Green fluorescent protein nanobody with high affinity to GFP, and preparation method and application thereof

Abstract

The application belongs to the field of biology and relates to a green fluorescent protein nanobody with high affinity for GFP as well as a preparation method and application thereof, a phage-assisted continuous evolution (PACE) system is successfully built, and the PACE system operation test is completed by using existing plasmids and strains. The GFPNb is preliminarily evolved by using the PACE system, and stable mutation sites are found by sequence alignment analysis. It is proved by an indirect enzyme-linked immunosorbent assay that the affinity of the evolved GFPNb for GFP is several times higher. It is proved by using existing split luciferase complementation analysis technology and non-natural amino acid site-specific coupling technology that the affinity of the evolved GFPNb for GFP is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a green fluorescent protein nanobody with high affinity for GFP, its preparation method and application. Background Technology

[0002] Directed evolution is a key strategy for improving protein function. Traditional directed evolution methods require the artificial construction of large-scale mutant libraries and multiple generations of screening to achieve the desired evolutionary outcome. Phage-assisted continuous evolution (PACE), as an emerging directed evolution technology, stands out due to its rapid evolution rate, excellent compatibility, and lack of human intervention. Currently, this system has achieved significant results in the directed evolution of proteins and DNA molecules, and between proteins, successfully evolving T7 RNA polymerase, proteases, BT toxin proteins, and Cas9 proteins, among others. Its potential evolutionary evolution continues to be explored and refined.

[0003] Green fluorescent protein (GFP) is a protein composed of approximately 238 amino acid residues with a molecular weight of about 26.9 kDa. GFP has a typical β-barrel structure, rich in β-sheets and α-helices, with the fluorescent group embedded within. The 65th, 66th, and 67th amino acids (Ser-Tyr-Gly) of GFP form the luminescent group, enabling it to be excited by light and emit fluorescence. GFP can be coupled with conventional antibodies to bind to target proteins and perform localization studies. However, when studying the function of certain proteins, the size and multivalent nature of conventional antibodies often limit the effectiveness of GFP. Nanobodies (Nb) are single-domain antibodies consisting only of heavy chains; they naturally lack light chains. With a molecular weight of approximately 12–15 kDa, they are the smallest naturally occurring fragments capable of binding to antigens. Thanks to their unique structure and small size, nanobodies can bind well to protein surfaces, even to sites such as folds and grooves that are difficult for conventional antibodies to bind to. However, there are no existing products that significantly improve the affinity between GFPNb and GFP. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a green fluorescent protein nanobody with high affinity for GFP, its preparation method, and its application. By utilizing the PACE system, GFPNb is subjected to directed evolution, resulting in directed mutations in the GFPNb gene sequence, thereby enhancing the interaction strength between GFPNb and GFP.

[0005] The technical solution provided by this invention is as follows:

[0006] A green fluorescent protein nanobody with high affinity for GFP, wherein the amino acid sequence of the green fluorescent protein nanobody with high affinity for GFP is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0007] Furthermore, the variable region of the green fluorescent protein nanobody with high affinity for GFP has three complementarity-determining regions CDR1, CDR2, and CDR3, the sequence of CDR1 is shown in SEQ ID NO. 2; the sequence of CDR2 is shown in SEQ ID NO. 3; and the sequence of CDR3 is shown in SEQ ID NO. 4.

[0008] The present invention also provides a method for preparing the green fluorescent protein nanobody with high affinity for GFP, wherein the GFPNb original is subjected to directed evolution to obtain the green fluorescent protein nanobody with high affinity for GFP, and the amino acid sequence of the GFPNb original is shown in SEQ ID NO. 1.

[0009] Furthermore, the directed evolution includes mutations at three sites: D3G, M71I, and A81V.

[0010] The present invention also provides a nucleic acid molecule that encodes the aforementioned green fluorescent protein nanobody with high affinity for GFP.

[0011] Furthermore, the nucleic acid molecule includes the sequence shown in SEQ ID NO. 9.

[0012] The present invention also provides an expression vector containing the above-described nucleic acid molecules.

[0013] The present invention also provides a host cell containing the above-described expression vector.

