Preparation method and application of shark nano antibody targeting HER2 (Human Epidermal Growth Factor Receptor 2)

Abstract

The invention belongs to the technical field of biology, and relates to a preparation method and application of a shark nano antibody (VNAR) targeting HER2 (human epidermal growth factor receptor 2). The shark nano antibody is named as VNAR-G4, and the amino acid sequence of the shark nano antibody is as shown in SEQ ID No. 10. The molecular weight of the VNAR-G4 is about 15kDa, which is far less than that of the traditional monoclonal antibody (-150kDa); the antibody has an extended CDR3 ring structure, can be specifically combined with hidden epitopes (such as a dimerization interface) of HER2, and overcomes the epitope recognition limitation of a traditional antibody; the structural integrity and binding activity can still be maintained under an extreme pH condition, and the catalyst is suitable for a harsh in-vivo environment; experiments prove that the kit has high affinity (KD < = nM level) and strong specificity with HER2 antigen, and has no cross reaction; soluble expression is realized by adopting an escherichia coli prokaryotic expression system, the process is simple, the cost is low, and large-scale production is easy; flexible genetic engineering modification (such as construction of bispecific antibodies or drug conjugates) is supported; the VNAR-G4 can be used for effectively inhibiting the activity of an HER2 signal channel and remarkably inhibiting the proliferation of HER2 positive tumor cells in vitro and in an animal model; and a novel targeted therapy tool is provided for treating HER2-driven malignant tumors such as breast cancer and gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the preparation of antibodies, in particular to a shark nanoantibody VNAR-G4 targeting HER2 and applications thereof. Background Art

[0002] While monoclonal antibodies (mAbs) are widely used in biotechnology and biomedicine, their large molecular size (approximately 150 kDa) limits their ability to penetrate dense tissues (such as solid tumors) and effectively bind to epitopes located in clefts on the antigen surface (e.g., receptor binding domains). Furthermore, stability challenges faced by mAbs under harsh conditions such as high temperature or high humidity can also impact their efficacy. To overcome these limitations, the exploration of novel binding molecules with smaller size and superior stability is crucial. Variable neoantigen receptors (VNARs), also known as shark nanobodies (molecular weight approximately 12-15 kDa), discovered in sharks in the 1990s and composed solely of heavy chains, offer a promising solution. The unique structure of VNARs (containing only CDR1 and CDR3, adapted through compensatory evolution) confers significant advantages: extremely high antigen affinity, excellent water solubility and stability, superior tissue penetration, and the ability to recognize hidden or conformational epitopes inaccessible to traditional antibodies. These properties make VNARs promising candidates for the development of a new generation of diagnostic and therapeutic agents. Given the overexpression of HER2 (human epidermal growth factor receptor 2) in various cancers (especially breast and gastric cancers) and its importance as a key therapeutic target, the development of innovative therapies that can efficiently and specifically target HER2 is crucial. Leveraging the unique advantages of shark nanobodies, such as their small size, strong penetrability, ability to recognize complex epitopes, and good stability, the design of HER2-targeting VNARs is expected to overcome the potential limitations of existing HER2-targeting antibodies (such as trastuzumab) in tissue penetration, stability, or recognition of specific HER2 epitopes, opening up new avenues for the development of more effective and penetrating anti-tumor drugs or diagnostic tools. Summary of the Invention

[0003] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a shark nanoantibody VNAR-G4 targeting HER2 and its application. The present invention obtains a new shark nanoantibody VNAR-G4 through screening, which has high affinity and high specificity for HER2.

[0004] The technical solution of the present invention is:

[0005] The present invention protects a shark nanobody VNAR-G4 targeting HER2, whose amino acid sequence is shown in SEQ ID No.10.

