A shark single-domain antibody VNAR-F10 targeting GPC3 and its applications

Abstract

The present invention discloses a shark single-domain antibody VNAR-F10 targeting GPC3 and its applications. The amino acid sequence of the shark single-domain antibody VNAR-F10 is as shown in SEQ ID No.9, and the nucleotide sequence of its encoding gene is as shown in SEQ ID No.10. The shark single-domain antibody VNAR-F10 prepared in the present invention has a small molecular weight, only about 18 kDa, which is smaller than traditional monoclonal antibodies; the shark single-domain antibody VNAR-F10 has high stability and good tissue permeability, is easier to store and transport than conventional antibodies, and has good affinity and specificity for GPC3; VNAR-F10 is expressed using the Escherichia coli prokaryotic expression system, is easier to express and genetically engineered, and finally a large amount of shark single-domain antibodies can be obtained. The shark single-domain antibody VNAR-F10 can be applied to drugs for diagnosing and treating liver cancer, and the gene encoding VNAR-F10, or a recombinant plasmid, or a recombinant cell containing the recombinant plasmid can be applied to the preparation of immunoassay diagnostic kits, flow cytometry, cellular immunofluorescence detection, and the treatment and diagnosis of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody preparation, and particularly relates to a shark single-domain antibody VNAR-F10 targeting GPC3 and its application. Background Art

[0002] Monoclonal antibodies have been the key driving force for the rapid development of biopharmaceuticals to date. However, traditional monoclonal antibodies are large in size, with limited penetration and accumulation in tissues, and they have problems such as insufficient solubility and stability during manufacturing, transportation, and storage, which easily lead to antibody aggregation and protein denaturation, restricting the further development of antibody drugs. With the development of antibody engineering technology, in order to make up for the limitations of traditional monoclonal antibodies, the size of antibodies has become smaller and smaller. They have a low molecular weight and are composed of a simple structure of only the heavy chain or light chain. In 1993, researchers first discovered in the blood of camelids an antibody that naturally lacks the light chain and only contains the heavy chain (Heavy chain antibodies, HcAbs). Its variable region (variable heavy chain domain, VHH) has a binding activity similar to that of the antigen-binding region of traditional IgG antibodies. At the same time, because its molecular weight is about 15 kDa, it is also called a nanobody. Two years later, researchers such as Andrew discovered another Ig subtype with only the heavy chain (new antigenreceptor, IgNAR) in nurse sharks ( Ginglymostoma cirratum ), and IgNAR was subsequently also found in the bodies of other cartilaginous fish. The antibody fragment that only contains the antibody variable region in cartilaginous fish IgNAR is called a single-domain antibody (Single-domain Variable New Antigen Receptor, VNAR). Its molecular weight is about 12 kDa, which is the smallest antigen-binding domain found in nature. It has the characteristics of small molecular weight, simple structure, strong stability, and good tissue permeability. They can overcome the deficiencies of traditional antibodies and are expected to treat more diseases, becoming a good source of therapeutic antibodies.

[0003] Hepatocellular carcinoma is one of the most common malignant tumors, with high incidence and mortality rates globally. The number of hepatocellular carcinoma cases in China ranks first in the world, and the burden of hepatocellular carcinoma is heavy. The early diagnosis rate of hepatocellular carcinoma is less than 15%. The vast majority of patients are found to be in the middle and late stages when diagnosed, and the cure rate of patients in the late stage is low due to the spread of cancer cells. Existing treatment methods for hepatocellular carcinoma are not sufficient to be effective ways to cure hepatocellular carcinoma. Therefore, it is of great significance to find effective targets for the diagnosis and treatment of hepatocellular carcinoma and reduce the mortality rate of hepatocellular carcinoma. Phosphatidylinositol proteoglycan 3 (glypican-3, GPC3) is one of the members of the heparan sulfate proteoglycan family, and it is anchored to the cell surface through a glycosylphosphatidylinositol anchor. The core protein of GPC3 consists of 580 amino acids and has a size of 70 kDa. GPC3 can promote the growth of hepatocellular carcinoma cells by stimulating the wnt / β-catenin signaling pathway. At the same time, GPC3 has been proven to interact with growth factors, act as a co-receptor, and regulate the activity of growth factors through its heparan sulfate chain, and ultimately stimulate cell growth. GPC3 is highly expressed in hepatocellular carcinoma tissues, not expressed in adult normal organs and hepatocytes of benign liver diseases such as hepatitis, and the high expression of GPC3 is positively correlated with the poor prognosis of hepatocellular carcinoma. In summary, GPC3 is an excellent target for the diagnosis and treatment of hepatocellular carcinoma. Summary of the Invention

[0004] The object of the present invention is to provide a shark single-domain antibody VNAR-F10 targeting GPC3 and its application. The present invention screens and obtains a brand-new shark single-domain antibody VNAR-F10, which has high affinity and high specificity for GPC3.

