in sequence, the protein sequence of the green fluorescent protein GFP is shown as SEQ ID NO: 7, and the protein sequence of the endoplasmic reticulum membrane protein CNX is shown as SEQ ID NO: 8.

[0015] A biosensor comprising a nanobody NbA and epitope AYD interaction system for plant cell research.

[0016] Beneficial Effects: This invention provides a nanobody NbA and epitope AYD interaction system and a biosensor for plant cell research. The nanobody and epitope interaction system includes a first carrier and a second carrier; the first carrier contains the nanobody NbA; the second carrier contains the epitope AYD; the gene sequence of the nanobody NbA is shown in SEQ ID NO:1, and the gene sequence of the epitope AYD is shown in SEQ ID NO:2. This invention, by applying the nanobody NbA and epitope AYD to plant cells for the first time, provides a new tool for protein-protein interactions and a highly specific protein tag for future plant research without affecting the original molecular solubility and biophysical properties, and without causing physiological effects on the linked proteins. The nanobody NbA with the above gene sequences and the expressed AYD achieve specific binding in plant cells. This characteristic can be used to study cutting-edge issues in the plant field, including but not limited to the tracing of specific compartments of the target protein and the sorting and transport of proteins. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the method for testing the NbA-epitope AYD interaction of nanobodies in plant cells, as described in Example 1.

[0018] Figure 2 This is a confocal microscope imaging result from Example 1;

[0019] Figure 3 This is a diagram showing the results of the immunoprecipitation in Example 1. Detailed Implementation

[0020] This invention provides a nanobody NBA and epitope AYD interaction system and a biosensor for plant cell research. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0021] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0022] This invention provides a nanobody NBA and epitope AYD interaction system for plant cell research, comprising a first carrier and a second carrier;

[0023] The first carrier contains the nanobody NbA; the second carrier contains the epitope AYD;

[0024] The gene sequence of the nanobody NbA is shown in SEQ ID NO:1, and the gene sequence of the epitope AYD is shown in SEQ ID NO:2.

[0025] In this embodiment, the nanobody NbA and epitope AYD are optimized for application in plant cells without causing physiological effects on the linked proteins, providing a new technical means and tool for future plant research. The nanobody NbA and expressed AYD, utilizing the aforementioned gene sequences, achieve specific binding within plant cells. This property can be used for research on cutting-edge issues in the future plant field, including but not limited to the tracing of specific compartments of target proteins and the sorting and transport of proteins.

[0026] In some embodiments, the protein sequence of the nanobody NbA (β-amyloid nanobody) is shown in SEQ ID NO:3; the epitope AYD (β-amyloid epitope) is from the NCBI database (PDB:2LMQ_R), and its protein sequence is shown in SEQ ID NO:4.

[0027] In some embodiments, the first carrier includes the nanobody NbA and a red fluorescent protein RFP linked to the nanobody NbA; the protein sequence of the red fluorescent protein RFP is shown in SEQ ID NO:5.

[0028] In some embodiments, the second vector includes epitope AYD and green fluorescent protein GFP linked to epitope AYD; the protein sequence of green fluorescent protein GFP is shown in SEQ ID NO:7.

[0029] Specifically, by linking the nanobody NbA and the epitope AYD to red fluorescent protein RFP and green fluorescent protein GFP, respectively, the specific binding of the nanobody NbA and the epitope AYD in plant cells can be detected.

[0030] In some embodiments, the first carrier comprises the nanobody NbA, red fluorescent protein RFP, and sequence-specific vacuole sorting signal Aleu connected in sequence; the protein sequence of the red fluorescent protein RFP is shown in SEQ ID NO:5; and the protein sequence of the sequence-specific vacuole sorting signal Aleu is shown in SEQ ID NO:6.

[0031] In some embodiments, the second vector comprises the epitope AYD, green fluorescent protein GFP, and endoplasmic reticulum membrane protein CNX connected in sequence; the protein sequence of the green fluorescent protein GFP is shown in SEQ ID NO:7; and the protein sequence of the endoplasmic reticulum membrane protein CNX is shown in SEQ ID NO:8.

[0032] Specifically, the endoplasmic reticulum membrane protein CNX (Calnexin) is used to localize the epitope AYD in the endoplasmic reticulum, while Aleurain (Aleu), a sequence-specific vacuolar sorting signal, is used to target the nanobody NbA to the vacuolar. This allows for a more direct detection of the interaction between the nanobody NbA and the epitope AYD in plant cells.

