Flow sorting assisted shark single-domain antibody screening method and application thereof

Abstract

According to the shark single-domain antibody screening method assisted by flow sorting, antigen-specific single-domain antibodies can be rapidly and effectively screened out according to the method, the method has the advantages of being easy and convenient to operate, rapid, high in throughput and the like, the screening method of the single-domain antibodies can be further enriched, and the screening efficiency of the shark single-domain antibodies is improved. The drug development and application of the single-domain antibody are greatly promoted, and reference is provided for screening of other nano antibodies.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for screening shark single-domain antibodies assisted by flow sorting and its application. Background Art

[0002] In the fields of biopharmaceuticals and antibody development, single-domain antibodies (also known as nanobodies) have gradually become important tools in research and clinical applications due to their advantages such as small size, stability, and strong specificity. Especially in the screening of shark-derived single-domain antibodies, these antibodies have unique structural and functional advantages, such as extremely high affinity and antigen recognition ability. Traditional antibody screening methods, such as phage display technology, yeast display technology, etc., although they can efficiently screen specific antibodies, usually require a large amount of time for the construction of the display system, screening steps, and in vitro verification process, which are often cumbersome and inefficient in practical applications. Summary of the Invention

[0003] Aiming at the above deficiencies in the prior art, the present invention provides a method for screening shark single-domain antibodies assisted by flow sorting and its application, and an enrichment strategy for shark single-domain antibodies, providing new ideas for screening specific single-domain antibodies.

[0004] Therefore, on the one hand, the present invention discloses a method for screening shark single-domain antibodies assisted by flow sorting, and the method includes the following steps:

[0005] (1) Isolate the spleen tissue and PBMC cells of immunized Chiloscyllium plagiosum, extract RNA and reverse transcribe it into cDNA; using the cDNA as a template, amplify the single-domain antibody VNAR library with the Chiloscyllium plagiosum library construction primers.

[0006] (2) Amplify the screened single-domain antibody VNAR library, digest it with SfiI enzyme, ligate the digested product into the pcDNA3.1-VP64 expression vector, transform the ligation product into DH5α competent cells, inoculate all the transformed cells into 100 ml of LB medium, and extract endotoxin-free plasmid using an endotoxin-free kit to obtain the pcDNA3.1-VNAR-VP64 plasmid.

[0007] (3) Co-transfect the above-prepared pcDNA3.1-VNAR-VP64 plasmid, pcDNA3.1-Cre plasmid, and pcDNA3.1-dLox-EGFP plasmid into 293T cells using Lipo2000, and the mass ratio of the three plasmids during transfection is 1:1:1.

[0008] (4) After 48 h of transfection, digest the cells for flow sorting, and sort 0.1-1% of the cells with the strongest EGFP fluorescence value for subsequent experiments.

[0009] (5) Using the amplified barred bamboo shark library construction primers, with cells as templates, amplify the screened VNAR sequences;

[0010] (6) Use seamless cloning to clone the amplified products into the pcDNA3.1 vector, transform the seamless cloning products into DH5α competent cells, spread them on Amp plates, culture overnight at 37 °C, and pick monoclonal colonies the next day for sequencing to obtain specific sequence information.

[0011] Preferably, the barred bamboo shark library construction primers in step (1) of the present invention are as follows:

[0012]

[0013] Preferably, the mass ratio of IgNAR1:IgNAR2:IgNAR3:IgNAR4 when used in the present invention is 15:1:3:1, where IgNAR1 includes IgNAR1_HF_Phage and IgNAR1_HR_Phage, IgNAR2 includes IgNAR2_HF_Phage and IgNAR2_HR_Phage, IgNAR3 includes IgNAR3_HF_Phage and IgNAR3_HR_Phage, and IgNAR4 includes IgNAR4_HF_Phage and IgNAR4_HR_Phage.

[0014] Preferably, the sequence of the pcDNA3.1-VP64 expression vector in step (2) of the present invention is as shown in SEQ ID NO.1.

