Shark source synthetic nano antibody targeting green fluorescent protein and application of shark source synthetic nano antibody

Abstract

The invention discloses a shark source synthetic nano antibody targeting green fluorescent protein and application of the shark source synthetic nano antibody. Nucleotide sequences of the nano antibody are shown as SEQ ID NO: 1-4, and amino acid sequences of the nano antibody are shown as SEQ ID NO: 5-8. The preparation method comprises the following steps: screening a nano antibody sequence with green fluorescent protein specificity from a shark source synthetic antibody library, constructing an eukaryotic expression vector, and then instantaneously transfecting HEK 293F cells for expression to finally obtain the shark source synthetic nano antibody targeting the green fluorescent protein. The nano antibody synthesized from the shark source is used for affinity purification of the green fluorescent protein. The nano antibody provided by the invention is derived from the spotted bamboo shark, has the advantages of small molecular weight, strong stability, strong tissue penetrating power and capability of recognizing hidden antigen epitopes, and can be used in the fields of affinity purification, immunological detection, molecular imaging, immunosensing probes and the like of green fluorescent proteins.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a shark-derived synthetic nano antibody targeting green fluorescent protein and an application thereof. Background Art

[0002] In 1993, antibodies containing only heavy chains (HcAbs) with natural light chains missing were first discovered in camelids. In 1995, immunoglobulins with a structure similar to heavy chain antibodies were found in nurse sharks (Ginglymostoma cirratum), which were called immunoglobulin new antigen receptors (IgNARs). The antigenic determinant cluster of IgNAR consists of only one domain, namely VNAR (Variable Domain of Immunoglobulin New Antigen Receptor). The antibody fragment composed of this single domain is called a single domain antibody. The diameter of a single domain antibody protein is less than 10 nanometers, so it is also called a nanobody. Unlike conventional antibodies and camel nanobodies, VNAR contains only two CDR regions (CDR1 and CDR3), and its CDR2 region is replaced by two high-edge regions. Due to the large loss of FR2-CDR2, VNAR has become the smallest antibody fragment with binding function discovered, with a molecular weight of about 12kDa. At the same time, the CDR3 region of VNAR is long and has a special finger-like structure, which makes it easier and more tightly bound to the hidden epitope of the protein. In addition, VNAR also has the characteristics of strong stability, high affinity and specificity, good solubility, and strong tissue penetration, which makes it have the advantages of low production cost and stable quality when applied. 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 jellyfish Aequorea victoria. The protein produced by its gene will emit green fluorescence when stimulated by light in the blue wavelength range. The molecular mass of GFP is 26kDa and it is composed of 238 amino acids. The amino acids 65 to 67 (Ser-Tyr-Gly) form a luminophore, which is the main location of light emission. 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. It can also be applied to the determination of transfected cells, the determination of gene expression in vivo, the localization of protein molecules, and the dynamic monitoring of molecular communication between cells. Anti-GFP nanobodies can bind to GFP in vivo and in vitro, and are widely used in subcellular localization, protein activity, protein interaction, etc. GFP nanobodies can be used as affinity ligands for purification to purify GFP-tagged proteins. High-affinity anti-GFP nanobodies can be coupled with horseradish peroxidase as secondary detection antibodies, which have higher sensitivity than existing secondary detection antibodies on the market. Nanobodies can also be used as new immunosensor probes. By combining immunoassays and fluorescence resonance energy transfer principles, a fluorescence resonance energy transfer nanosensor based on fluorescent nanobodies has been developed. Currently, there are no shark-derived synthetic nanobodies that bind to green fluorescent protein. Summary of the invention

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

[0005] The technical solution adopted by the present invention is as follows:

[0006] 1. A shark-derived synthetic nanobody targeting green fluorescent protein:

[0007] The nucleotide sequences of the shark-derived nanobodies are shown in SEQ ID NO.1-4.

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

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

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

[0011] 2. Application of a shark-derived synthetic nanobody targeting green fluorescent protein:

[0012] The shark-derived synthetic nano-antibody is used in the preparation of reagents related to detecting / tracking green fluorescent protein.

[0013] The shark-derived synthetic nano-antibody is used in preparing a green fluorescent protein affinity purification reagent.

