Shark synthetic nanobodies targeting green fluorescent protein and uses thereof

Abstract

The application discloses a shark-derived synthetic nanobody targeting green fluorescent protein and an application thereof. The nucleotide sequence of the nanobody is as shown in SEQ ID NO:1-4, and the amino acid sequence is as shown in SEQ ID NO:5-8. The preparation method comprises the following steps: screening a green fluorescent protein specific nanobody sequence from a shark-derived synthetic antibody library, performing eukaryotic expression vector construction, then performing transient transfection in HEK 293F cells for expression, and finally obtaining the shark-derived synthetic nanobody targeting the green fluorescent protein. The shark-derived synthetic nanobody is used for affinity purification of the green fluorescent protein. The nanobody of the application is derived from Chlamydoselachus anguineus, has small molecular weight, strong stability, strong tissue penetration ability and can recognize hidden antigen epitopes, and can be used in the fields of affinity purification, immunological detection, molecular imaging, immunosensing probes and the like of the green fluorescent protein.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a shark-derived synthetic nanobody targeting green fluorescent protein and application thereof. BACKGROUND

[0002] In 1993, heavy chain-only antibodies (HcAbs) with natural light chain deletion were first discovered in camelids. In 1995, immunoglobulins with similar heavy chain antibody structure were found in Ginglymostoma cirratum, which were called immunoglobulin new antigen receptors (IgNAR). The antigenic determinant of IgNAR is composed of only one domain, namely VNAR (Variable Domain of Immunoglobulin New Antigen Receptor), and this antibody fragment composed of a single domain is called a single-domain antibody. The single-domain antibody protein is less than 10 nanometers in diameter, and is therefore also called a nanobody. Different from conventional antibodies and camel nanobodies, VNAR only contains two CDR regions (CDR1 and CDR3), and the CDR2 region is replaced by two high flanking regions. Due to the large deletion of FR2-CDR2, VNAR becomes the smallest antibody fragment with binding function ever discovered, with a molecular weight of about 12 kDa. At the same time, the CDR3 region of VNAR is long and has a special finger-like structure, which makes it easier and tighter to bind to the hidden epitope of proteins. In addition, VNAR also has the characteristics of high stability, high affinity and specificity, good solubility, and strong tissue penetration ability, so that it has the advantages of low production cost and stable quality in application. It has potential application prospects in the fields of molecular imaging, disease diagnosis, immune detection, environmental monitoring, etc.

[0003] Green fluorescent protein (GFP) was discovered by Osamu Shimomura et al. in 1962 in the Victoria multiple tube luminescent jellyfish. The protein produced by the gene emits green fluorescence under the excitation of light in the blue wavelength range. The molecular weight of GFP is 26 kDa, which is composed of 238 amino acids. The amino acids at positions 65-67 (Ser-Tyr-Gly) form a luminophore, which is the main light-emitting position. Green fluorescent protein is basically non-toxic and harmless to biological cells and tissues, and its fluorescence signal is stable and easy to detect. Therefore, it is often used to study the skeleton and cell division, dynamics and vesicle transport, developmental biology, etc., and can be applied to the determination of transfected cells, the determination of in vivo gene expression, the positioning of protein molecules, the dynamic monitoring of intercellular molecular communication, etc. Anti-GFP nanobodies can bind to GFP in vivo and in vitro, and can be widely used in subcellular localization, protein activity, protein interaction, etc. GFP nanobodies can be used as affinity ligands for purifying GFP-tagged proteins. Anti-GFP nanobodies with high affinity can be coupled with horseradish peroxidase to serve as a detection secondary antibody, which has higher sensitivity than existing detection secondary antibodies on the market. Nanobodies can also be used as new immunosensing probes. By comprehensively using immunological analysis and fluorescence resonance energy transfer principles, a fluorescence resonance energy transfer nanosensor based on fluorescent nanobodies can be developed. Currently, there is no shark-derived synthetic nanobody that binds to green fluorescent protein. SUMMARY

[0004] In order to solve the problems in the background art, the purpose of the present application is to provide a shark-derived synthetic nanobody targeting green fluorescent protein and its application. The present application provides a shark-derived heavy chain antibody variable region sequence (VNAR) capable of binding to green fluorescent protein with high affinity, which is also called nanobody, and can be used in the fields of affinity purification, immunological detection, cell imaging, immunosensing probes, etc.