[0014] The present invention also provides the application of the above-mentioned green fluorescent protein nanobody with high affinity for GFP in the preparation of ultrasensitive sandwich ELISA or homogeneous immunoassay reagents.

[0015] The present invention also provides a detection reagent comprising the above-mentioned green fluorescent protein nanobody with high affinity for GFP.

[0016] Beneficial effects

[0017] This invention presents a scheme for evolving GFPNb based on the PACE system. First, nanobodies with affinity for the antigen are preliminarily screened using a yeast / phage display library of nanobodies. Then, a PACE system is constructed for directed evolution. After evolution, site mutation comparison analysis reveals stable mutation sites in the evolved GFPNb. Subsequently, the affinity of different antibody mutation types with the antigen is tested, and experiments (such as split-Luciferase and indirect enzyme-linked immunosorbent assay) are used to verify the interaction strength between the evolved GFPNb and GFP. Finally, based on the evolved antibody, modifications or tandem conjugation of multiple antibodies are performed to further enhance antibody affinity.

[0018] This invention successfully constructed a phage-assisted continuous evolution (PACE) system and conducted operational tests using existing plasmids and strains. GFPNb was initially evolved using the PACE system, and stable mutation sites were identified through sequence alignment analysis. Indirect enzyme-linked immunosorbent assay (ELISA) demonstrated that the affinity of the evolved GFPNb for GFP was increased by approximately 10-fold. Existing luciferase complementation analysis and site-directed non-natural amino acid conjugation techniques further confirmed the significant improvement in the affinity of the evolved GFPNb for GFP.

[0019] This invention uses immunofluorescence experiments on the D3G mutant and the original GFPNb vector. It can be observed that the negative control group did not show Cy5 staining, and the red color of the D3G-pAzF-Cy5 group was significantly more pronounced than that of the GFPNb-pAzF-Cy5 group, indicating that the affinity of the D3G mutant is significantly improved compared with that of the original GFPNb vector.

[0020] The green fluorescent protein nanobodies described in this invention have strong interactions with GFP, making them applicable in various fields. In the preparation of detection reagents, they can amplify signals, improve sensitivity, widen the detection window, and reduce background. These nanobodies can be used as capture or detection antibodies to detect trace amounts of GFP-labeled reporter proteins, viral particles, or exosomes in blood / cell lysates, and can be applied in ultrasensitive sandwich ELISA or homogeneous immunoassay. The green fluorescent protein nanobodies described in this invention can be pre-coated onto 96-well plates with HRP- or fluorescently labeled detection antibodies; or they can be fabricated into homogeneous wash-free microbeads (AlphaLISA / AlphaScreen) for the detection of trace amounts of GFP-labeled reporter proteins. Attached Figure Description

[0021] Figure 1 The results are from the Split-LUC (luciferase complementation) assay.

[0022] Figure 2It is a D3G sequence;

[0023] Figure 3 The sequences are M71I and A81V.

[0024] Figure 4 This is an indirect ELISA result;

[0025] Figure 5 Photographs showing the comparison of ELISA results between GFPNb natively loaded and D3G and D3G-M71I-A81V with an antigen coating amount of 20ng.

[0026] Figure 6 Comparison of ELISA results between GFPNb natively loaded and D3G and D3G-M71I-A81V with an antigen coating amount of 20ng;

[0027] Figure 7 A schematic diagram of the translational extension of pAzF-labeled nanobodies;

[0028] Figure 8 Immunofluorescence results for GFPNb-pAzF-Cy5, D3G-pAzF-Cy5, and the Cy5 control group. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0030] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0031] The amino acid sequence originally GFPNb

[0032] MADVQLQESGGGLVQAGGSLRLSCAASGGTFSILSLGWFRQAPGKEREFVAAISRSEGSTDYADFVKGRFMISRENAKNTAYLQMNSLKPEDTAVYFCAASYARRLSTTASRVLYWGQGTQVTVSS (SEQ ID NO. 1);

[0033] The variable region of the nanobody has three complementarity-determining regions, CDR1, CDR2, and CDR3, with the sequences of CDR1, CDR2, and CDR3 as follows:

[0034] CDR1: GTFSILS (SEQ ID NO. 2);

[0035] CDR2: SRSEGST (SEQ ID NO. 3);

[0036] CDR3: AASYARRLSTTASRVLYWGQGTQVTVSS (SEQ ID NO. 4).