[0014] 3. A method for applying shark-derived synthetic nanoantibodies in affinity purification of green fluorescent protein, comprising the following steps:

[0015] Step S1, first, using agarose gel coupled with shark-derived synthetic nano-antibodies as a filler of an affinity chromatography column to prepare an affinity chromatography column;

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

[0017] In the process of coupling the shark-derived synthetic nano-antibody and agarose gel, 5-10 mg of the shark-derived synthetic nano-antibody is coupled to each milliliter of agarose gel.

[0018] Agarose gel activation treatment: Use cyanogen bromide CNBr to activate under alkaline conditions (pH 10-11) to make agarose gel with active groups that can react with the amino groups of GFP antibodies. When a mixed sample containing green fluorescent protein GFP passes through the affinity chromatography column, GFP will specifically bind to the shark-derived synthetic nanoantibody, while other impurities cannot bind, thereby achieving the initial separation and purification of GFP. GFP can be dissociated from the antibody by changing the elution conditions, such as adjusting the pH value and ionic strength of the buffer or adding specific elution reagents, and then purified GFP can be obtained.

[0019] Step S2: Load the mixed sample containing green fluorescent protein GFP onto an affinity chromatography column, and extract the purified green fluorescent protein from the eluate of the affinity chromatography column.

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

[0021] During the GFP purification process, shark-derived synthetic nanoantibodies can be used to detect the presence, content, molecular weight, and other information of GFP in each purification step through methods such as Western blot, so as to monitor the effect and progress of purification and ensure that high-purity GFP is obtained.

[0022] 4. 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 as follows:

[0025] S11. First, the existing shark-derived nanoantibody synthetic library is inoculated into a liquid culture medium, and then a helper phage is added to the liquid culture medium to promote the proliferation of the phage. After static culture and centrifugal collection of the phage, the phage is shaken and cultured overnight to obtain more phages, and then the phages are enriched by PEG precipitation method to obtain the titer of the enriched phages;

[0026] S12, dissolving green fluorescent protein in a protein-free solution GFBE to prepare a green fluorescent protein solution, then coating 0.1 mg / mL of the green fluorescent protein solution in an immunoplate as a green fluorescent protein group, and using an immunoplate not coated with the green fluorescent protein solution as a negative control group, respectively adding S11-enriched phages to the immunoplates of the green fluorescent protein group and the negative control group, so that the phages bind to the green fluorescent protein in the green fluorescent protein group, and then washing the immunoplate with PBST (phosphate buffer), and after washing, obtaining the detached phages in the green fluorescent protein group and the negative control group, respectively;

[0027] S13. The phages of the green fluorescent protein group and the negative control group were used to infect TG1 bacteria (Escherichia coli), and then the phages infected with TG1 bacteria were spread on a plate. After culturing for 12-24 hours, single colonies were obtained. The number of single colonies of the green fluorescent protein group and the negative control group was counted and compared:

[0028] If the number of single colonies in the green fluorescent protein group is 10 times greater than that in the negative control group, it means that the number of single colonies in the green fluorescent protein group meets the requirement, and the process goes to S14;

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

[0030] S14, selecting a single colony in the green fluorescent protein group and placing it in an immune well plate, adding a helper phage, and amplifying to obtain a monoclonal phage;

[0031] S15. Use green fluorescent protein solution to coat the immunoplate where the monoclonal phage is located, then add horseradish peroxidase-labeled phage-specific antibodies to the immunoplate, and then add TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution to develop the monoclonal phage. Measure the absorbance OD 450 The monoclonal phage with S1>1 was sequenced, and the repeated sequences were removed by sequence alignment software. The nucleotide sequence obtained after sequencing and removing the repeated sequences was used as the nucleotide sequence of the coding gene of the shark-derived nanobody. Among them, the nucleotide sequence obtained by S1 has green fluorescent protein specificity.

[0032] Step S2: recombinant expression of nanobodies;

[0033] The S2 is specifically:

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

[0035] Step S3: Characterization of shark-derived nanobodies.