[0015] Preferably, when screening single-domain antibodies against other antigen proteins in the present invention, the antigen to be screened is optimized according to human codons and then constructed into the pcDNA3.1-Cre plasmid in step (2) for fusion expression with Cre, thereby obtaining the pcDNA3.1-antigen-Cre plasmid. During transfection, replacing the pcDNA3.1-Cre plasmid in step (2) with the pcDNA3.1-antigen-Cre plasmid can achieve the screening of single-domain antibodies against other antigen proteins;

[0016] Preferably, the nucleotide sequence of dLox in the pcDNA3.1-dLox-EGFP plasmid in step (3) of the present invention is as shown in SEQ ID NO.8.

[0017] In one aspect, the present invention also discloses the application of the described method in the preparation of Cre-specific shark-derived single-domain antibodies.

[0018] Preferably, 6 Cre-specific shark single-domain antibodies of the present invention were co-harvested, and their amino acid sequences are shown in SEQ ID NO.2 to SEQ ID NO.7 respectively.

[0019] In one aspect, the present invention also discloses the application of the described method in the preparation of specific shark single-domain antibodies.

[0020] Beneficial effects of the present invention: Flow cytometry (FACS), as an efficient and precise cell analysis technique, can be used in cell screening and antibody identification. Through flow cytometry, rapid and high-throughput analysis can be achieved at the single-cell level, directly obtaining information on the binding of antigens and antibodies in cells, and avoiding the common steps of expression, purification, and in vitro detection in traditional screening methods. By using a system that simultaneously expresses antigens and antibodies in mammalian cells such as 293T, the antigen-binding ability of antibodies can be directly verified. This method not only simplifies the traditional screening process but also more realistically simulates the functional performance of antibodies in cells, greatly improving the screening efficiency and success rate.

[0021] By directly expressing antigens and single-domain antibodies in 293T cells and using flow cytometry to perform real-time analysis of the cells, antibodies that can specifically bind to antigens can be screened out. This method not only optimizes the screening process of shark single-domain antibodies but also improves the accuracy and efficiency of antibody screening, providing new ideas and technical support for the research and application of single-domain antibodies. Description of the Drawings

[0022] Figure 1 is the SDS-PAGE result of Cre antigen purification.

[0023] Figure 2 is the nucleic acid gel diagram of PCR amplification of the shark single-domain antibody library.

[0024] Figure 3 is the cell fluorescence diagram at 48 h after transfection.

[0025] Figure 4 is the flow sorting diagram.

[0026] Figure 5 is the nucleic acid gel diagram of PCR amplification of the sorted cell library.

[0027] Figure 6 is the single-domain antibody sequence obtained by sanger sequencing.

[0028] Figure 7 is the flow cytometry detection result.

[0029] Figure 8 is the schematic diagram of the flow sorting-assisted shark single-domain antibody screening method. Detailed Embodiments

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] The present invention will be specifically described or further illustrated by the following examples. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention.

[0032] In the embodiments of the present invention, those not described in detail are all completed by conventional experimental methods. For the processes involved in the embodiments that are not described in detail, those skilled in the art can understand and easily implement them according to the product specifications or the basic knowledge in the art. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase, so they will not be described in detail.

[0033] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the protection scope of the present invention.

[0034] Example 1: Purification of Antigen and Immunization of Chiloscyllium plagiosum

[0035] I. Construction of Recombinant Expression Plasmid: The gene sequence (Gene ID: 2777477) was inserted into the expression vector pET-28a-TrxA-SUMO by double digestion to obtain the pET-28a-Cre recombinant plasmid, and then the recombinant plasmid was transferred into BL21Star(DE3) cells.

[0036] II. Induced Expression and Purification of Recombinant Protein

[0037] 1. The recombinant expression bacterium BL21 Star(DE3)-pET-28a-Cre was inoculated into LB medium containing Amp resistance at a ratio of 1:100 and cultured in a shaker at 37 °C and 220 rpm for 4 h.

[0038] 2. Then IPTG was added to make the final concentration of IPTG 0.1 - 0.5 mM, preferably 0.2 mM, and the culture was continued at 37 °C for 4 - 6 h (preferably 5 h) and at 16 °C for 14 - 16 h (preferably 16 h) respectively.