[0037] D3G, M71I, and A81V are three mutation sites of GFPNb that have evolved. The amino acid sequence of the D3G mutant is as follows:

[0038] MAGVQLQESGGGLVQAGGSLRLSCAASGGTFSILSLGWFRQAPGKEREFVAAISRSEGSTDYADFVKGRFMISRENAKNTAYLQMNSLKPEDTAVYFCAASYARRLSTTASRVLYWGQGTQVTVSS (SEQ ID NO. 5);

[0039] The amino acid sequence of the D3G+M71I+A81V mutant (i.e., the green fluorescent protein nanobody) is as follows:

[0040] MAGVQLQESGGGLVQAGGSLRLSCAASGGTFSILSLGWFRQAPGKEREFVAAISRSEGSTDYADFVKGRFIISRENAKNTVYLQMNSLKPEDTAVYFCAASYARRLSTTASRVLYWGQGTQVTVSS (SEQ ID NO. 6);

[0041] The original nucleic acid sequence of GFPNb is as follows:

[0042] ATGGCAGATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCTTCAGTATCTTGTCCTTGGGCTGGTTTCGCCAGGCGCCAGGGAAGGAGCGCGAATTTGTAGCAGCTATTAGCCGGAGTGAAGGTAGCACAGACTATGCAGACTTCGTGAAGGGCCGATTCATGATCTCCAGAGAGAACGCCAAGAATACGGCGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCTTCATACGCGCGCAGACTATCTACTACAGCGTCTCGCGTTTTATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC (SEQ ID NO. 7);

[0043] The nucleic acid sequence of the D3G mutant is as follows:

[0044] ATGGCAGGTGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCTTCAGTATCTTGTCCTTGGGCTGGTTTCGCCAGGCGCCAGGGAAGGAGCGCGAATTTGTAGCAGCTATTAGCCGGAGTGAAGGTAGCACAGACTATGCAGACTTCGTGAAGGGCCGATTCATGATCTCCAGAGAGAACGCCAAGAATACGGCGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCTTCATACGCGCGCAGACTATCTACTACAGCGTCTCGCGTTTTATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC (SEQ ID NO. 8);

[0045] The nucleic acid sequence of the D3G+M71I+A81V mutant (i.e., the nanobody of green fluorescent protein) is as follows:

[0046] ATGGCAGGTGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCTTCAGTATCTTGTCCTTGGGCTGGTTTCGCCAGGCGCCAGGGAAGGAGCGCGAATTTGTAGCAGCTATTAGCCGGAGTGAAGGTAGCACAGACTATGCAGACTTCGTGAAGGGCCGATTCATAATCTCCAGAGAGAACGCCAAGAATACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCTTCATACGCGCGCAGACTATCTACTACAGCGTCTCGCGTTTTATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC (SEQ ID NO. 9).

[0047] GFP gene:

[0048] TCACTTGTACAGCTCGTCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAAGACCATACGCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACT (SEQ ID NO.10).

[0049] Example 1

[0050] 1.1 Vector construction

[0051] (1) Add the GFP gene (SEQ ID NO.10) to the helper plasmid AP (purchased from Addgene, catalog number Plasmid #79218) to construct the complete pAB107a - 434cI - Flag - GFP plasmid.

[0052] (2) The GFPNb gene (SEQ ID NO. 1) was inserted into the SP plasmids (purchased from Addgene, catalog numbers Plasmid #138523 and Plasmid #138521) to generate the complete SP-rpoZ-Myc-GFPNb plasmid. Primer design is shown in Table 1:

[0053] Table 1

[0054]

[0055] 1.2 Plaque Assay

[0056] (1) S2208 bacteria (obtained by transforming pJC175e into S2060, both S2060 and pJC175e were purchased from Addgene, catalog numbers Bacterial strain #105064 and Plasmid #79219 respectively) were grown in a biological shaker at 37°C until OD. 600 It is 0.2-0.6;

[0057] (2) Set up a negative control: Mix 100 µL of S2208 bacterial culture, 40 µL of 2% X-galactoside (X-gal) and 4 µL of 1 MIPTG in a PCR tube by inverting and pouring it into 4 ml of upper agar (2×YT, agar 7 g / L) that has been incubated in a 50℃ oven beforehand. Mix again by inverting and pouring it into the solidified lower agar (2×YT, agar 20 g / L).