[0005] The technical solutions adopted by the present application are as follows:

[0006] One, a shark-derived synthetic nanobody targeting green fluorescent protein:

[0007] The nucleotide sequence of the shark-derived nanobody is shown as SEQ ID NO. 1-4.

[0008] The amino acid sequence of the shark-derived nanobody is shown as SEQ ID NO. 5-8.

[0009] Specifically, the shark-derived synthetic nanobodies include green fluorescent protein shark-derived nanobody 14A-1A, green fluorescent protein shark-derived nanobody 14A-3H, green fluorescent protein shark-derived nanobody 14A-4D, and green fluorescent protein shark-derived nanobody 15A-5D; the amino acid sequence of the green fluorescent protein shark-derived nanobody 14A-1A is shown as SEQ ID NO. 5, the amino acid sequence of the green fluorescent protein shark-derived nanobody 14A-3H is shown as SEQ ID NO. 6, the amino acid sequence of the green fluorescent protein shark-derived nanobody 14A-4D is shown as SEQ ID NO. 7, and the amino acid sequence of the green fluorescent protein shark-derived nanobody 15A-5D is shown as SEQ ID NO. 8.

[0010] The nucleotide sequence of the gene encoding the green fluorescent protein shark-derived nanobody 14A-1A is shown as SEQ ID NO. 1, the nucleotide sequence of the gene encoding the green fluorescent protein shark-derived nanobody 14A-3H is shown as SEQ ID NO. 2, the nucleotide sequence of the gene encoding the green fluorescent protein shark-derived nanobody 14A-4D is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene encoding the green fluorescent protein shark-derived nanobody 15A-5D is shown as SEQ ID NO. 4.

[0011] II. Use of a shark-derived synthetic nanobody targeting green fluorescent protein:

[0012] The use of the shark-derived synthetic nanobody in the preparation of a reagent for detecting / tracking green fluorescent protein.

[0013] The use of the shark-derived synthetic nanobody in the preparation of a green fluorescent protein affinity purification reagent.

[0014] III. A method for using a shark-derived synthetic nanobody in green fluorescent protein affinity purification, comprising the following steps:

[0015] Step S1, first, the agarose gel coupled with the shark-derived synthetic nanobody is used as the filler of the affinity chromatography column to prepare the affinity chromatography column;

[0016] The step S1 is specifically: selecting Sepharose 4B as an affinity chromatography medium, coupling the shark-derived synthetic nanobody and the pre-activated Sepharose, coupling 5-10 mg of the shark-derived synthetic nanobody per milliliter of Sepharose, slowly stirring at 4°C overnight to ensure sufficient coupling, then loading the Sepharose coupled with the shark-derived synthetic nanobody into an affinity chromatography column, avoiding the generation of bubbles, then washing the affinity chromatography column with PBS buffer (pH 7.4) at a flow rate of 2-3 mL / min, washing for 5-6 column volumes until the pH and ionic strength of the effluent of the affinity chromatography column are consistent with the PBS buffer.

[0017] In the process of coupling the shark-derived synthetic nanobody and the Sepharose, 5-10 mg of the shark-derived synthetic nanobody is coupled per milliliter of Sepharose.

[0018] The activated treatment of Sepharose: using cyanogen bromide CNBr to activate under alkaline conditions (pH 10-11) to make the Sepharose have active groups that can react with the amino groups of the GFP antibody. When the mixed sample containing green fluorescent protein GFP passes through the affinity chromatography column, the GFP specifically binds to the shark-derived synthetic nanobody, while other impurities cannot bind, thereby achieving the preliminary separation and purification of GFP. By changing the elution conditions, such as adjusting the pH and ionic strength of the buffer or adding specific elution reagents, the GFP can be dissociated from the antibody, and then the purified GFP can be obtained.

[0019] Step S2: loading the mixed sample containing green fluorescent protein GFP into the affinity chromatography column, and obtaining the purified green fluorescent protein from the eluate of the affinity chromatography column.