[0039] 3. After the induction, centrifuge the bacterial cells to obtain a pellet, and resuspend it in 1×PBS (40 ml of PBS per 1 L of cells). Lyse the cells using a high-pressure cell disruptor at a pressure of 800 bar. After centrifugation, incubate the lysate supernatant with Ni-NTA beads at 4°C for 3 hours, wash it three times with 50 mM imidazole solution, and elute it with 300 mM imidazole solution. Add SUMO protease (Ulp1) and digest at 30°C for 2 hours. Remove the digested solution using a Ni-NTA column. The SDS-PAGE detection results of the purified Cre antigen are as Figure 1 shown, and its purity is above 90%, which is suitable for subsequent immunization experiments.

[0040] Dilute the Cre antigen to 1 μg / ml, mix it thoroughly with an equal volume of biphasic adjuvant, and immunize Chiloscyllium plagiosum. The inoculation site is the intersection of the two side fins and the abdomen. The control group uses BSA to immunize the sharks. Immunize once every two weeks, with a mass of 100 μg of the antigen each time, for a total of 8 times.

[0041] Example 2: Construction of a single-domain antibody library to be screened

[0042] 1. Using primers specific for shark single-domain antibodies, and taking the cDNA obtained by reverse transcription of the RNA from the spleen and PBMC of the immunized sharks in Example 1 as a template, perform PCR amplification by adopting two rounds of PCR to increase the specificity of amplification. The amplification primers in the first round do not contain SfiI restriction sites, while the amplification primers in the second round contain restriction sites at both ends. The amplification results are as Figure 2 shown. The amplified band is approximately 400 - 500 bp, and the band is single, indicating good amplification specificity.

[0043] In the first-round amplification, use primers without SfiI restriction sites with cDNA as a template for specific amplification for 27 cycles, and recover the amplification product; the PCR amplification program and system are as described below:

[0044] PCR amplification reaction program: 98°C for 3 min; 98°C for 10 sec; 55°C for 15 sec; 72°C for 30 sec; 27 cycles; 72°C for 5 min.

[0045] PCR amplification reaction system: 1 μl of template, 25 μl of 2× amplification reaction mixture, 1 μl of mixed upstream primer, 1 μl of mixed downstream primer, and supplement with ddH 2 O to 50 μl.

[0046] In the second-round amplification, use the recovered product of the first round as a template, and amplify for 5 cycles using primers with SfiI restriction sites, and recover the amplification product. The PCR amplification program and system are as described below:

[0047] Reaction program for PCR amplification: 98°C for 3 min; 98°C for 10 sec; 55°C for 15 sec; 72°C for 30 sec; 5 cycles; 72°C for 5 min.

[0048] Reaction system for PCR amplification: X μl of template, 25 μl of 2× amplification reaction mixture, 1 μl of upstream primer after mixing, 1 μl of downstream primer after mixing, supplemented with ddH 2 O to 50 μl.

[0049] 2. Digest the amplified product with SfiI at 50°C for 16 h, and recover the digested product after digestion. The digestion system is as follows:

[0050] Digestion system: 10 μg of VNAR; 15 μl of SfiI; 50 μl of 10× M buffer; supplemented with ddH 2 O to 500 μl.

[0051] 3. Use T4 ligase to ligate the digested VNAR into the pcDNA3.1-VP64 vector (vector sequence see SEQ ID NO.1), and transform the digested product into DH5α. Ligation system: 2.5 μg of VNAR, 5 μg of pcNDA3.1-VP64, 42.5 μl of T4 buffer, 25.5 μl of T4 DNA ligase, supplemented with ddH 2 O to 425 μl.