[0005] To achieve the above object, the present invention adopts the following technical solutions to implement:

[0006] The present invention provides a shark single-domain antibody VNAR-F10 targeting GPC3, and its amino acid sequence is as shown in SEQ ID No.9.

[0007] Furthermore, the shark single-domain antibody VNAR-F10 comprises fixed amino acid sequences FR1, FR2, FR3a, FR3b and FR4, hypervariable regions HV2 and HV4, and complementary determining regions CDR1 and CDR3.

[0008] Furthermore, the amino acid sequence of CDR3 is KAYIGRYTPGCPYRWIFY.

[0009] The present invention also provides the coding gene of the shark single-domain antibody VNAR-F10, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.10.

[0010] Furthermore, the forward primer sequence for cloning the coding gene is as shown in SEQ ID NO.11, and the reverse primer sequence is as shown in SEQ ID NO.12.

[0011] The present invention also provides a recombinant plasmid containing the coding gene described above.

[0012] The present invention also provides a recombinant strain containing the coding gene described above.

[0013] The present invention also provides the application of the shark single-domain antibody described above in the preparation of a GPC3 protein targeting agent.

[0014] The present invention also provides the application of the shark single-domain antibody VNAR-F10 or the coding gene described above in the preparation of a tumor detection reagent and a drug for treating tumors.

[0015] Furthermore, the tumor is liver cancer.

[0016] The single-domain antibody of the present invention has the following advantages:

[0017] 1. The shark single-domain antibody VNAR-F10 prepared in the present invention has a small molecular weight, only about 18 kDa, which is smaller than traditional monoclonal antibodies.

[0018] 2. The shark single-domain antibody VNAR-F10 has high stability and good tissue permeability, is easier to store and transport than conventional antibodies, and has good affinity and specificity for GPC3.

[0019] 3. It is easier to express and genetically engineered. VNAR-F10 is expressed using the Escherichia coli prokaryotic expression system, and a large amount of shark single-domain antibodies are finally obtained.

[0020] The sequence of the shark single-domain antibody VNAR-F10 consists of FR1, FR2, FR3a, FR3b and FR4, HV2, HV4, CDR1 and CDR3. Among them, FR1 and FR4 are fixed amino acid sequences, HV2 and HV4 are hypervariable regions, which affect the antigen-binding site, and CDR1 and CDR3 are the complementarity-determining regions of the antibody, which determine the binding to different antigens. In this case, it has the ability to recognize glypican-3 (GPC3). At the same time, due to its special variable domain, VNAR is the antibody structure with the smallest molecular weight. Therefore, VNAR overcomes the disadvantages and deficiencies of traditional antibodies in application. In addition, the present invention selects the barred bamboo shark (Chiloscyllium plagiosum) as the model animal for antibody preparation to prepare VNAR. It does not belong to endangered shark species, has a small body size, is easy to breed artificially, and is suitable for antibody development.

[0021] The shark antibody VNAR-F10 can be applied to drugs for diagnosing and treating liver cancer. The gene encoding VNAR-F10, recombinant plasmid or recombinant cell containing the gene can be applied to prepare immune detection diagnostic kits, flow cytometry, cellular immunofluorescence detection, and the treatment and diagnosis of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the result of the determination of the serum titer after shark immunization;

[0023] Figure 2 It is for PCR amplification of shark nanobody gene. Lanes 1 and 4: Primer combination one; Lanes 2 and 5: Primer combination two; Lanes 3 and 6: Primer combination three;

[0024] Figure 3 It is the result of the library capacity and positive rate of the single-domain antibody phage display library; a is the library capacity of the single-domain antibody phage display library. In the figure, A and B: diluted 10 8 times; C: diluted 10 7 times; D: diluted 10 6 times; b is the result of the determination of the positive rate of 24 randomly selected monoclonal antibodies in the single-domain antibody phage library;