[0006] Furthermore, the shark nanobody VNAR-G4 includes a framework region, a complementarity determining region and a hypervariable region;

[0007] The framework regions are FR1, FR2, FR3a, FR3b and FR4;

[0008] The amino acid sequence of FR1 is shown in SEQ ID No. 14;

[0009] The amino acid sequence of FR2 is shown in SEQ ID No. 15;

[0010] The amino acid sequence of FR3a is shown in SEQ ID No. 16;

[0011] The amino acid sequence of FR3b is shown in SEQ ID No. 17;

[0012] The amino acid sequence of FR4 is shown in SEQ ID No. 18;

[0013] The complementary determining regions are CDR1 and CDR3;

[0014] The amino acid sequence of the CDR1 is shown in SEQ ID No 21;

[0015] The amino acid sequence of the CDR3 is shown in SEQ ID No 22;

[0016] The hypervariable regions are HV2 and HV4;

[0017] The amino acid sequence of HV2 is shown in SEQ ID No. 19;

[0018] The amino acid sequence of HV4 is shown in SEQ ID No. 20.

[0019] The present invention also protects the gene encoding the shark nanobody VNAR-G4, and the nucleotide sequence of the gene is shown in SEQ ID No.11.

[0020] The present invention further protects a recombinant vector containing the above-mentioned encoding gene.

[0021] The present invention further protects a recombinant strain containing the above-mentioned encoding gene or the recombinant vector.

[0022] The present invention also protects the use of the shark nanobody VNAR-G4 in preparing HER2 protein inhibitors.

[0023] The present invention also protects the use of the shark nanobody VNAR-G4 in the preparation of anti-cancer drugs.

[0024] Advantages of the present invention:

[0025] (1) The shark nanoantibody VNAR-G4 prepared by the present invention has the ability to recognize HER2, and its sequence composition is FR1, FR2, FR3a, FR3b and FR4, HV2, HV4, CDR1 and CDR3; due to the special variable domain of VNAR-G4, it is the antibody structure with the smallest molecular weight, only about 15kDa, which is smaller than traditional monoclonal antibodies. Therefore, it overcomes the shortcomings and deficiencies of traditional antibodies in application and can better bind to epitopes in cracks on the surface of antigens (such as the active site of enzymes).

[0026] (2) The shark nanoantibody VNAR-G4 of the present invention is expressed using the Escherichia coli prokaryotic expression system, which is easier to express and genetically engineer, and can be obtained in large quantities. The prepared shark nanoantibody VNAR-G4 has high stability and good tissue permeability, is easier to store and transport than conventional antibodies, and has good affinity and specificity for HER2.

[0027] (3) The shark nanobody VNAR-G4 provided by the present invention has the potential to inhibit proteins and can be used to prepare HER2 protein inhibitors, providing new HER2 protein inhibitory drugs for the treatment of cancer.

[0028] The gene or recombinant plasmid encoding VNAR-G4 or the recombinant cell containing the recombinant plasmid of the gene can be used for preparing HER2 protein inhibitors and treating cancer.

[0029] (4) The present invention selects the striped bamboo shark as a model animal for antibody preparation to prepare VNAR. It is not an endangered shark species, is small in size, and is easy to artificially breed, making it suitable for antibody development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 PCR amplification of shark nanobody genes; lane description: M: DNA Marker, 1: PCR product of primer combination 1, 2: PCR product of primer combination 2, 3: PCR product of primer combination 3;

[0031] Figure 2 The results are for phage polyclonal ELISA;

[0032] Figure 3 The results of VNAR-G4 purification are shown in Figure 1. Lane description: M: Protein Marker, 1-3: 200 mM imidazole eluent, 4-6: 250 mM imidazole eluent.

[0033] Figure 4 ELISA was used to detect the binding of shark nanobodies to recombinant human HER2 protein; DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] In order to further understand the present invention, the present invention will be further described with reference to the accompanying drawings and embodiments.