[0052] 4. Inoculate DH5α-pcDNA3.1-VNAR-VP64 into 100 ml of LB medium and culture for 14 - 16 h. Centrifuge at 5000×g for 10 min at room temperature and collect the bacterial cells. Add 500 μl of Solution I / RNase A mixture to the bacterial cell pellet and resuspend the pellet thoroughly. Add 500 μl of Solution II and gently mix 8 - 10 times. Add 250 μl of pre-cooled N3 Buffer and gently mix several times until a white flocculent precipitate forms. Centrifuge at 13000×g for 10 min at room temperature. Transfer the supernatant to a new centrifuge tube, add 0.1 volume of the ETR Solution based on the volume of the supernatant to the lysate, mix by inverting the tube up and down 10 times, and then incubate on ice for 10 min. Incubate the lysate in a water bath at 42 °C for 5 min and centrifuge at 12000×g for 3 min at room temperature. Transfer the supernatant to a new centrifuge tube, add 0.5 volume of absolute ethanol based on the volume of the supernatant, mix by inverting the tube up and down 6 - 7 times, and let it stand at room temperature for 1 - 2 min. After equilibration of the adsorption column, transfer the entire mixture to the centrifugal column successively. Add 500 μl of HBC Buffer to the centrifugal column and centrifuge at 12000×g for 1 min at room temperature, then discard the filtrate. Add 700 μl of DNA Washing Buffer and centrifuge at 12000×g for 1 min at room temperature, then discard the filtrate. Repeat this three times. Add 50 - 100 μl of Endo-Free-Buffer to the adsorption column, let it stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min to elute the plasmid.

[0053] Example 3: Plasmid co-transfection

[0054] 1. Plasmid co-transfection is performed using Lipofectamine 2000 (Thermo). One day before transfection, passage 293T cells into a 6 cm dish so that the cell density before transfection is 70% - 90%.

[0055] 2. For each transfection sample, prepare the complex in two EP tubes as follows: Take 4 μg of each plasmid DNA to be transfected and add it to 500 μl of serum-free Opti-MEM I reduced serum medium (or other serum-free medium) and mix gently. Take 12 μl of Lipo2000 and dilute it in 488 μl of Opti-MEM I medium. Incubate at room temperature for 5 minutes. After incubating for 5 minutes, gently mix the diluted DNA with the diluted Lipo2000 (total volume = 1000 μl) and then incubate at room temperature for 20 minutes. During the 20-minute incubation at room temperature, change the cell culture medium. Drop 1000 μl of the complex into the dish. Gently mix by shaking the dish back and forth. Place the cells in a cell culture incubator and culture for 48 hours. The fluorescence image of the cells at 48 h is as shown in Figure 3As shown, the overall transfection efficiency is relatively high. EGFP expression can be observed in some cells under a fluorescence microscope, indicating that the constructed VNAR library contains Cre-specific VNAR. Meanwhile, differences in fluorescence intensity can be seen among different cells from the fluorescence results, suggesting that there are also antibodies with different specificities in the VNAR library.

[0056] Example 4: Flow sorting

[0057] 1. Preheat the cell culture medium and 1×PBS in a 37°C water bath, and place them in a sterilized ultra-clean workbench for standby. 2. Discard the medium in the cell culture dish, add 3 mL of 1×PBS to rinse the cells, and repeat once. 3. Add an appropriate amount of 0.25% trypsin solution and incubate at 37°C until the intercellular spaces of the adherent cells become larger and the cells tend to become round, then add 1 mL of cell culture medium and shake the culture dish quickly to terminate the reaction. 4. Carefully pipette the adherent cells and transfer them to a 15 mL centrifuge tube. Centrifuge at 1000 rpm at room temperature for 3 min and discard the supernatant. 5. Resuspend the cells with 1 mL of 1×PBS containing 2% FBS, filter the cell suspension through a 200-mesh cell sieve, and place the filtered cell suspension in a flow tube for flow sorting.

[0058] The sorting process is as Figure 4 shown. The results are consistent with the cell fluorescence results. EGFP expression can be detected in some cells by a flow cytometer, and there are differences in different fluorescence intensities. During the sorting process, we selected about 0.2% of the cells with the strongest EGFP expression for sorting and collection. The VNAR expressed in these cells has better specificity.

[0059] Example 5: Obtaining and identifying the library after screening

[0060] 1. Centrifuge the sorted cell suspension at 600×g for 3 min, remove the supernatant, and resuspend the cell pellet with the PCR reaction solution for amplification. The amplification results are as Figure 5 shown. The amplified bands are single and highly specific. The PCR amplification program and system are as follows:

[0061] PCR amplification reaction program: 98°C for 3 min; 98°C for 10 sec; 55°C for 15 sec; 72°C for 30 sec; 32 cycles; 72°C for 5 min.

[0062] PCR amplification reaction system: template X μl, 2× amplification reaction mixture 25 μl, mixed upstream primer 1 μl, mixed downstream primer 1 μl, supplemented with ddH 2 O to 50 μl.