[0058] (3) Experimental group: SP-GFPNb was serially diluted 10-fold to a final concentration of 10. -12 Transfer 10 µL of each dilution to a PCR tube. To each phage dilution, add 100 µL of S2208 bacterial culture, 40 µL of 2% X-galactoside (X-gal), and 4 µL of 1 M IPTG. Mix by inverting and pour in 4 ml of upper agar (2×YT, 7 g / L agar). Mix by inverting again and pour into the solidified lower agar (2×YT, 20 g / L agar).

[0059] (4) Incubate overnight upside down at 37°C;

[0060] (5) The titer of the phage stock solution can be determined by the following formula: titer (in pfu / ml) = (number of blue plaques) × (dilution factor) × 100.

[0061] 1.3 Phage titer standard curve construction and qPCR detection of phage titers

[0062] (1) Transfer 25-50 µL of phage of unknown titer (phage supernatant from PACE and PANCE systems) into a PCR tube;

[0063] (2) Using phages of known titers, from ~10 9 Or 10 10 Start with phage at pfu / mL and perform eight 1:10 serial dilutions to create a titration profile. Transfer 25–50 µL of each dilution to a PCR tube;

[0064] (3) Heating at 80℃ for 30 minutes to destroy multivalent bacteriophages;

[0065] (4) To digest the multivalent phage genome, 5 µL of heat-treated phage was diluted in 45 µL of 1× deoxyribonuclease I buffer (DNase I buffer);

[0066] (5) Heat at 37℃ for 20 minutes, then at 95℃ for 20 minutes to establish a qPCR reaction system. Add the corresponding template and primers to the eight-tube strip, and set three replicates for each group. After brief centrifugation, place the tubes in a PCR instrument and set the following program: 1. Pre-denaturation stage: 95℃, 3 min; 2. Cyclic reaction: 95℃, 10 s; 60℃, 30 s; for a total of 40 cycles; 3. Melting curve: 95℃, 15 s; 60℃, 60 s; 95℃, 15 s.

[0067] (6) Plot a standard curve based on the CT value and dilution factor, and determine the titer of the unknown phage based on the standard curve.

[0068] 1.4 Phage-Assisted Discontinuous Evolution (PANCE)

[0069] (1) The host bacteria (S2060, which has been transformed into helper plasmid pAB107a-434cI-Flag-GFP and MP6 (purchased from Addgene, catalog number Plasmid #69669) mutant plasmid) were inoculated into 2×YT liquid medium supplemented with ampicillin, streptomycin, tetracycline, chloramphenicol and 100 mM glucose, and cultured overnight at 37°C with shaking.

[0070] (2) Take 25-50 μL of overnight cultured host bacteria and add it to 2.5 mL of 2×YT medium containing the corresponding resistance and 50 mM arabinose;

[0071] (3) Take 50 µL of GFPNb original vector phage (obtained by transforming S2208 bacteria with SP-rpoZ-Myc-GFPNb plasmid) to infect the host bacteria;

[0072] (4) Incubate in a biological shaker at 37°C for 12-16 hours;

[0073] (5) Centrifuge at 8000 g for 2 min to precipitate the cells, filter with a 0.22 µm filter and collect the supernatant to remove residual cells.

[0074] (6) Take the phage supernatant for PCR, and determine the amount of phage to be added for the next generation based on the band brightness after agarose gel electrophoresis;

[0075] (7) Repeat steps 2-6, using phage supernatant to infect the next generation of fresh host cells, adjusting the initial titer and incubation time as needed to improve the selectivity;

[0076] (8) When the SP-rpoZ-Myc-GFPNb plasmid generated by the PANCE system showed high activity in the phage amplification experiment (e.g., the supernatant PCR band was bright and the qPCR showed a high phage titer), it indicated that they could be used to start the PACE experiment. After 168 hours of repetition, the band brightness in the agarose gel increased significantly. After sequencing by the company, it was found that the D3G mutant (sequence shown in SEQ ID NO. 1) was generated from the original GFPNb (sequence shown in SEQ ID NO. 5). The significant increase in band brightness indicated that the interaction level between the D3G mutant and GFP was much higher than that between the original GFPNb and GFP.