[0020] The step S2 is specifically: centrifuging (12000 rpm, 4°C, 10 min) the mixed sample containing GFP, taking the supernatant, filtering with a 0.45 μm filter membrane, loading the filtered mixed sample into the affinity chromatography column at a flow rate of 2 mL / min to allow the GFP to fully bind to the shark-derived synthetic nanobody; then washing the affinity chromatography column with PBS buffer containing 0.1 M NaCl at a flow rate of 2 mL / min to wash away the unbound impurity green fluorescent protein, then eluting the green fluorescent protein with glycine-HCl buffer at pH 3.0-3.5, collecting the eluate containing GFP at 2 mL per tube, and adding 1 M Tris-HCl (pH 9.0) to neutralize the eluate to prevent GFP denaturation, thereby achieving affinity purification of the green fluorescent protein.

[0021] In the process of purifying GFP, the presence or absence of GFP, the content and the molecular weight of GFP in each purification step can be detected by Western blotting and other methods using shark-derived synthetic nanobodies, so as to monitor the effect and progress of purification and ensure that high-purity GFP is obtained.

[0022] Four, a method for preparing and characterizing a shark-derived synthetic nanobody targeting green fluorescent protein, comprising the following steps:

[0023] Step S1: screening specific nanobodies;

[0024] The step S1 is specifically:

[0025] S11, first inoculate the existing shark nanobody synthesis library into a liquid culture medium, then add helper phages to the liquid culture medium to promote the proliferation of the phages, after standing culture and centrifugal collection of the phages, the phages are cultured overnight by shaking to obtain more phages, then the phages are enriched by PEG precipitation method to obtain the titer of the enriched phages;

[0026] S12, dissolve the green fluorescent protein in a protein-free solution GFBE to obtain a green fluorescent protein solution, then coat 0.1 mg / mL of the green fluorescent protein solution in an immunoplate as a green fluorescent protein group, and coat the immunoplate without the green fluorescent protein solution as a negative control group, add the phages enriched in S11 to the immunoplates of the green fluorescent protein group and the negative control group respectively, so that the phages combine with the green fluorescent protein in the green fluorescent protein group, then wash the immunoplates with PBST (phosphate buffer), and obtain the phages shed from the green fluorescent protein group and the negative control group after washing respectively;

[0027] S13, infect the TG1 bacteria (Escherichia coli) with the phages of the green fluorescent protein group and the negative control group, then coat the phages of the infected TG1 bacteria on a plate, and after 12-24h of culture, obtain single colonies, count and compare the number of single colonies of the green fluorescent protein group and the negative control group:

[0028] If the number of single colonies of the green fluorescent protein group is more than 10 times the number of the negative control group, it indicates that the number of single colonies of the green fluorescent protein group meets the requirements and enters S14;

[0029] Otherwise, return to S12 to prepare new phages until the number of single colonies of the green fluorescent protein group meets the requirements;

[0030] S14, select single colonies in the green fluorescent protein group and place them in an immunoplate, add helper phages, and amplify to obtain monoclonal phages;

[0031] S15, the single-stranded phage is coated with green fluorescent protein solution, and then horseradish peroxidase-labeled phage-specific antibody is added to the immunoplate, followed by TMB (3,3',5,5'-tetramethylbenzidine) color developing solution for color developing reaction of the single-stranded phage, and then the OD 450 value of the single-stranded phage with OD 450 >1 is measured, and the sequence comparison software is used to remove repeated sequences, and the nucleotide sequence obtained after sequencing and removing the repeated sequences is used as the nucleotide sequence of the coding gene of the shark-derived nanobody. The nucleotide sequence obtained in S1 has green fluorescent protein specificity.

[0032] Step S2: nanobody recombinant expression;

[0033] The S2 is specifically:

[0034] The coding gene of the shark-derived nanobody is constructed into a eukaryotic expression vector pTT5-TEV-Fc, and then expressed by HEK293F (human embryonic kidney cells), and the shark-derived synthetic nanobody (i.e. nanobody-Fc fusion protein specific to green fluorescent protein) is obtained by purification with agarose gel rProtein A affinity chromatography column.