[0036] The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art and were performed according to the techniques described in the literature in the art or according to the product instructions; the various biological materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0037] Example 1 Construction of a Shark Phage Antibody Library Targeting HER2

[0038] 1. Striped Bamboo Shark Immunity

[0039] (1) First immunization: 200 μg of human HER2 recombinant protein was dissolved in PBS and then mixed with Freund's complete adjuvant in a 1:1 ratio to emulsify the mixture and then immunize the striped bamboo shark;

[0040] (2) Second immunization: Three weeks after the first immunization, 150 μg of antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1 and emulsified to immunize the striped bamboo shark for the second time;

[0041] (3) Third immunization: Three weeks after the second immunization, 150 μg of antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1 and emulsified to immunize the bamboo shark for the third time;

[0042] (4) Fourth immunization: 3 weeks after the third immunization, 150 μg of antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1.

[0043] Example: Immunize striped bamboo shark for the fourth time after mixed emulsification;

[0044] (5) Fifth immunization: Three weeks after the fourth immunization, 3 μg of antigen was injected intravenously to immunize the bamboo shark for the fifth time.

[0045] 2. Construction of VNAR immune library

[0046] (1) Obtaining cDNA

[0047] Peripheral blood lymphocytes (PBMCs) and spleen tissues of immunized striped bamboo sharks were collected, and RNA was extracted using the TRIZOL method and then reverse transcribed into cDNA.

[0048] (2) Cloning of target genes

[0049] The cDNA was amplified by PCR using primer combination 1 (upstream NewVF1+NewVF2, downstream NewVR1+NewVR2), primer combination 2 (upstream NewVF2+BamVF3, downstream BamVR3+BamVR4), and primer combination 3 (upstream BamVF4, downstream BamVR5). The resulting PCR products were digested with SfiI and ligated to the phagemid vector pComb3xss.

[0050] The sequences of the primer combinations are as follows:

[0051] Upstream primer NewVF1 (SEQ ID NO.1)

[0052] CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC

[0053] Upstream primer NewVF2 (SEQ ID NO.2)

[0054] CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAAAAACACCG

[0055] Downstream primer NewVR1 (SEQ ID NO.3)

[0056] CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC

[0057] Downstream primer NewVR2 (SEQ ID NO.4)

[0058] CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC

[0059] Upstream primer BamVF3 (SEQ ID NO.5)

[0060] CAATTTGATTGGGCCCAGGCGGCCCAATGGGTTGAACAAACACCGA

[0061] Upstream primer BamVF4 (SEQ ID NO.6)

[0062] CAATTTGATTGGGCCCAGGCGGCCGCATGGGTTGACCAAACAC

[0063] Downstream primer BamVR3 (SEQ ID NO.7)

[0064] CAATTTGATTGGGCCGGCCTGCCAGGTTTCACAGTCAGAATGGTG

[0065] Downstream primer BamVR4 (SEQ ID NO.8)

[0066] CAATTTGATTGGGCCGGCCTGGCCGGGTTTTACACTCAGAATGGTG

[0067] Downstream primer BamVR5 (SEQ ID NO.9)

[0068] CAATTTGATTGGGCCGGCCTGCCAGGTTTCACTGCCAGAAAAGTGC

[0069] The primer combinations were used to perform PCR reactions as shown in Table 1 PCR reaction system and Table 2 PCR reaction program.

[0070] Table 1 PCR reaction system

[0071]

[0072]

[0073] Table 2 PCR reaction procedure

[0074]

[0075] The results of nucleic acid electrophoresis of the VNAR region of the bamboo shark obtained by PCR amplification are as follows: Figure 1 .

[0076] (3) Construction of phagemid

[0077] The PCR product digestion system is as follows:

[0078] Table 3 PCR product enzyme digestion system

[0079]

[0080] Prepare the enzyme digestion reaction system (Table 3) in 13 tubes on ice and digest at 50°C for 2 h in a PCR instrument. Run the digestion products on a 1% agarose gel. Recover all digestion products using a kit and store at -20°C until needed.