[0025] Figure 4 It is the panning result; A is the result of phage polyclonal ELISA, and B is the result of phage monoclonal ELISA;

[0026] Figure 5 It is the result of single-domain antibody purification. Lanes 1-9 are in turn: supernatant, precipitate, flow-through, 100 mM imidazole eluate once, 100 mM imidazole eluate twice, 200 mM imidazole eluate, 300 mM imidazole eluate, 400 mM imidazole eluate, 500 mM imidazole eluate;

[0027] Figure 6 It is for ELISA to detect the binding of shark nanobody and recombinant human GPC3 protein;

[0028] Figure 7 It is the result of the immunofluorescence experiment of antibody VNAR-F10 and HepG2 / HUVEC;

[0029] Figure 8 It is the result of the flow cytometry experiment of antibody VNAR-F10 and HepG2 / HUVEC. DETAILED DESCRIPTION OF THE INVENTION

[0030] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection required by the present invention is not limited to the scope described in the examples. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the kit biological materials can be obtained from commercial sources unless otherwise specified.

[0031] Example 1: Construction of a Shark Phage Antibody Library Targeting Recombinant Human GPC3 Protein

[0032] 1. Immunization of Chiloscyllium plagiosum

[0033] (1) First immunization: Take 300 μg of human GPC3 recombinant protein (purchased from Novoprotein Scientific Inc.) and dissolve it in PBS, and mix it with complete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the first time;

[0034] (2) Second immunization: Two weeks after the first immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the second time;

[0035] (3) Third immunization: Two weeks after the second immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the third time;

[0036] (4) Fourth immunization: Two weeks after the third immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the fourth time;

[0037] (5) Fifth immunization: Three weeks after the fourth immunization, take 200 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the fifth time;

[0038] (6) Sixth immunization: Two weeks after the fifth immunization, take 300 μg of the antigen and mix it with incomplete Freund's adjuvant at a ratio of 1:1 to immunize Chiloscyllium plagiosum for the sixth time.

[0039] As Figure 1 shown, after six immunizations, the shark had a good immune response to the antigen GPC3, and the titer reached 1:1600.

[0040] 2. Construction of the VNAR library

[0041] (1) Obtaining of cDNA

[0042] Immunize sharks with human GPC3 recombinant protein, collect peripheral blood lymphocytes PBMC and spleen tissues, extract RNA using the TRIZOL method, and then reverse transcribe it into cDNA.

[0043] (2) Cloning of the target gene

[0044] The cDNA was amplified by PCR using primer sequences (SEQ ID NO.1 - 8) (see Figure 2 ), and the resulting PCR product was digested with Sfi I and ligated to the phagemid vector pComb3xss.

[0045] For combination one (upstream primers NewVF1 + NewVF2, downstream primers NewVR1 + NewVR2) and combination two (upstream primers NewVF2 + BamVF3, downstream primers BamVR3 + BamVR4), the following reaction systems were used (Tables 1 and 2). The sequence of primer NewVF1 is shown as SEQ ID NO.1, the sequence of primer NewVF2 is shown as SEQ ID NO.2, the sequence of primer BamVF3 is shown as SEQ ID NO.3, the sequence of primer BamVF5 is shown as SEQ ID NO.4, the sequence of primer NewVR1 is shown as SEQ ID NO.5, the sequence of primer NewVR2 is shown as SEQ ID NO.6, the sequence of primer BamVR3 is shown as SEQ ID NO.7, and the sequence of primer BamVR4 is shown as SEQ ID NO.8.

[0046] Table 1 PCR Reaction System One

[0047]

[0048] Table 2 PCR Reaction Program One

[0049]

[0050] For combination three (upstream primer BamVF5, downstream primer BamVR3), the following reaction systems were used (Tables 3 and 4):

[0051] Table 3 PCR Reaction System Two

[0052]

[0053] Table 4 PCR Reaction Program Two

[0054]

[0055] The nucleic acid electrophoresis results of the VNAR region of Chiloscyllium plagiosum obtained by PCR amplification are as Figure 2 .

[0056] (3) Construction of phagemid

[0057] The PCR product digestion system is as follows:

[0058] Table 5 PCR Product Digestion System

[0059]

[0060] After thorough mixing, digest at 50 °C for 3 h. After verifying complete digestion by agarose gel electrophoresis, perform gel extraction to recover the digested PCR product, obtaining the shark nanobody gene fragment for subsequent construction of the pComb3XSS recombinant plasmid.