[0035] Step S3: characterization of the shark-derived nanobody.

[0036] The step S3 is specifically:

[0037] S31, green fluorescent protein antigen is coated on an immunoplate at a concentration of 1 μg / mL, and a protein-free solution is used as a negative control group, and the coating is performed at 4°C overnight, the next day, the liquid is removed, and the immunoplate is washed with PBS (phosphate buffer), and then blocked with MPBS (phosphate buffer plus 5% skim milk), and then the expressed antibody is added to the well at multiple concentrations for incubation, after incubation, the well is washed with PBST (phosphate buffer plus 0.1% Tween-20), and then goat anti-human IgG Fc-HRP antibody is added for incubation, and then washed, and then TMB is used for color development, and H2SO4 is used for termination, and then the OD 450 value is measured, and the half-effective concentration EC 50 value of the antibody is fitted.

[0038] S32, green fluorescent protein is biotinylated, and at the same time, the SA biosensor is pre-wetted in PBST buffer containing 0.01% Tween 20 for 10 min, and then the biotinylated green fluorescent protein is loaded on the SA biosensor, and the solidification value is higher than 1.0 nm, and then the VNAR-Fc fusion protein is incubated at room temperature for 240 s, and then dissociated for 240 s. The kinetic process of the binding interaction is monitored in real time, and BLItz Pro TMThe software performs data acquisition and fitting analysis of the binding dissociation curves based on a 1:1 binding model to obtain the binding constant k. a dissociation constant k d And calculate the equilibrium dissociation constant K. D .

[0039] This invention utilizes a pre-constructed shark-derived nanobody synthesis library and, through biopanning of green fluorescent protein, ultimately isolates four nanobodies, such as... Figure 2 As shown, they are named 14A-1A, 14A-3H, 14A-4D and 15A-5D respectively.

[0040] The nanobody of this invention was fused with the human IgG1 Fc fragment, cloned into the pTT5 vector, and expressed secretoriously in mammalian 293F cells. After 5 days of expression, the nanobody fusion protein in the culture supernatant was purified using an rProtein A affinity chromatography column. Figure 3 As shown, this invention yields a high-purity green fluorescent protein nanobody Fc fusion protein.

[0041] The nanobodies in this invention can bind to green fluorescent protein with high affinity. Non-competitive ELISA assays show that... Figure 4 As shown, the affinity of the four nanobodies of this disclosure for green fluorescent protein is ranked in the following order: 14A-3H > 15A-5D > 14A-1A > 14A-4D, with a half-maximal effective concentration (COP) of EC50. 50 The values ​​are 0.027, 0.05, 0.056, and 0.45 nM, respectively.

[0042] The affinity between the nanobody and green fluorescent protein was determined using biolayer interferometry (BLI), and the results are as follows: Figure 5 As shown, the binding affinity of the four nanobodies to green fluorescent protein is in the following order: 14A-3H > 14A-1A > 15A-5D > 14A-4D, and their affinity constants K0 are... D The values ​​are 4.56, 6.36, 7.64, and 9.54 nM, respectively.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] 1. The nanobody of the present invention is derived from the striped bamboo shark. It has a small molecular weight, strong stability, strong tissue penetration ability and can recognize hidden antigen epitopes. It can be used in the fields of affinity purification of green fluorescent protein, immunological detection, molecular imaging, and immunosensing probes.

[0045] 2、The nanobody protein can be obtained in large quantities through a recombinant expression system, has high antibody expression, low preparation cost and strong stability, and solves the problems of large molecular weight, difficult preparation and complex operation of traditional human monoclonal antibodies.

[0046] 3、The nanobody of the application is derived from Chiloscyllium plagiosum and has strong tissue penetration ability, and the shark-derived single-domain antibody can purify, detect the concentration of and locate green fluorescent protein. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is a monoclonal phage ELISA result graph.