[0063] 2. Recover the amplified product, digest it with SfiI, and then ligate it into a vector with SfiI sticky ends using T4 ligase. Add the recombinant plasmid into DH5α competent cells, gently pipette to mix well, and place on ice for 30 min.

[0064] 3. Heat shock in a 42 °C water bath for 45 s, and quickly return it to ice after heat shock. Add 1 mL of LB liquid medium to the competent cells, culture in a shaker at 37 °C and 220 rpm for 1 h. Centrifuge at 12,000×g at room temperature for 1 min, remove part of the supernatant bacterial liquid, and spread the remaining bacterial liquid on the plate.

[0065] 4. Pick monoclonal colonies for Sanger sequencing the next day. The obtained single-domain antibody sequences are as Figure 6 shown. By this method, a total of 6 Cre-specific shark single-domain antibodies were obtained, and their amino acid sequences are shown in SEQ ID NO.2 to SEQ ID NO.7 respectively.

[0066] Example 6: Construction of eukaryotic expression vector for single-domain antibody

[0067] Optimize the obtained VNAR sequence according to human codons, synthesize the optimized DNA sequence, and clone the synthesized DNA into the expression vector pcDNA3.1-VP64 using the HieffClone One Step Cloning Kit (the vector sequence is shown in SEQ ID NO.1), transform it into DH5α cells, pick colonies and sequence to obtain positive clones. Specifically as follows:

[0068] The DH5α-pcDNA3.1-VNAR-VP64 was inoculated into 100 ml of LB medium and cultured for 14 - 16 h. Centrifuge at 5000×g for 10 min at room temperature to collect the bacterial cells. Add 500 μl of Solution I / RNase A mixture to the bacterial cell pellet and resuspend the pellet thoroughly. Add 500 μl of Solution II and gently mix 8 - 10 times. Add 250 μl of pre-cooled N3 Buffer and gently mix several times until a white flocculent precipitate is formed. Centrifuge at 13000×g for 10 min at room temperature. Transfer the supernatant to a new centrifuge tube, add 0.1 volume of ETR Solution to the lysate, mix by inverting up and down 10 times, and then incubate on ice for 10 min. Incubate the lysate in a water bath at 42 °C for 5 min and centrifuge at 12000×g for 3 min at room temperature. Transfer the supernatant to a new centrifuge tube, add 0.5 volume of absolute ethanol based on the supernatant volume, mix by inverting up and down 6 - 7 times, and let stand at room temperature for 1 - 2 min. After equilibration of the adsorption column, transfer the mixture to the centrifugal column in portions. Add 500 μl of HBC Buffer to the centrifugal column and centrifuge at 12000×g for 1 min at room temperature, discard the filtrate. Add 700 μl of DNA Washing Buffer and centrifuge at 12000×g for 1 min at room temperature, discard the filtrate. Repeat this three times. Add 50 - 100 μl of Endo-Free-Buffer to the adsorption column, let stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min to elute the plasmid.

[0069] Example 7: Verification of Antibody Function

[0070] 1. Co-transfect 293T cells with pcDNA3.1-VNAR-VP64 plasmid, pcDNA3.1-Cre plasmid and pcDNA3.1-dLox-EGFP plasmid (the mass ratio of plasmids is 1:1:1), and then detect the reporting of the reporter gene 48 h after transfection. The co-transfection of plasmids is carried out using Lipofectamine 2000 (Thermo). One day before transfection, passage the cells into a 6 cm dish so that the cell density before transfection is 70% - 90%.

[0071] 2. For each transfected sample, prepare the complex in two EP tubes as follows: Take 4 μg of the plasmid DNA to be transfected and add it to 500 μl of serum-free Opti-MEM I reduced serum medium (or other serum-free medium) and mix gently. Take 12 μl of Lipo2000 and dilute it in 488 μl of Opti-MEM I medium. Incubate at room temperature for 5 minutes. After incubating for 5 minutes, gently mix the diluted DNA with the diluted Lipo2000 (total volume = 1000 μl) and then incubate at room temperature for 20 minutes. During the 20-minute incubation at room temperature, change the cell medium.