[0077] 1.5 Setting and Operating Parameters of the PACE System

[0078] The PACE system primarily relies on two peristaltic pumps and one syringe pump. One peristaltic pump continuously delivers fresh culture medium to the chemostat, while the other transports the host cells growing in the chemostat to the lagoon, ensuring continuous flow and renewal within the system. The syringe pump quantitatively injects arabinose into the lagoon to control the mutation efficiency of the MP6 mutant plasmid. Specific parameter settings and operating procedures are as follows:

[0079] (1) All components, including culture medium bottles, waste liquid containers, 1×2mm silicone tubing, syringes, needles, chemistosensors, lagoons, filters and magnetic rotors, are autoclaved (121°C, 20min) to ensure aseptic operation.

[0080] (2) Prepare 5L of 2×YT liquid culture medium and autoclave it. At the same time, prepare 20mL of host bacteria transformed with pAB107a-434cI-Flag-GFP and MP6 plasmids in advance.

[0081] (3) The D3G mutant was subjected to plaque and qPCR titer determination. The sequence of the D3G mutant is shown in SEQ ID NO. 5. 10 mL was stored at 4°C for subsequent inoculation.

[0082] (4) After the 2×YT liquid medium has cooled to room temperature, add antibiotics at the final concentrations: ampicillin (50 μg / mL), streptomycin (50 μg / mL), tetracycline (10 μg / mL), chloramphenicol (25 μg / mL), and glucose (100 mM). Assemble the PACE system tubing in a clean bench. Connect the host bacterial culture to the chemostat, and simultaneously add 10 mL of GFPNb phage to the lagoon. Then connect all tubing to the peristaltic pump system.

[0083] (5) Start the peristaltic pump of the chemostat and maintain the culture medium input in the 37°C incubator until the volume in the chemostat is stable and the OD600nm is stable in the range of 0.2~0.6, indicating that the cell growth has entered the exponential phase.

[0084] (6) After 2 hours of incubation, adjust the flow rate of the peristaltic pump: increase the inlet flow rate of the chemistostat and the outlet flow rate of the lagoon together to maintain stable system updates.

[0085] (7) Load 1M arabinose solution into a syringe and fix it on the injection pump. Set the injection speed to 0.1 mL / h to control the activity of the induction promoter and adjust the selection pressure.

[0086] (8) During system operation, the flow rate of the chemotherapeutic device and the lagoon and the arabinose injection rate can be dynamically adjusted according to the bacterial growth status, phage concentration or evolution process to optimize evolution conditions.

[0087] (9) During the operation of the PACE system, samples were taken every 12 hours. After PCR amplification, agarose gel electrophoresis was performed. Samples with increased band brightness were sent to the company for sequencing. When the PACE system had been running for 120 hours, the PCR band brightness was higher than that of the D3G mutant. Sequencing revealed the production of the D3G+M71I+A81V mutant (sequence shown in SEQ ID NO. 6).

[0088] 1.6 Experimental Results:

[0089] Phage-assisted sequential evolution (PACE) has mediated the rapid evolution of various protein classes, including polymerases, proteases, and genome editing proteins, producing variants with significantly altered activity and specificity. To evolve the affinity between antigens and antibodies, this experiment will be based on the principle of the bacterial two-hybrid system, utilizing the interaction of two fusion proteins to activate the transcription of a reporter gene. In this experiment, the reporter gene is gIII of filamentous phage M13. During PACE, the host *E. coli* infected with the phage carries two plasmids: an accessory plasmid (AP) containing the complete gIII but subject to selectively regulated expression; and a mutagenesis plasmid (MP) capable of high-level mutations induced by arabinose during PACE.