[0048] Figure 2 is a nanobody amino acid sequence alignment result graph;

[0049] Figure 3 is an SDS-PAGE gel electrophoresis result graph of the nanobody Fc fusion protein. Lane M is a standard protein;

[0050] Figure 4 is an enzyme-linked immunosorbent assay (ELISA) for determining the binding affinity of specific antibodies to green fluorescent protein;

[0051] Figure 5 is a graph of the affinity between the nanobody and green fluorescent protein detected by BLI using antibody affinity assay. The solid line is the real-time monitoring kinetic curve, and the dashed line is the software fitting curve. The kinetic curves of different green fluorescent protein concentration gradients from top to bottom correspond to the concentrations from top to bottom on the right side. DETAILED DESCRIPTION

[0052] The application will be further described in detail below in combination with the drawings and specific embodiments. The following examples are an explanation of the application, and the application is not limited to the following examples.

[0053] The embodiments of the application are as follows:

[0054] Example 1, screening and characterization of shark-derived single-domain antibodies binding to green fluorescent protein

[0055] I. Screening of nanobody targeting green fluorescent protein

[0056] Step S1, inoculate the shark-derived nanobody synthesis library into 2×TY liquid medium containing 100 μg / mL ampicillin (Amp) and 2% glucose (G) (100 mL in volume) to make the initial OD 600 about 0.1. Culture at 37℃, 200 rpm, until the OD 600about 0.5-0.6 (OD 600 The value refers to the absorbance of the solution at a wavelength of 600 nm). Then 10 μL of 10 12 pfu of KM13 helper phage was added and incubated at 37°C for 45 min. After the incubation, the bacterial solution was centrifuged at 37°C for 10 min at 3500 g, and the supernatant was removed. The precipitated bacteria were resuspended in 200 mL of 2TY / Amp / Kanamycin / Kana / 0.1% G medium and incubated at 25°C for 16 h with shaking at 200 rpm. After the incubation, the whole bacterial solution was centrifuged at 4°C for 30 min at 3500 g. The whole supernatant was added to 20% PEG / NaCl (polyethylene glycol / sodium chloride) at a ratio of 4:1, mixed and incubated in an ice bath for 1 h, and then centrifuged for 30 min at 4°C at 3500 g. The supernatant was discarded, and the precipitate was resuspended in PBS (phosphate buffered saline) that had been sterilized by high-temperature and high-pressure sterilization. The precipitate was vortexed and then transferred to a centrifuge tube, which was centrifuged for 10 min at 4°C at 12000 rpm. The supernatant was transferred to a new centrifuge tube, and the OD 260 value was measured.

[0057] Step S2, panning. The green fluorescent protein antigen was coated in the immunoplate at a concentration of 0.1 mg / mL, and a protein-free solution GFBE negative control was set. After incubation at 4°C overnight, the coating solution was discarded, and 280 μL of PBS was used for washing three times. Then 280 μL of MPBS (phosphate buffer solution) was added, and the blocking reaction was performed at room temperature for 2 h. Subsequently, 280 μL of PBST (phosphate buffer) was used for washing twice, and 1 x 10 11 pfu of the phage solution prepared above was added to the green fluorescent protein group Hb and the negative control group, and incubated at 80 rpm for 1 h. The waste liquid was discarded, and 280 μL of PBST was used for washing 20 times. 100 μL of trypsin (0.5 mg / mL) was added to the green fluorescent protein group Hb and the negative control group, and incubated at 80 rpm for 1 h to elute the phage. Then 100 μL of the eluted phage was used to infect 900 μL of TG1 bacterial solution with an OD 600 about 0.5, and incubated at 37°C for 45 min. 50 μL and 5 μL of the infected bacterial solution were respectively plated on LB / Amp plates and incubated overnight.

[0058] Step S3, phage ELISA screening of green fluorescent protein nanobody monoclonal:

[0059] Step S31, preparation of monoclonal phage: 95 single clones were picked from the overnight culture plate, inoculated into 100 μL 2xTY / Amp / 2% G respectively, another hole as negative control, and cultured in a 96-well culture plate for 6-8 h (37°C, 250 rpm). Then, 2.5 μL of the bacterial solution was taken from each hole and inoculated into 100 μL 2xTY / Amp / 2% G / KM13 culture medium (2.5 μL KM13 was added to 12.5 mL 2xTY / Amp / 2% G), and cultured again for 1.5 h (37°C, 250 rpm), and then left at 37°C for 45 min. Equal amount of 30% glycerol was added to the remaining 97 μL bacterial solution, and the mixture was stored in a refrigerator at -80°C. After completion of the standing, the bacterial solution was thoroughly mixed and 50 mL was discarded, and the precipitate was centrifuged at 3200 g for 20 min, and the supernatant was thoroughly removed. The precipitate was resuspended with 200 μL 2TY / Amp / Kana / 0.1% G culture medium, and cultured for 20 h (25°C, 250 rpm). After completion of the culture, the precipitate was centrifuged at 4°C for 40 min, and the supernatant of the monoclonal phage was transferred to a new 96-well plate and stored at 4°C.

[0060] Step S32, ELISA detection of monoclonal phage: the green fluorescent protein was diluted with GFBE to 1 μg / mL, and 100 μL was taken for coating a 96-well immunoplate respectively, and a negative control of no protein solution GFBE was set, and the coating was performed at 4°C overnight. The plate was washed with PBS for 3 times, 300 μL MPBS was added to each hole, and the blocking was performed at room temperature for 2 h. 100 μL of the phage MPBS mixture prepared above was added to each hole, and incubated at room temperature for 1 h. The plate was washed with PBST for 4 times. The HRP-anti M13 antibody was diluted with MPBS at a ratio of 1:8000, and 100 μL was added to each hole of the above immunoplate, and incubated at room temperature for 1 h. The plate was washed with PBST for 4 times. 100 μL TMB color developing substrate was added to each hole, and the plate was wrapped with aluminum foil to avoid light, and reacted at room temperature for 5 min. 50 μL of 1M H2SO4 was added to each hole to terminate the reaction, and the OD value was measured 450 Figure 1

[0061] Step S33, the holes with OD 450 value greater than 1 were picked for sequencing analysis, and the sequencing primer was 5'-CCCTCATAGTTAGCGTAACGA-3'.

[0062] Step S34, after alignment and analysis of the measured antibody sequences, and after exclusion of repeated clones, a total of 4 different nanobody sequences were obtained Figure 2 ​​). SEQ ID NO. 1-4 are the nucleotide sequences of the Nanobodies, from which the amino acid sequences of the Nanobodies as shown in SEQ ID NO. 5-8 are derived.

[0063] II. Expression and purification of Nanobodies and Fc fusion proteins thereof

[0064] The primers were designed to fuse the N-terminal of the Nanobodies with the signal peptide of IFNα protein to guide the secretion expression, and to fuse the C-terminal of the Nanobodies with human IgG1 Fc, while introducing a TEV enzyme cutting site between them, and then cloned into the mammalian expression vector pTT5. The constructed recombinant vector was transiently transfected into mammalian cells HEK293F using PEI, and the supernatant was collected after 5 days of culture. The antibody fusion protein in the supernatant was purified using rProtein A affinity chromatography column, and subjected to SDS-PAGE electrophoresis analysis. The results are shown in Figure 3 , indicating that high-purity Nanobodies were obtained after purification.

[0065] III. Characterization of Nanobodies

[0066] Step S1, in the immunoplate, the green fluorescent protein antigen was coated at a concentration of 1 μg / mL, and the non-protein solution GFBE was used as a negative control, both at 4°C overnight. The next day, the protein coating solution and the liquid in the non-protein control wells were removed, and after two washes with PBS, the purified antibody was added at an initial concentration of 10 3 nM, and then serially diluted to 10 -6 nM, a total of 22 gradient concentrations were set. All dilution steps were performed in MPBS. After the blocking was completed, the antibody protein solution at each concentration gradient was added to the pre-coated wells (100 μL per well), and incubated at 80 rpm for 1 h. The control wells only added an equal amount of MPBS. To ensure data reliability, three parallel wells were set for each concentration. After incubation, the wells were washed 4 times with PBST. 100 μL of anti-IgG Fc-HRP antibody (diluted 1:10000 in MPBS) was added to each well, incubated at 80 rpm for 1 h, and washed again 3 times with PBST. The OD 450 value was determined after TMB color development and termination with 1 moL / L H2SO4 solution. The results are shown in Figure 4 , indicating that the four purified Nanobodies have good affinity to green fluorescent protein, and the order of their binding affinity to green fluorescent protein is 14A-3H > 15A-5D > 14A-1A > 14A-4D, and the half-effect concentration EC 50 values are 0.027, 0.05, 0.056, and 0.45 nM, respectively.