[0072] 3. Pipette 1000 μl of the complex into the dish. Gently mix by rocking the plate back and forth, and place the cells in the cell culture incubator for 48 hours. Cre can bind to the dLox site, and the selected VNAR can bind to the Cre protein, and VP64 fused with VNAR will activate the EGFP reporter.

[0073] 4. Perform flow cytometry on the transfected cells (as Figure 7 shown), and it can be seen that the selected VNAR can achieve binding to Cre and report EGFP.

[0074] The method of the present invention has significant advantages, especially in terms of the efficiency and reliability of antibody screening. Compared with traditional in vitro display techniques (such as phage display or yeast display), flow sorting can directly verify the binding ability of antibodies to target molecules within living cells. This method avoids the cumbersome antibody expression, purification, and subsequent in vitro functional verification steps in the traditional screening process, significantly shortening the screening cycle. In addition, this method can also retain the native conformation and functional characteristics of antibodies under physiological conditions, improving the biological relevance and practicality of the screening results.

[0075] Example 8: Summary of the flow sorting-assisted method for screening shark single-domain antibodies, specifically as Figure 8 shown

[0076] (1) Isolate the spleen tissue and PBMC cells of the immunized bamboo shark, extract RNA and reverse transcribe it into cDNA; using the cDNA as a template, amplify the single-domain antibody VNAR library using the bamboo shark library construction primers; the bamboo shark library construction primers are shown in Table 1:

[0077] Table 1 Sequences of bamboo shark library construction primers

[0078]

[0079] The underlined sequence is the SfiI restriction enzyme site;

[0080] Among them, IgNAR1 (including IgNAR1_HF_Phage and IgNAR1_HR_Phage): IgNAR2 (including IgNAR2_HF_Phage and IgNAR2_HR_Phage): IgNAR3 (including IgNAR3_HF_Phage and IgNAR3_HR_Phage): IgNAR4 (including IgNAR4_HF_Phage and IgNAR4_HR_Phage) have a mass ratio of 15:1:3:1 when used.

[0081] It should be noted that when the researchers in this study used other ratios for amplification, the amplification effect was not as good as the above-mentioned usage ratio.

[0082] (2) Amplify the screened single-domain antibody VNAR library, use SfiI for digestion, ligate the digestion product into the pcDNA3.1-VP64 expression vector (the vector sequence is shown in SEQ ID NO.1), transform the ligation product into DH5α competent cells, inoculate all the transformed cells into 100 ml of LB medium, and use an endotoxin-free kit to extract endotoxin-free plasmid to obtain pcDNA3.1-VNAR-VP64 plasmid;

[0083] (3) Co-transfect the pcDNA3.1-VNAR-VP64 plasmid, pcDNA3.1-Cre plasmid and pcDNA3.1-dLox-EGFP plasmid (where the nucleotide sequence of dLox is shown in SEQ ID NO.8) prepared above into 293T cells using Lipo2000. When transfecting, the mass ratio of the three plasmids is 1:1:1;

[0084] It should be noted that when screening single-domain antibodies against other antigen proteins, the antigen to be screened is optimized according to human codons and then constructed into the pcDNA3.1-Cre plasmid for fusion expression with Cre, so as to obtain the pcDNA3.1-antigen-Cre plasmid. When transfecting, replacing the pcDNA3.1-Cre plasmid above with the pcDNA3.1-antigen-Cre plasmid can achieve the screening of single-domain antibodies against other antigen proteins.

[0085] (4) 48 hours after transfection, digest the cells for flow sorting, and sort 0.1 - 1% of the cells with the strongest EGFP fluorescence value for subsequent experiments;

[0086] (5) Use the amplified striped bamboo shark library construction primers, use the cells as a template, and amplify the screened VNAR sequence;

[0087] (6) The amplified product was seamlessly cloned into the pcDNA3.1 vector, and the seamless cloned product was transformed into DH5α competent cells. Amp plates were coated and cultured at 37°C overnight. The next day, a single clone was picked for sequencing to obtain specific sequence information.