[0061] The restriction enzyme digestion system of the pComb3XSS plasmid is as follows:

[0062] Table 6 Restriction enzyme digestion system of the pComb3XSS plasmid

[0063]

[0064] After thorough mixing, digest at 50 °C for 3 h. After verifying complete digestion by agarose gel electrophoresis, perform gel extraction to recover the digested pComb3XSS plasmid, obtaining the linearized pComb3XSS plasmid for subsequent construction of the pComb3XSS recombinant plasmid.

[0065] The ligation system of T4 DNA ligase is as follows:

[0066] Table 7 Ligation system of T4 DNA ligase

[0067]

[0068] Incubate at 16 °C for 12 h. Inactivate T4 DNA Ligase at 65 °C for 10 min and then recover the ligation product, which is the pComb3XSS recombinant plasmid.

[0069] 3. Library construction

[0070] Electrotransform the ligation product into competent Escherichia coli TG1 to form the original phage library. Subsequently, dilute the bacterial solution by 10 6 , 10 7 , 10 8 times. Take 100 μL of the serially diluted bacterial solution and spread it on freshly prepared 2×YT / A100 (16 g tryptone, 10 g yeast extract, 5 g NaCl, dissolved in 900 mL of double-distilled water and cultured overnight at 37 °C). According to the colony growth situation, count the number of colonies on the plate diluted 10 7 times to calculate the library capacity of the constructed shark single-domain antibody library. The quality evaluation of the VNAR library is shown in Table 8, including library capacity, gene insertion rate, and gene diversity as Figure 3 shown.

[0071] Table 8: Quality evaluation of the phage library

[0072]

[0073] Example 2: Panning of GPC3 shark single-domain antibody phage

[0074] The phage was prepared by adding helper phage to amplify the original phage library in Example 1 and used for panning.

[0075] I. First-round panning:

[0076] 1. Coating: Two wells of a 96-well plate, 100 μL / well, overnight at 4°C.

[0077] ① Coat with human GPC3 recombinant protein at 10 μg / mL;

[0078] ② Coat with 5% MPBS.

[0079] 2. Phage collection

[0080] Centrifuge the bacterial solution shaken overnight in the previous round at 12000g for 5 min, filter the supernatant through a 0.22 μm filter membrane, add PEG at 1 / 4 of the bacterial solution volume, and incubate on ice for 15 - 30 min; centrifuge at 10000g at 4°C for 15 min, discard the supernatant, invert and air-dry on absorbent paper, resuspend with PBS, and measure OD280 (OD280 = 1 is equivalent to 2.5x10 12 pfu / mL).

[0081] 3. Wash the plate three times

[0082] Wash the plate with PBST (PBS containing 0.1% Tween-20), fill the entire well (about 300 μL), and tap the plate.

[0083] 4. Block at 37°C for 1 h.

[0084] ① Add 3% BSA or 5% skim milk powder, the same as the coating, 300 μL / well;

[0085] ② Add the diluted phage, 100 μL / well;

[0086] Calculation method: Make the 400 μL phage added contain 100 times the library capacity to ensure complete panning.

[0087] 5. Activate TG1:

[0088] Take 10 μL of TG1 bacterial strain shaken overnight and add it to 2 mL of fresh 2x YT medium, culture at 37°C with 200 rpm for 3 h to reach the logarithmic growth phase (OD600 = 0.5 - 0.6).

[0089] 6. Incubation: Take out the phage in well ② and transfer it into an EP tube. Wash the plate 3 times, add the above phage into well ①, and incubate at 37 °C for 1 - 1.5 h.

[0090] 7. Wash the plate 6 times.

[0091] 8. Infection: Add the activated TG1 into well ①, 100 μL / well, let it stand at 37 °C for 30 min, collect the infected TG1, replace it with fresh TG1, and repeat this 3 times.

[0092] 9. Verification by plating

[0093] Dilute the bacterial solution to 1000 - fold, 10000 - fold, and 100000 - fold (1 mL volume) by the method of limited dilution, then take 100 μL from each dilution and plate them (Amp + ). Incubate overnight at 37 °C, observe the number of colonies grown, which are the successfully infected TG1. When the same amount of TG1 is inoculated, a significant increase in the number of colonies grown indicates enrichment, and the following experiment can be carried out.