[0036] The step S3 is specifically as follows:

[0037] S31. Green fluorescent protein antigen was coated on the immunoplate at a concentration of 1 μg / mL. The negative control group was a protein-free solution. The plate was coated overnight at 4°C. The next day, the liquid was removed, the plate was washed with phosphate buffer PBS, and blocked with MPBS (phosphate buffer plus 5% skim milk). Subsequently, the expressed antibody was diluted to multiple concentrations and added to the wells for incubation. After incubation, the wells were washed with PBST (phosphate buffer plus 0.1% Tween-20), incubated with goat anti-human IgG Fc-HRP antibody, washed again, developed with TMB, terminated with sulfuric acid H2SO4, and the OD was measured. 450 The half effective concentration (EC) of the antibody was obtained by fitting. 50 value.

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

[0039] The present invention utilizes the constructed shark-derived nanoantibody synthetic library and performs bio-panning on green fluorescent protein to finally separate and obtain 4 nanoantibodies, such as Figure 2 As shown, they are named 14A-1A, 14A-3H, 14A-4D and 15A-5D, respectively.

[0040] After the nanobody of the present invention is fused with the human IgG1 Fc segment, it is cloned into the pTT5 vector and secreted by mammalian cells 293F. After 5 days of expression, the nanobody fusion protein in the culture supernatant is purified by rProtein A affinity chromatography column. Figure 3 As shown, the present invention obtains a high-purity green fluorescent protein nanobody Fc fusion protein.

[0041] The nanobody of the present invention can bind to green fluorescent protein with high affinity. The non-competitive ELISA test shows that Figure 4 As shown, the affinity of the four nanobodies disclosed in the present invention for binding to green fluorescent protein is ranked as follows: 14A-3H>15A-5D>14A-1A>14A-4D, and the half effective concentration EC 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 measured by biolayer interferometry (BLI). Figure 5 As shown in the figure, the binding strength of the four nanobodies to green fluorescent protein is as follows: 14A-3H>14A-1A>15A-5D>14A-4D, and their affinity constants K D The values ​​are 4.56, 6.36, 7.64, and 9.54 nM, respectively.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The nanoantibody 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 immune sensor probes.

[0045] 2. The nano antibody protein of the present invention can be obtained in large quantities through a recombinant expression system, with high antibody expression, low preparation cost, and strong stability. The present invention solves the shortcomings of existing traditional human monoclonal antibodies such as large molecular weight, difficult preparation, and complex operation.

[0046] 3. Since the nanoantibody of the present invention is derived from the striped bamboo shark, it has strong tissue penetration ability, and the shark-derived single-domain antibody can purify green fluorescent protein, detect the concentration of green fluorescent protein, and locate green fluorescent protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the result of monoclonal phage ELISA.

[0048] Figure 2 is a graph showing the alignment of the amino acid sequences of the Nanobodies;

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

[0050] Figure 4 This is the binding affinity diagram of specific antibody and green fluorescent protein measured by ELISA;

[0051] Figure 5 This is an affinity diagram between nanoantibodies and green fluorescent protein detected by BLI using antibody affinity determination. The solid line is the kinetic curve monitored in real time, and the dotted line is the curve fitted by the software. The kinetic curves of different green fluorescent protein concentration gradients correspond to the concentrations marked on the right from top to bottom. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are for explanation of the present invention and the present invention is not limited to the following embodiments.

[0053] Embodiments of the present invention are as follows:

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

[0055] 1. Screening of nanoantibodies targeting green fluorescent protein

[0056] Step S1: Inoculate the shark-derived nanobody synthetic library into 2×TY liquid culture medium containing 100 μg / mL ampicillin (Amp) and 2% glucose (G) (volume: 100 mL) to make the initial OD 600 The culture was shaken at 37°C and 200 rpm until the OD 600About 0.5~0.6(OD 600 The value refers to the absorbance value of the solution at 600nm wavelength). Then add 10μL 10 12 pfu of KM13 helper phage, and let it stand at 37°C for 45 minutes. After the standing, centrifuge the bacterial solution for 10 minutes (37°C, 3500g), and remove the supernatant. Resuspend the precipitated bacteria in 200mL of 2TY / Amp / Kanamycin sulfate (Kana) / 0.1%G medium, and shake and culture at 25°C and 200rpm for 16 hours. After the end, centrifuge the entire bacterial solution at 4°C and 3500g for 30 minutes. Take all the supernatant, add 20% PEG / NaCl (polyethylene glycol / sodium chloride) in a ratio of 4:1, mix well, let it stand in an ice bath for 1 hour, and centrifuge for 30 minutes (4°C, 3500g). Discard the supernatant, resuspend the precipitate with PBS (phosphate buffered saline) sterilized by high temperature and high pressure, vortex the precipitate and place it in a centrifuge tube, and centrifuge it for 10 minutes (4°C, 12000rpm). Transfer the supernatant to a new centrifuge tube and measure the OD 260 value.