[0088] It should be noted that the system of the present invention is also applicable to the preparation and screening of single domain antibodies of other proteins (such as MUC1, CD16, etc.). The researchers successfully screened suitable single domain antibodies, which have good sensitivity and specificity after verification. Therefore, the screening method has wide applicability. The specific method is briefly described as follows:

[0089] The antigen to be screened was optimized according to the codon of the people and then constructed into the pcDNA3.1-Cre plasmid and fused with Cre for expression. The constructed antigen-Cre plasmid was substituted for the Cre plasmid transfected 293T cells in the previous embodiment for screening. The pcDNA3.1-VNAR-VP64 plasmid, pcDNA3.1-antigen-Cre plasmid and pcDNA3.1-dLox-EGFP plasmid were co-transfected into 293T cells, and then the 293T cells were digested with trypsin for flow sorting after 48h of transfection. The subsequent specific screening process is described in the above embodiment.

[0090] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A flow sorting-assisted shark single domain antibody screening method, characterized in that: The method comprises the following steps: (1) Isolating spleen tissue and PBMC cells from immunized striped bamboo sharks, extracting RNA and reversely transcribing it into cDNA; using cDNA as a template, amplifying the single-domain antibody VNAR library using striped bamboo shark library construction primers; (2) Amplifying the screened single-domain antibody VNAR library, digesting it with SfiI, connecting the digestion product to the pcDNA3.1-VP64 expression vector, transforming the ligation product into DH5α competent cells, inoculating all transformed cells into 100 ml LB medium, and extracting the endotoxin-free plasmid using an endotoxin-free kit to obtain the pcDNA3.1-VNAR-VP64 plasmid; (3) The pcDNA3.1-VNAR-VP64 plasmid, pcDNA3.1-Cre plasmid and pcDNA3.1-dLox-EGFP plasmid prepared above were co-transfected into 293T cells using Lipo2000. The mass ratio of the three plasmids during transfection was 1:1:1; (4) 48 h after transfection, the cells were digested and flow sorted, and the 0.1-1% of cells with the strongest EGFP fluorescence values ​​were selected for subsequent experiments; (5) using primers for library construction of striped bamboo shark and cells as templates to amplify and obtain the screened VNAR sequence; (6) The amplified product was seamlessly cloned into the pcDNA3.1 vector, and the seamless cloned product was transformed into DH5α competent cells. Amp plates were coated and cultured at 37°C overnight. The next day, a single clone was picked for sequencing to obtain specific sequence information.

2. The method according to claim 1, characterized in that The primers for constructing the library of the bamboo shark in step (1) are as follows:

3. The method according to claim 2, characterized in that The mass ratio of IgNAR1:IgNAR2:IgNAR3:IgNAR4 when used is 15:1:3:1, wherein IgNAR1 includes IgNAR1_HF_Phage and IgNAR1_HR_Phage, IgNAR2 includes IgNAR2_HF_Phage and IgNAR2_HR_Phage, IgNAR3 includes IgNAR3_HF_Phage and IgNAR3_HR_Phage, and IgNAR4 includes IgNAR4_HF_Phage and IgNAR4_HR_Phage.

4. The method according to claim 1, characterized in that: The sequence of the pcDNA3.1-VP64 expression vector in step (2) is shown in SEQ ID NO.

1.

5. The method according to claim 1, characterized in that: When screening single-domain antibodies against other antigenic proteins, the antigen to be screened is optimized according to human codons and then constructed into the pcDNA3.1-Cre plasmid in step (2) for fusion expression with Cre, thereby obtaining a pcDNA3.1-antigen-Cre plasmid. During transfection, the pcDNA3.1-antigen-Cre plasmid is replaced with the pcDNA3.1-Cre plasmid in step (2) to achieve screening of single-domain antibodies against other antigenic proteins.

6. The method according to claim 1, characterized in that The nucleotide sequence of dLox in the pcDNA3.1-dLox-EGFP plasmid in step (3) is shown in SEQ ID NO.

8.

7. Use of the method according to claim 1 in preparing Cre-specific shark-derived single-domain antibodies.

8. The use according to claim 7, characterized in that: A total of 6 strains of the Cre-specific shark-derived single-domain antibodies were harvested, and their amino acid sequences are shown in SEQ ID NO.2 to SEQ ID NO.7, respectively.

9. Use of the method according to claim 1 in preparing specific shark-derived single domain antibodies.

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