[0067] Step S2, the affinity between the nanobody and green fluorescent protein is characterized by using biofilm interference technology BLI

[0068] In the experiment, the SA sensor is used, and the antigen is immobilized by biotinizing the green fluorescent protein. First, in order to ensure the effective work of the sensor, 200 μL of phosphate buffer (PBST) is used to pre-wet the sensor for 10 min. Then, the bio-green fluorescent protein is loaded on the SA biosensor, and the solidification value is higher than 1.0 nm. The gradient-diluted VNAR-Fc fusion protein is incubated at room temperature for 240 s, and dissociated for 240 s. According to the formula: Kd= (Rmax-R) / Rmax, the affinity constant Kd of the four nanobodies to the green fluorescent protein can be calculated. Figure 5 It can be known that the binding force of the four nanobodies to the green fluorescent protein is in the order of 14A-3H>14A-1A>15A-5D>14A-4D, and the affinity constant Kd is 4.56, 6.36, 7.64, 9.54 nM respectively. D

[0069] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

[0070] The sequence involved in the present application is as follows:

[0071] SEQ ID NO. 1:

[0072] Name: nucleotide sequence of green fluorescent protein shark nanobody 14A-1A

[0073] DNA type: other DNA

[0074] Organism source: Cihiloscyllium plagiasum

[0075] ​ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGCGGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGACATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCTATTAGTACCTCGCCCCCTTACACTATCGGCCCTCTCGTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0076] SEQ ID NO. 2:

[0077] Name: Nucleotide sequence of green fluorescent protein shark nanobody 14A-3H

[0078] DNA type: other DNA

[0079] Organism: Cihiloscyllium plagiasum

[0080] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGAGTTTATCAAATGCCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATAACTACCCGACGGGGACTCTTTTTCGCGATCTTCGCTACAACGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0081] SEQ ID NO. 3:

[0082] Name: Nucleotide sequence of green fluorescent protein shark nanobody 14A-4D

[0083] DNA type: other DNA

[0084] Organism: Cihiloscyllium plagiasum

[0085] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAACAAGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCTATTACTACCGCGATCCCTTACTATGACGGTCCTCTCGTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0086] SEQ ID NO. 4:

[0087] Name: Nucleotide sequence of green fluorescent protein shark nanobody 15A-5D

[0088] DNA type: other DNA

[0089] Organism: Cihiloscyllium plagiasum

[0090] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAACAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGCCCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCGCTATCCGCCGAGCGGCGAGTACGCGGCCGATCTCGCCTCTATCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0091] SEQ ID NO. 5:

[0092] Name: Amino acid sequence of green fluorescent protein shark nanobody 14A-1A

[0093] Sequence type: AA

[0094] Organism: Cihiloscyllium plagiasum

[0095] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKRASLSTGGRYSDTKNTTSKSFSLRISDLRVEDSGTYHCKAYPISTSPPYTIGPLVEGGGTILTVKP

[0096] SEQ ID NO. 6:

[0097] Name: Amino acid sequence of green fluorescent protein shark nanobody 14A-3H

[0098] Sequence type: AA

[0099] Organism: Cihiloscyllium plagiasum

[0100] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSNAGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYNYPTGTLFRDLRYNEGGGTILTVKP

[0101] SEQ ID NO. 7:

[0102] Name: Amino acid sequence of green fluorescent protein shark nanobody 14A-4D

[0103] Sequence type: AA

[0104] Organism: Cihiloscyllium plagiasum

[0105] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSTGGRYSDTKNKASKSFSLRISDLRVEDSGTYHCKAYPITTAIPYYDGPLVEGGGTILTVKP

[0106] SEQ ID NO. 8:

[0107] Name: Amino acid sequence of green fluorescent protein shark nanobody 15A-5D

[0108] Sequence type: AA

[0109] Organism source: Cihiloscyllium plagiasum

[0110] TQRVEQTPTTTTKEAGESLTINCVLRDSSYALGSTYWYFTKKGATNKES LSNGGRYAETVNKASKSFSLRISALRVEDSGTYHCKAYRYPPSGEYAADLASI EGGGTILTVKP.