[0033] In practical applications, if NbA-AYD nanobody-epitope interaction is successfully generated in plant cells, the vacuolar-targeted NbA nanobody will co-localize with the epitope AYD. Simultaneously, nanobody-epitope interaction can be detected by introducing co-immunoprecipitation (Co-IP) technology. This invention, by utilizing the successful and specific binding of NbA nanobody to the epitope AYD in plant cells, provides a novel tool for protein-protein interaction research in the field of plant science. It can also serve as a highly specific protein tag, providing a research foundation for future studies of other physiological processes in plants without affecting the original molecular solubility and biophysical properties.

[0034] By optimizing the gene sequences of nanobodies NbA and epitopes AYD and applying them in plant cells without causing physiological effects on the linked proteins, new technical means and tools are provided for future plant research.

[0035] In addition, the present invention also provides a biosensor comprising a nanobody and an epitope interaction system for plant cell research.

[0036] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0037] Example 1

[0038] This embodiment verifies the interaction between the nanobody NbA and the epitope AYD in plant cells, as detailed below:

[0039] 1) Protoplast isolation and gene expression

[0040] Electroporation-capable protoplasts were isolated according to a predetermined procedure. Tobacco leaves, after being perforated for 6-8 weeks, were incubated overnight 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.) at 25°C in the dark. Tobacco leaf protoplasts were filtered through a 100 μm nylon filter and washed with EPB electrotransfection 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). The buffer was centrifuged at 80 g for 15 minutes and resuspended. The precipitate and excess EPB solution were removed using a peristaltic pump. This process was repeated five times. Each electrotransfection used approximately 2-5 × 10⁻⁵ protoplasts. 6 500 μl of protoplast suspension was mixed with 10–50 μg of plasmid DNA. Transfection was performed by applying a 160V single-wave pulse for 10 ms (Gene Pulser Xcell™, Bio-Rad Laboratories Co., Ltd., Shanghai). The transfection system was: Sample 1 (AYD-RFP-CNX and Aleu-RFP-NbA), Sample 2 (AYD-RFP-CNX and Aleu-RFP). After transfection, 2 ml of TEX buffer was added to each sample, and the samples were incubated in the dark at 25°C for 18–24 hours. NbA is a β-amyloid nanobody. AYD is a β-amyloid epitope. CNX is an endoplasmic reticulum membrane protein, Calnexin. Aleu is Aleurain, one of the sequence-specific vacuolar sorting signals. GFP is green fluorescent protein. RFP is red fluorescent protein.

[0041] Specifically, a schematic diagram of the method for testing the interaction between the nanobody NbA-epitope AYD and plant cells is shown below. Figure 1As shown, NbA-labeled red fluorescent protein RFP is linked to Aleu, one of the vacuolar sorting signals, to form an Aleu-RFP-NbA fusion protein targeting vacuoles. The corresponding epitope AYD-labeled green fluorescent protein GFP is linked to the endoplasmic reticulum membrane protein CNX, causing the fusion protein AYD-GFP-CNX to localize to the endoplasmic reticulum membrane. When both are co-expressed, if I) NbA-AYD nanobody-epitope interaction occurs in plants, the vacuolar protein Aleu-RFP-NbA will co-localize with the endoplasmic reticulum membrane protein AYD-GFP-CNX and will prevent it from being sorted into the vacuoles. If II) no nanobody-epitope interaction occurs, the NbA-deficient vacuolar protein Aleu-RFP will be sorted into the vacuoles and will not co-localize with the endoplasmic reticulum membrane protein AYD-GFP-CNX.

[0042] 2) Confocal microscopy imaging

[0043] Confocal images of Sample 1 and Sample 2 were acquired using a Leica TCS-SP8 confocal laser scanning microscope with a ×63 (1.2 numerical aperture) water immersion objective. Fluorophore excitation (ex) and emission (em) were detected by sequential mode switching using a HyD detector: GFP (ex / em, 488nm / 500-530nm), RFP (ex / em, 552nm / 580-640nm). 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), the results of which are shown below. Figure 2 As shown.

[0044] from Figure 2 As shown in section a, the green channel indicates the localization of the endoplasmic reticulum membrane protein AYD-GFP-CNX, and the red channel indicates the vacuolar protein Aleu-RFP-NbA. The two show co-localization, indicating that the nanobody NbA and the epitope AYD can interact in plant cells, causing the vacuolar protein Aleu-RFP-NbA to remain on the endoplasmic reticulum membrane instead of being sorted into the vacuoles. From... Figure 2 As shown in b, when there is no interaction between the nanobody NbA and the epitope AYD, the vacuolar protein Aleu-RFP shown in the red channel will reach the vacuoles and will not co-localize with AYD-GFP-CNX shown in the green channel.