[0090] The PACE system consists of the ω subunit of *E. coli* RNA polymerase (rpoZ) as the activation domain, phage cI repressor protein 434 (434cI) as the DNA-binding domain, and an optimized PlacZ-derived promoter (PlacZ-opt) to drive the transcription of the reporter gene gIII. We fused 434cI with the antigen GFP and expressed it on the helper plasmid AP, while fused rpoZ with a nanobody and expressed it on the selected phage SP. When the antigen and antibody interact, 434cI and rpoZ approach each other, driving the expression of phage gIII on AP. With pIII protein, the SP phage has the ability to package and reproduce, allowing it to survive and multiply in the system. Conversely, if the antibody and antigen do not interact or the interaction is weak, gIII cannot be expressed, the SP cannot survive, and it will be discharged with the waste liquid. In the first stage, using PACE technology, the original GFPNb vector (sequence shown in SEQ ID NO. 1) was directed to evolve for 168 hours to generate the D3G mutant (sequence shown in SEQ ID NO. 5). In the second stage, the D3G mutant was placed in the PACE system, and after 120 hours of directed evolution, the D3G+M71I+A81V mutant (sequence shown in SEQ ID NO. 6) was generated. Subsequently, multiple repetitions of the PACE experiment yielded the same result: GFPNb natively induced (sequence shown in SEQ ID NO. 1) generated the D3G mutant (sequence shown in SEQ ID NO. 5), and multiple repetitions of the PACE experiment also yielded the same result. Figure 1As shown, the results indicate that the relative luciferase activities of the D3G mutant and the D3G+M71I+A81V mutant were significantly higher than those of the original GFPNb mutant, while the positive and negative controls were normal. This suggests that the interaction activity between the D3G mutant and the D3G+M71I+A81V mutant and GFP was significantly enhanced compared to GFPNb. These results suggest that under certain selection pressure, the occurrence of these mutations in the original GFPNb mutant is a reasonable outcome, indicating evolution. The affinity of both the D3G mutant and the D3G+M71I+A81V mutant for green fluorescent protein (GFP) is higher than that of the original GFP mutant, and the affinity of the D3G+M71I+A81V mutant should be higher than that of the D3G mutant, but further verification is needed.

[0091] Example 2

[0092] 2.1 Carrier Construction

[0093] The prokaryotic expression vector pET28a-GFP (for expressing GFP protein) and the eukaryotic expression vector pEGFP-N1-GFPNb-HRP were constructed. The pET28a vector was purchased from Addgene. Primer designs are shown in Table 2.

[0094] Table 2

[0095]

[0096] 2.2 Split-luciferase Complementation Assay (Split-LUC)

[0097] The split-luciferase complementation assay is widely used in mammalian cells to investigate protein-protein interactions. Specifically, it involves splitting firefly luciferase into two functional fragments, N-terminus and C-terminus (Nfluc and Cfluc). Recombinant antigens and antibodies are fused to these two fragments (Nfluc and Cfluc), respectively. When the antigen and antibody interact, the two luciferase fragments (Nfluc and Cfluc) become spatially close and correctly assembled, thus activating luciferase activity—the breakdown of the substrate to produce fluorescence.

[0098] 2.3 Chemiluminescence determination

[0099] (1) Remove the well plate from the incubator, discard the old culture medium and wash twice with PBS, then add 200 µl of 1× Cell Lysis Buffer to each well and lyse at room temperature for 10-15 min;

[0100] (2) Collect the cell lysate, centrifuge at 12,000 rpm for 5 min, and collect the supernatant;

[0101] (3) The experiment was conducted using an opaque 96-well plate. 20 µl of the supernatant from the control group and the experimental group was taken and 100 µl of luciferase substrate (Luciferase Substrate solution) was added under light-protected conditions. The plate was then quickly placed into a multi-functional microplate reader to read the activity of the firefly luciferase reporter gene.

[0102] (4) Add 100 µl of Renilla luciferase substrate (Renilla Substrate solution) under light-protected conditions, and read the Renilla luciferase activity in the same manner;

[0103] (5) The activity of firefly luciferase was standardized using the activity of Renali luciferase to correct for differences between experiments. The standardized fluorescence values ​​of the experimental group and the control group were compared and plotted for analysis.