[0057] Step S2, selection. Coat the green fluorescent protein antigen at a concentration of 0.1 mg / mL in the immunowell plate, and set up a negative control of protein-free solution GFBE. After standing overnight at 4°C, discard the coating solution and wash three times with 280 μL of PBS. Then add 280 μL of MPBS (phosphate buffer solution) and block the reaction at room temperature for 2 hours. Then wash twice with 280 μL of PBST (phosphate buffer), add 1×10 11 pfu The phage solution prepared above was incubated at 80 rpm for 1 h, the waste liquid was discarded, and 280 μL of PBST was used for 20 washes. 100 μL of trypsin (0.5 mg / mL) was added to the green fluorescent protein group Hb and negative control wells, and the phages were eluted at 80 rpm for 1 h. Then 100 μL of the eluted phage was taken to infect 900 μL OD 600 About 0.5% TG1 bacterial solution was incubated in a 37°C water bath for 45 min. 50 μL and 5 μL of the infected bacterial solution were spread on LB / Amp plates respectively and cultured overnight.

[0058] Step S3, phage ELISA screening of anti-green fluorescent protein nanoantibody monoclonal clones:

[0059] Step S31, preparation of monoclonal phage: 95 monoclones were picked from the overnight culture plate and inoculated into 100 μL 2×TY / Amp / 2% G respectively. Another well was used as a negative control and cultured in a 96-well culture plate with shaking for 6-8 hours (37°C, 250 rpm). Subsequently, 2.5 μL of bacterial solution was taken from each well and re-inoculated into 100 μL 2×TY / Amp / 2% G / KM13 culture medium (2.5 μL KM13 was added to 12.5 mL 2×TY / Amp / 2% G), and cultured again with shaking for 1.5 hours (37°C, 250 rpm), and then allowed to stand at 37°C for 45 minutes. An equal amount of 30% glycerol was added to the remaining 97 μL bacterial solution wells, and the mixed solution was stored in a -80°C refrigerator. After standing, the bacterial solution was thoroughly mixed and 50 mL was discarded, centrifuged at 3200g for 20 minutes, and the supernatant was completely removed. The pellet was resuspended in 200 μL 2TY / Amp / Kana / 0.1% G medium and cultured for 20 h (25°C, 250 rpm). After the culture, the plate was centrifuged for 40 min (4°C, 3200 g), and the monoclonal phage supernatant was transferred to a new 96-well plate and stored at 4°C.

[0060] Step S32, monoclonal phage ELISA detection: dilute green fluorescent protein to 1 μg / mL with GFBE, take 100 μL to coat the 96-well immunoplate, and set up a protein-free solution GFBE as a negative control, and coat overnight at 4°C. Wash 3 times with PBS, add 300 μL MPBS to each well, and block at room temperature for 2 hours. Add 100 μL of the phage MPBS mixture prepared above to each well and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Use MPBS to moderately dilute the HRP-anti M13 antibody (diluted in MPBS at a ratio of 1:8000), add 100 μL to each well of the above immunoplate, and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Add 100 μL TMB colorimetric substrate to each well, wrap it with aluminum foil to avoid light, and react at room temperature for 5 minutes. Add 50 μL of 1M H2SO4 to each well to terminate the reaction and measure OD 450 Value, the result is Figure 1 shown.

[0061] Step S33, picking OD 450 Wells with values ​​greater than 1 were subjected to sequencing analysis, and the sequencing primer was: 5'-CCCTCATAGTTAGCGTAACGA-3'.

[0062] Step S34: Compare and analyze the antibody sequences measured, and after excluding duplicate clones, a total of 4 different nanobody sequences are obtained ( Figure 2). SEQ ID NO.1-4 are the nucleotide sequences of the nanobody, from which the amino acid sequences of the nanobody as shown in SEQ ID NO.5-8 can be obtained.