[0045] 3) Protein extraction and co-immunoprecipitation (Co-IP)

[0046] Protoplasts from samples 1 and 2 (step 1) and the blank control group were diluted five-fold with 250 mM NaCl and centrifuged at 80 g for 10 minutes to precipitate the cells. The supernatant was aspirated using a peristaltic pump, and the cell pellet was resuspended. A 1:1 mixture of 2× binding buffer (40 mM HEPES, 300 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.1) was added, and the mixture was incubated at 4 °C for 30 minutes. The supernatant was then collected by centrifugation at 12,000 g for 15 minutes. Magnetic beads containing GFP antibody were fused with the collected supernatant at 4 °C for 60-120 minutes. The magnetic beads were then collected using a magnetic plate, washed with binding buffer, and then mixed with loading buffer. The mixture was denatured at 95 °C for 10 minutes before loading. The antibodies used for subsequent incubation were as follows: mouse monoclonal anti-RFP (ChromoTek, CAS NO. 6g6, 1:2,000) and mouse monoclonal anti-GFP (ChromoTek, CAS NO. 7g9, 1:2,000). Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) (Immunoway, CAS NO. RS0001, 1:20,000) was used as the secondary antibody.

[0047] The results of immunoprecipitation are as follows Figure 3 As shown, co-immunoprecipitation (Co-IP) demonstrated the specificity of the NbA-AYD nanobody-epitope interaction. Immunoprecipitation (anti-GFP antibody-coated magnetic beads; IP, α-GFP), and Western blotting (IB) were performed using antibody probes to detect the anchor sites (α-GFP and α-RFP). M, mock, and blank control were also included. These results demonstrate that the NbA nanobody and the AYD epitope not only successfully generated a specific interaction in plant cells but also did not cause any physiological effects on the linked proteins, proving the value and potential of the NbA-AYD nanobody interaction for future research in the plant field.

[0048] In summary, this invention provides a nanobody NbA and epitope AYD interaction system and a biosensor for plant cell research. The nanobody and epitope interaction system includes a first carrier and a second carrier; the first carrier contains the nanobody NbA; the second carrier contains the epitope AYD; the gene sequence of the nanobody NbA is shown in SEQ ID NO:1, and the gene sequence of the epitope AYD is shown in SEQ ID NO:2. This invention, by applying the nanobody NbA and epitope AYD in plant cells, provides a novel tool for protein-protein interactions and a highly specific protein tag for future plant research without affecting the original molecular solubility and biophysical properties. The specific binding of the nanobody NbA and epitope AYD within plant cells using the above gene sequences provides a research foundation for future research on other cutting-edge issues in the plant field, including but not limited to the tracing of specific compartments of the target protein and the sorting and transport of proteins.

[0049] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A nanobody NbA and epitope AYD interaction system for plant cell research, characterized in that, The first vector and the second vector are used for the study of the interaction between the nanobody NbA and the epitope AYD. The first vector contains the nanobody NbA; and the second vector contains the epitope AYD. The genetic sequence of the nanobody NbA is shown as SEQ ID NO: 1, and the genetic sequence of the epitope AYD is shown as SEQ ID NO:

2.

2. The Nanobody NbA and epitope AYD interaction system for plant cell studies according to claim 1, characterized in that, The protein sequence of the nanobody NbA is shown as SEQ ID NO: 3, and the protein sequence of the epitope AYD is shown as SEQ ID NO:

4.

3. The Nanobody NbA and epitope AYD interaction system for plant cell studies according to claim 1, characterized in that, The first vector contains the nanobody NbA and the red fluorescent protein RFP connected to the nanobody NbA; and the protein sequence of the red fluorescent protein RFP is shown as SEQ ID NO:

5.

4. The Nanobody NbA and epitope AYD interaction system for plant cell studies according to claim 1, characterized in that, The first vector contains the nanobody NbA, the red fluorescent protein RFP and the sequence-specific vacuole sorting signal Aleu connected in sequence; the protein sequence of the red fluorescent protein RFP is shown as SEQ ID NO: 5; and the protein sequence of the sequence-specific vacuole sorting signal Aleu is shown as SEQ ID NO:

6.

5. The Nanobody NbA and epitope AYD interaction system for plant cell studies according to claim 1, characterized in that, The second vector contains the epitope AYD and the green fluorescent protein GFP connected to the epitope AYD; and the protein sequence of the green fluorescent protein GFP is shown as SEQ ID NO:

7.

6. The Nanobody NbA and epitope AYD interaction system for plant cell studies according to claim 1, characterized in that, The second vector contains the epitope AYD, the green fluorescent protein GFP and the endoplasmic reticulum membrane protein CNX connected in sequence; the protein sequence of the green fluorescent protein GFP is shown as SEQ ID NO: 7; and the protein sequence of the endoplasmic reticulum membrane protein CNX is shown as SEQ ID NO:

8.

7. A biosensor characterized by The biosensor contains the nanobody NbA and the epitope AYD interaction system for the study of plant cells according to any one of claims 1-6.

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