[0104] The results of the Split-LUC experiment are as follows: Figure 2 As shown in the figure, five samples are included: negative control, GFPNb, D3G, D3G+M71I+A81V, and positive control. Among them, the relative luciferase activity of the negative control is almost 0, while the relative luciferase activity of the positive control is close to 0.8. Compared with the original GFPNb, the activities of the two mutated GFP nanobodies, D3G and D3G+M71I+A81V, are significantly improved.

[0105] 2.4 iELISA verification showed that the evolved D3G mutant and D3G+M71I+A81V mutant had a stronger affinity for GFP protein.

[0106] The successfully constructed recombinant plasmid pEGFP-N1-Nbs-HRP (Nbs refers to GFPNb, D3G, and D3G+M71I+A81V) was transfected into HEK 293T cells. Supernatants were collected 24-48 hours after transfection, and the collected cell supernatants were serially diluted. Expression and titer were determined by indirect ELISA. Supernatants from 293T cells transfected with the empty vector were used as a control (NC). The results showed that the recombinant plasmid pEGFP-N1-Nbs-HRP was successfully expressed and secreted into the supernatant in 293T cells, and reacted well with the recombinant GFP protein. The control group showed a weaker reaction with the GFP protein, indicating that the reaction between Nbs-HRP and GFP is specific. The concentrations of the original GFPNb and the evolved Nbs-HRP were adjusted to maintain consistency according to the standard curve.

[0107] 2.5 Indirect ELISA results are as follows Figures 4-6As shown, the darker the color of the ELISA, the more antibodies bind to the antigen, and the stronger the affinity. Compared with the original, the evolved nanobodies all showed stronger affinity for GFP protein. Furthermore, the D3G+M71I+A81V mutant nanobodies evolved through the PACE system for 120 hours exhibited stronger interaction with the antigen than the D3G mutant evolved solely through the PACE system for 168 hours. This further demonstrates that the affinity between antigen and antibody gradually increases with the evolution of the PACE system. This enhanced affinity can be applied in multiple fields, providing signal amplification, increased sensitivity, wider detection window, and reduced background in the preparation of detection reagents. Green fluorescent protein nanobodies can be used as capture or detection antibodies to detect trace amounts of GFP-labeled reporter proteins, viral particles, or exosomes in blood / cell lysates, and can be applied in ultrasensitive sandwich ELISA or homogeneous immunoassay. The green fluorescent protein nanobody described in this invention can be pre-coated in a 96-well plate and paired with HRP- or fluorescently labeled detection antibody; or it can be made into homogeneous wash-free microbeads (AlphaLISA / AlphaScreen) to detect trace amounts of GFP-labeled reporter protein.

[0108] Example 3

[0109] 3.1 Carrier Construction

[0110] The pET28a vector was modified by changing the UAG stop codons at three specific sites to UAA (using primers SEQ ID NO. 28-33). The original GFPNb gene and the D3G mutant gene were cloned into the pET28a vector, respectively. The stop codons of both the original GFPNb gene and the D3G mutant gene were then modified to UAG, constructing the plasmids pET28a-GFPNb-pAzF-UAA and pET28a-D3G-pAzF-UAA. Primer designs are shown in Table 3.

[0111] Table 3

[0112]

[0113] 3.2 Click chemistry reactions

[0114] The pET28a-GFPNb-pAzF-UAA and pET28a-D3G-pAzF-UAA plasmids were transformed into two competent cell lines (Ochre, rEcΔ2.ΔA.B3.tW*, purchased from Addgene, catalog number Bacterial strain #234622), and co-transformed with the pEvol-pAzFRS.2.t1 plasmid (purchased from Addgene, catalog number Plasmid #73546). The cells were picked and cultured overnight at 37°C in LB medium supplemented with chloramphenicol and kanamycin. The overnight culture was then transferred to 50 mL of medium at a 1:20 ratio and cultured at 37°C until OD600nm = 0.5. Then, pAzF, arabinose, and IPTG were added at final concentrations of 1 mM, 0.2%, and 0.5 mM, respectively. Induction was performed overnight at 30°C. After sonication of the cells, the supernatant was used for AKTA purification to obtain pAzF-labeled GFPNb and D3G proteins. The pAzF labeling process is as follows: Figure 7 As shown.