Claims

1. A shark-sourced synthetic nanobody targeting green fluorescent protein, characterized in that: the shark-sourced synthetic nanobody comprises green fluorescent protein shark-sourced nanobody 14A-1A, green fluorescent protein shark-sourced nanobody 14A-3H, green fluorescent protein shark-sourced nanobody 14A-4D and green fluorescent protein shark-sourced nanobody 15A-5D; the amino acid sequence of the green fluorescent protein shark-sourced nanobody 14A-1A is shown as SEQ ID NO. 5, the amino acid sequence of the green fluorescent protein shark-sourced nanobody 14A-3H is shown as SEQ ID NO. 6, the amino acid sequence of the green fluorescent protein shark-sourced nanobody 14A-4D is shown as SEQ ID NO. 7, and the amino acid sequence of the green fluorescent protein shark-sourced nanobody 15A-5D is shown as SEQ ID NO.

8. The nucleotide sequence of the gene encoding the green fluorescent protein shark-sourced nanobody 14A-1A is shown as SEQ ID NO. 1, the nucleotide sequence of the gene encoding the green fluorescent protein shark-sourced nanobody 14A-3H is shown as SEQ ID NO. 2, the nucleotide sequence of the gene encoding the green fluorescent protein shark-sourced nanobody 14A-4D is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene encoding the green fluorescent protein shark-sourced nanobody 15A-5D is shown as SEQ ID NO.

4.

2. The shark synthetic nanobody targeting green fluorescent protein according to claim 1, characterized in that: The shark-sourced synthetic nanobody is used for preparing a green fluorescent protein affinity purification reagent.

3. Use of a shark-derived synthetic nanobody targeting green fluorescent protein according to any one of claims 1-2, characterized in that: The shark-sourced synthetic nanobody is used for preparing a green fluorescent protein related tracking reagent.

4. Use of a shark-derived synthetic nanobody targeting green fluorescent protein according to any one of claims 1-2, characterized in that: The method comprises the following steps:

5. A method for the affinity purification of green fluorescent protein using a shark-derived synthetic nanobody according to any one of claims 1 to 2, characterized in that, Step S1, first, agarose gel coupled with the shark-sourced synthetic nanobody is used as a filler to prepare an affinity chromatography column; Step S2, a mixed sample containing green fluorescent protein GFP is loaded into the affinity chromatography column, and the purified green fluorescent protein is obtained from the eluate of the affinity chromatography column. The step S1 specifically comprises the following steps: coupling the shark-sourced synthetic nanobody with agarose gel, then loading the agarose gel coupled with the shark-sourced synthetic nanobody into the affinity chromatography column, and then rinsing the affinity chromatography column with PBS equilibration buffer until the pH value and ionic strength of the effluent are consistent with those of the PBS equilibration buffer.

6. The method for green fluorescent protein affinity purification using shark synthetic nanobodies according to claim 5, characterized in that: The step S2 specifically comprises the following steps: centrifuging the mixed sample containing GFP to obtain the supernatant, filtering the supernatant, loading the filtered mixed sample into the affinity chromatography column to allow the GFP to fully bind with the shark-sourced synthetic nanobody, then washing the affinity chromatography column with PBS washing buffer to wash away the unbound impurity green fluorescent protein, and then eluting the green fluorescent protein with glycine-HCl buffer with a pH value of 3.0-3.5, and collecting the eluate containing the GFP to realize affinity purification of the green fluorescent protein.

7. The method for green fluorescent protein affinity purification using shark synthetic nanobodies according to claim 5, characterized in that: In the process of coupling the shark-sourced synthetic nanobody with agarose gel, 5-10 mg of the shark-sourced synthetic nanobody is coupled with every milliliter of agarose gel.

8. The method for green fluorescent protein affinity purification using shark synthetic nanobodies according to claim 6, characterized in that: ​

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