[0094] 10. Superinfection with helper phage: Add M13K07 at 20 times the bacterial cell amount to the infected TG1, let it stand at 37 °C for 1 h (mix it at 30 min).

[0095] 11. Amplification of progeny phage

[0096] Centrifuge the bacterial solution at 3000 g at 25 °C for 5 min, discard the supernatant, resuspend it in 10 mL of fresh 2xYT, add Kana (50 μg / mL), Amp (100 μg / mL), IPTG (0.1 mM), and culture overnight at 30 °C with shaking at 200 rpm.

[0097] II. Second - round panning

[0098] The steps of the second - round panning are the same as those of the first - round panning, except that the concentration of the coated antigen in step 1 is decreased to 4 μg / mL; in step 4, use PBS containing 3% BSA to block the plate and incubate overnight at 4 °C; in step 7, wash the plate 8 times with PBST, and the rest of the experimental operation steps are the same as those of the first - round panning.

[0099] III. Third - round panning

[0100] The steps of the third - round panning are the same as those of the first - round panning, except that the concentration of the coated antigen in step 1 is decreased to 2 μg / mL; in step 4, use PBS containing 5% skim milk powder to block the plate and incubate overnight at 4 °C; in step 7, wash the plate 10 times with PBST, and the rest of the experimental operation steps are the same as those of the first - round panning.

[0101] The titer of the selected phages increased significantly in the second round, and specific phages were also enriched as detected by polyclonal ELISA (see Table 9 and Figure 4 ).

[0102] Table 9: Titre results of the original and three-round selected phage single-domain antibody libraries

[0103]

[0104] To obtain a single phage with high affinity, 94 single colonies were picked from the plates obtained in the third round of selection for identification. Among the 94 picked monoclonal clones, 35 were positive, with a positive rate of 37%. All 35 picked single colonies met the requirements, and the bacterial solutions of these 35 single colonies were sent to a sequencing company for sequencing. After translation and sequence alignment of the sequencing results of the positive monoclonal clones, shark nanobody sequences were obtained and named VNAR-F10.

[0105] The amino acid sequence of VNAR-F10 is shown in SEQ ID NO.9, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.10; when the VNAR-F10 sequence was aligned with the NCBI database, the results indicated that these sequences were all single-domain antibody gene sequences derived from sharks.

[0106] The shark single-domain antibody VNAR-F10 includes a framework region FR and complementarity-determining regions CDR. The framework region FR includes the amino acid sequences of FR1, CDR1, FR2, FR3a, HV2, HV4, FR3b, CDR3, and FR4, which are as follows respectively:

[0107] FR1: AARVEQTPTTTTKEAGESLTINCVLR;

[0108] FR2: TYWYFTKKGATKKE;

[0109] HV2: SLSNG;

[0110] FR3a: GRYAETV;

[0111] HV4: NKASK;

[0112] FR3b: SFSLRISDLRVEDSGTYHC;

[0113] FR4: EGGDTILTVKP;

[0114] The complementarity-determining regions CDR include the amino acid sequences of CDR1 and CDR3. Corresponding to the above respective FRs, the CDR amino acid sequences of the shark single-domain antibody VNAR-F10 are respectively:

[0115]

[0116] Example 3: In Vitro Recombinant Expression and Purification of Single-Domain Antibody

[0117] Using the positive clone phage plasmid as a template, according to the sequence information of the sequencing results, forward primer shown in SEQ ID NO.11 and reverse primer shown in SEQ ID NO.12 were designed, and the VNAR-F10 sequence was amplified by PCR. The pET28a vector was digested with Not I、 Nco I. The VNAR-F10 fragment of the shark single-domain antibody gene was ligated to the pET28a vector, and the above ligation product was transformed into competent E. coli BL21 (DE3) cells. Purification was carried out using nickel ion chelating packing with his tag. Each purified protein was aliquoted and stored at -80 °C. The results were as Figure 5 shown. The position of the target band was consistent with the theoretical molecular weight of VNAR-F10, proving that VNAR-F10 was successfully expressed and purified, and VNAR-F10 could be eluted using a 200 mM imidazole eluent.