[0115] GFPNb-pAzF and D3G-pAzF proteins (in PBS) underwent click chemistry reactions with diphenylcyclooctyn-Cy5 (DBCO-Cy5) dye. DBCO-Cy5 dye was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 5 mg / mL. Based on the protein concentration, an appropriate amount of DBCO-Cy5 dye was added, and the mixture was incubated at room temperature for 45 minutes. The incubation was then passed through a desalting column to obtain GFPNb-pAzF-Cy5 and D3G-pAzF-Cy5.

[0116] 3.3 Immunofluorescence assay

[0117] The pcDNA3.1-GFP plasmid was constructed. The pcDNA3.1 plasmid (purchased from Addgene, catalog number Plasmid #10842) was transfected into HeLa cells in 24-well plates (with a 10 mm round coverslip added to each well). After culturing for 24 hours, the culture medium was discarded, and the cells were washed three times with PBS for 1 minute each time. Then, 300 μL of 4% paraformaldehyde was added, and the cells were incubated at room temperature for 15 minutes. The cells were then washed three times with PBS. Add 200 μL of Triton permeation buffer (5 mL PBS + 5 μL Triton X-100 + 50 μL 3% hydrogen peroxide) to each well and permeate at room temperature for 20 minutes. Wash 3 times with PBS. Block with 5% sheep serum blocking buffer (4 mL PBS + 200 μL sheep serum) for 1 hour. Add GFPNb-pAzF-Cy5 and D3G-pAzF-Cy5 to the sealing film respectively. Invert a round coverslip onto the antibody and incubate overnight at 4°C. Wash 3 times with PBS. Add DAPI staining solution to the sealing film and stain at room temperature in the dark for 5 minutes. Wash 3 times with PBS. Add fluorescent anti-quenching mounting medium to the slide. Invert the coverslip and seal with nail polish. Air dry at 4°C for 20 minutes.

[0118] Immunofluorescence results as follows Figure 8 As shown, images were taken using a confocal microscope. The images are divided into GFPNb-pAzF-Cy5, D3G-pAzF-Cy5, and a negative control group containing only Cy5. It can be observed that the negative control group did not show Cy5 staining, and the D3G-pAzF-Cy5 group showed a significantly more pronounced red color than the GFPNb-pAzF-Cy5 group, indicating that the D3G mutant has a significantly improved affinity compared to the original GFPNb.

Claims

1. A green fluorescent protein nanobody with high affinity for GFP, characterized in that, The amino acid sequence of the green fluorescent protein nanobody with high affinity for GFP is shown in SEQ ID NO. 5 or SEQ ID NO.

6.

2. The green fluorescent protein nanobody with high affinity for GFP according to claim 1, characterized in that, The variable region of the green fluorescent protein nanobody with high affinity for GFP has three complementarity-determining regions CDR1, CDR2, and CDR3, the sequence of CDR1 is shown in SEQ ID NO. 2; the sequence of CDR2 is shown in SEQ ID NO. 3; and the sequence of CDR3 is shown in SEQ ID NO.

4.

3. The method for preparing green fluorescent protein nanobodies with high affinity for GFP according to claim 1, characterized in that, Directed evolution was performed on the original GFPNb to obtain a green fluorescent protein nanobody with high affinity for GFP. The amino acid sequence of the original GFPNb is shown in SEQ ID NO.

1.

4. The method for preparing green fluorescent protein nanobodies with high affinity for GFP according to claim 3, characterized in that, The mutation sites for the directed evolution are the three sites: D3G, M71I, and A81V.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the green fluorescent protein nanobody with high affinity for GFP as described in claim 1.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid molecule includes a sequence as shown in SEQ ID NO. 8 or SEQ ID NO.

9.

7. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 5 or 6.

8. A host cell, characterized in that, The host cell contains the expression vector as described in claim 7.

9. The application of the green fluorescent protein nanobody with high affinity for GFP as described in claim 1 in the preparation of ultrasensitive sandwich ELISA or homogeneous immunoassay reagents.

10. A detection reagent, characterized in that, This includes the green fluorescent protein nanobody with high affinity for GFP as described in claim 1.

Images