[0063] 2. Expression and purification of nanobodies and their Fc fusion proteins

[0064] Primers were designed to fuse the IFNα protein signal peptide to the N-terminal of the gene sequence of the nanobody to guide secretory expression, and the human IgG1 Fc was fused to the C-terminal of the gene sequence of the nanobody. A TEV restriction site was introduced between them, and then cloned into the mammalian expression vector pTT5. The constructed recombinant vector was transiently transfected into mammalian cells HEK293F with PEI, and the supernatant was collected after 5 days of culture. The antibody fusion protein in the supernatant was purified using an rProtein A affinity chromatography column and analyzed by SDS-PAGE electrophoresis. The results are shown in Figure 3 As shown, highly pure nanobodies were obtained by purification.

[0065] 3. Characterization of Nanobodies

[0066] Step S1: On the immunowell plate, the green fluorescent protein antigen was coated at a concentration of 1 μg / mL, and the protein-free solution GFBE was used as a negative control, and both were coated overnight at 4°C. The next day, the protein coating solution and the liquid in the protein-free control wells were removed, and the wells were washed with PBS twice and blocked with MPBS for 2 hours. The purified antibody was added at an initial concentration of 10 3 nM as the starting point, and serial dilution to 10 -6 nM, a total of 22 gradient concentrations were set. All dilution steps were performed in MPBS. After blocking, each concentration gradient antibody protein solution was added to the pre-coated wells (100 μL per well) and incubated at 80 rpm for 1 hour. Only an equal amount of MPBS was added to the control wells. 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 in MPBS at a ratio of 1:10000) was added to each well, incubated at 80 rpm for 1 hour, and washed again with PBST 3 times. After TMB color development and termination with 1moL / L sulfuric acid H2SO4 solution, OD was measured. 450 The result is Figure 4 As shown in the figure, the four purified nanobodies have good affinity for green fluorescent protein, and their affinity for green fluorescent protein is ranked as follows: 14A-3H>15A-5D>14A-1A>14A-4D, and the half effective concentration EC 50 The values ​​are 0.027, 0.05, 0.056, and 0.45 nM respectively.

[0067] Step S2: Characterizing the affinity between nanoantibodies and green fluorescent protein using biomembrane interferometry (BLI)

[0068] The SA sensor was used in the experiment, and the antigen was immobilized by biotinylating green fluorescent protein. First, to ensure the effective operation of the sensor, the sensor was pre-wetted with 200 μL of phosphate buffered saline (PBST) for 10 minutes. Then, the biotinylated green fluorescent protein was loaded onto the SA biosensor with a solidification value higher than 1.0 nm, and incubated with gradient diluted VNAR-Fc fusion protein at room temperature for 240 seconds and dissociated for 240 seconds. Figure 5 It can be seen that the binding strength of the four nanobodies to green fluorescent protein is as follows: 14A-3H>14A-1A>15A-5D>14A-4D, and their affinity constants K D The values ​​are 4.56, 6.36, 7.64, and 9.54 nM, respectively.

[0069] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

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

[0071] SEQ ID NO.1:

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

[0073] DNA type: other DNA

[0074] Organism source: Cihiloscyllium plagiasum

[0075] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGCGGGCGAGCTTATCAACTGGCGGACGATAC TCGGACACAAAGAATACGACATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCTATTAGTACCTCGCCCCTTACACTATCGGCCCTCTCGTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0076] SEQ ID NO.2:

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

[0078] DNA type: other DNA

[0079] Organism source: Cihiloscyllium plagiasum

[0080] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGAGTTTATCAAATGCCGGACGATAC GCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATAACTACCCGACGGGGACTCTTTTCGCGATCTTCGCTACAACGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0081] SEQ ID NO.3:

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

[0083] DNA type: other DNA

[0084] Organism source: Cihiloscyllium plagiasum

[0085] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAACAAGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCTATTACTACCGCGATCCCTTACTATGACGGTCCTCTCGTCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0086] SEQ ID NO.4:

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

[0088] DNA type: other DNA

[0089] Organism source: Cihiloscyllium plagiasum

[0090] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCTACAAACAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGCCCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCGCTATCCGCCGAGCGGCGAGTACGCGGCCGATCTCGCCTCTATCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0091] SEQ ID NO.5:

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

[0093] Sequence Type: AA

[0094] Organism source: Cihiloscyllium plagiasum

[0095] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKRASLSTGGRYSDTKNTTSKSFSLRISDLRVEDSGTYHCKAYPISTSPPYTIGPLVEGGGTILTVKP

[0096] SEQ ID NO.6:

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

[0098] Sequence Type: AA

[0099] Organism source: Cihiloscyllium plagiasum

[0100] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSNAGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYNYPTGTLFRDLRYNEGGGTILTVKP

[0101] SEQ ID NO.7:

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

[0103] Sequence Type: AA

[0104] Organism source: Cihiloscyllium plagiasum

[0105] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSTGGRYSDTKNKASKSFSLRISDLRVEDSGTYHCKAYPITTAIPYYDGPLVEGGGTILTVKP

[0106] SEQ ID NO.8:

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

[0108] Sequence Type: AA

[0109] Organism source: Cihiloscyllium plagiasum

[0110] TQRVEQTPTTTTKEAGESLTINCVLRDSSYALGSTYWYFTKKGATNKES LSNGGRYAETVNKASKSFSLRISALRVEDSGTYHCKAYRYPPSGEYAADLASI EGGGTILTVKP.

Claims

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

8.

2. The shark-derived synthetic nanobody targeting green fluorescent protein according to claim 1, characterized in that: The nucleotide sequence of the green fluorescent protein shark-derived nanoantibody 14A-1A encoding gene is shown in SEQ ID NO.1, the nucleotide sequence of the green fluorescent protein shark-derived nanoantibody 14A-3H encoding gene is shown in SEQ ID NO.2, the nucleotide sequence of the green fluorescent protein shark-derived nanoantibody 14A-4D encoding gene is shown in SEQ ID NO.3, and the nucleotide sequence of the green fluorescent protein shark-derived nanoantibody 15A-5D encoding gene is shown in SEQ ID NO.

4.

3. The use of a shark-derived synthetic nanobody targeting green fluorescent protein as claimed in any one of claims 1 to 2, characterized in that: The shark-derived synthetic nano-antibody is used in preparing a green fluorescent protein affinity purification reagent.

4. The use of a shark-derived synthetic nanobody targeting green fluorescent protein as claimed in any one of claims 1 to 2, characterized in that: The shark-derived synthetic nano-antibody is used in the preparation of reagents related to tracking green fluorescent protein.

5. A method for affinity purification of green fluorescent protein using shark-derived synthetic nanobodies as described in any one of claims 1-2, characterized in that: The following steps are involved: Step S1, first, using agarose gel coupled with shark-derived synthetic nanobodies as a filler to prepare an affinity chromatography column; Step S2: Load the mixed sample containing green fluorescent protein GFP onto an affinity chromatography column, and extract the purified green fluorescent protein from the eluate of the affinity chromatography column.

6. The method for affinity purification of green fluorescent protein using shark-derived synthetic nanobodies according to claim 5, characterized in that: The step S1 is specifically as follows: coupling the shark-derived synthetic nanoantibody and agarose gel, then loading the agarose gel coupled with the shark-derived synthetic nanoantibody into an affinity chromatography column, and then washing 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.

7. The method for affinity purification of green fluorescent protein using shark-derived synthetic nanobodies according to claim 5, characterized in that: The step S2 is specifically as follows: centrifuging the mixed sample containing GFP to obtain the supernatant, filtering, and loading the filtered mixed sample onto an affinity chromatography column to allow GFP to fully bind to the shark-derived synthetic nanoantibody; 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 a glycine-HCl buffer at pH 3.0-3.5, collecting the eluate containing GFP, and then achieving affinity purification of the green fluorescent protein.

8. The method for affinity purification of green fluorescent protein using shark-derived synthetic nanobodies according to claim 6, characterized in that: In the process of coupling the shark-derived synthetic nano-antibody and agarose gel, 5-10 mg of the shark-derived synthetic nano-antibody is coupled to each milliliter of agarose gel.

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