[0118] Example 4: ELISA Detection of the Binding of Shark Nanobody to Recombinant Human GPC3 Protein

[0119] Add 100 μL / well of 2 mg / mL recombinant human GPC3 protein to the ELISA plate and incubate overnight at 4 °C; discard the supernatant, and wash 3 times with 300 μL / well of phosphate buffered saline with 0.1% Tween 20 (PBST); add 200 μL / well of PBS containing 5% skim milk powder and incubate at 37 °C for 1 h, then wash 3 times with 300 μL / well of PBST; add 100 μL / well of shark nanobodies at different concentrations (100, 10, 1, 0.1, 0.01, 0.001, 0.0001 μM) and incubate at 37 °C for 1 h, then wash 3 times with 300 μL / well of PBST; add 100 μL / well of HRP-anti-HA tag antibody (diluted 1:10000 in PBS containing 5% skim milk powder) and incubate at 37 °C for 1 h, then wash 3 times with 300 μL / well of PBST; add 100 μL of TMB substrate to each well and incubate in the dark at 37 °C for 10 - 15 min. After color development, add 50 μL of 1 mol / L H2SO4 to each well to terminate the reaction, and detect the OD450 value. According to the ELISA results, the EC 50 value of VNAR-F10 was calculated to be 48.88 nM, indicating that VNAR-F10 could specifically bind to recombinant human GPC3 protein.

[0120] Example 5: Detection of the binding of shark nanobody to HepG2 cells by immunofluorescence and flow cytometry

[0121] Immunofluorescence experiment: According to the instructions of FLUORESCEIN ISOTHIOCYANATE, the purified shark nanobody was labeled with FITC; the 96-well plate was coated with polylysine, washed 3 times with PBS, and then seeded with the human hepatocellular carcinoma cell line HepG2. The human umbilical vein endothelial cell line HUVEC was used as a control, and the seeding density was 10,000 cells / well; after culturing overnight, the medium was discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, and washed 3 times with PBS; 3% BSA dissolved in PBS was used as a blocking solution, 200 μL / well, incubated at room temperature for 1 h, and washed 3 times with PBS; the FITC-labeled shark nanobody was diluted with the blocking solution, and the final concentration was about 200 μg / mL, 20 μL / well, incubated at room temperature for 1 h, and washed 3 times with PBS; stained with DAPI for 5 min, and washed 3 times with PBS; after adding an anti-fluorescence quencher and mounting the coverslip, pictures were taken using a fluorescence microscope.

[0122] Flow cytometry: Digest HUVEC and HepG2 cells respectively; resuspend the cells with FACS buffer, centrifuge horizontally at 1000 rpm at 4°C for 5 min; resuspend the cells with FACS buffer, count them, and aliquot them into 2 tubes of 1.5 million cells / 200 μL each for HUVEC cells and HepG2 cells, centrifuge horizontally at 1000 rpm at 4°C for 5 min; the PBS group resuspended the cells with 1 mL PBS, and the nanobody group resuspended the cells with FACS buffer, centrifuge horizontally at 1000 rpm at 4°C for 5 min; discard the supernatant, the PBS group resuspended with 300 - 400 μL PBS for detection by flow cytometry; the nanobody group resuspended the cells with FACS solution containing VNAR-F10 (VNAR-F10 after FITC labeling was diluted to 200 μg / mL with FACS buffer), incubated in the dark on ice for 45 min, and gently flicked and mixed every 15 min; after the incubation on ice ended, centrifuge horizontally at 1000 rpm at 4°C for 5 min; resuspend with 1 mL PBS, centrifuge at 1000 rpm at 4°C for 5 min, wash 3 times with PBS, and finally resuspend with 300 - 400 μL PBS for detection by flow cytometry.

[0123] The immunofluorescence results are as Figure 7 shown. VNAR-F10 can specifically bind to the HepG2 cell line with high expression of GPC3, but does not bind to the GPC3-negative cell line HUVEC. The flow cytometry results are as Figure 8 shown, further demonstrating that VNAR-F10 has good binding specificity to HepG2 cells.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A shark single-domain antibody VNAR-F10 targeting GPC3, characterized in that, Its amino acid sequence is as shown in SEQ ID No.

9.

2. The encoding gene of the shark single-domain antibody VNAR-F10 according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is as shown in SEQ ID No.

10.

3. A recombinant vector containing the encoding gene according to claim 2.

4. An engineered strain containing the encoding gene according to claim 2.

5. Use of the shark single-domain antibody VNAR-F10 according to claim 1 in the preparation of a GPC3 protein targeting agent.

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