[0081] The pComb3XSS plasmid enzyme digestion system is as follows:

[0082] Table 4 pComb3XSS plasmid enzyme digestion system

[0083]

[0084] Prepare eight tubes of the pComb3XSS plasmid digestion reaction system (Table 4) on ice and digest at 50°C for 2 hours in a PCR instrument. Run the digestion products on a 1% agarose gel. Recover the digested pComb3XSS plasmid using a kit and store at -20°C until use for subsequent pComb3XSS recombinant plasmid construction.

[0085] The T4 DNA ligase ligation system is as follows:

[0086] Table 5T4 DNA ligase ligation system

[0087]

[0088] Prepare 20 tubes of the ligation system shown in Table 5 on ice and place them in a PCR machine at 16°C overnight. The next day, inactivate the ligation system in a PCR machine at 65°C for 10 minutes. Collect all ligation products, i.e., the pComb3XSS recombinant plasmid, and store at 4°C until needed.

[0089] 2. Library construction

[0090] The ligation products were electroporated into E. coli TG1 competent cells to form the primary phage library. The primary library capacity was calculated using the limiting dilution plating method: Shark Nanobody Primary Library Capacity (cfu) = Number of colonies × 10 × Dilution gradient × Library volume. Colony counts were counted based on colony growth to calculate the capacity of the constructed shark nanobody library. The quality evaluation of the VNAR library, including library capacity, gene insertion rate, and gene diversity, is shown in Table 6.

[0091] Table 6: Evaluation of the quality of phage libraries

[0092]

[0093] Example 2 Panning of shark nanobody phage targeting human HER2

[0094] The original phage library of Example 1 was amplified by adding helper phage M13KO7 to prepare phage for panning.

[0095] (1) First round of selection:

[0096] (1) Antigen coating: Dilute the human HER2 recombinant protein stock solution to 10 μg / mL with PBS solution and add 100 μL per well to a 96-well microtiter plate, for a total of 8 wells. At the same time, add 200 μL of 3% BSA solution to the wells not coated with antigen, for a total of 8 wells, for negative panning of the antibody library. Incubate the microtiter plate at 4°C overnight.

[0097] (2) Washing: The next day, discard the coating solution and wash the ELISA plate with 0.1% PBST solution at a volume of 300 μL / well for a total of three washes.

[0098] (3) Blocking: Add 200 μL / well of 3% BSA solution to the antigen wells and incubate at 37°C for 1 hour. Add 100 μL / well of phage antibody library (previously diluted with 3% BSA to 1000 times the library volume) to the BSA wells and incubate at 37°C for 1 hour.

[0099] (4) Discard the blocking solution in the antigen wells and wash the ELISA plate with 0.1% PBST solution at 300 μL / well for three washes. Subsequently, transfer the phage antibody library pre-incubated in the BSA wells to the antigen wells at 100 μL / well and incubate at 37°C for 1 hour.

[0100] (5) Activation of TG1 strain: Take an appropriate amount of overnight cultured TG1 bacterial solution and transfer it to fresh 2×YT liquid medium at a ratio of 1:100. Place it in a shaking incubator at 37°C and 180 rpm for about 2 hours to reach the logarithmic growth phase.

[0101] (6) Washing: Wash the antigen wells with 0.1% PBST solution at a volume of 300 μL / well for a total of three washes.

[0102] (7) Infection: Add TG1 bacterial solution in the logarithmic growth phase to the antigen wells at 100 μL / well and incubate at 37°C for 30 min. After infection, collect all TG1 bacterial solution. Repeat the above infection steps twice. Combine the three collected bacteria and take 50 μL for limiting dilution coating. The dilution multiples are 10 2 , 10 3 , 10 4 , 10 5 , spread 100 μL on a 2×YT agar plate (Amp+) and culture inverted at 37°C overnight.

[0103] (8) Helper phage superinfection: Add 20 times more helper phage M13KO7 to the remaining TG1 bacterial solution, incubate at 37°C for 1 hour, and mix once every 30 minutes.

[0104] (9) Progeny phage amplification: After incubation, the phage suspension was centrifuged at 8000 rpm for 5 min. The supernatant was discarded and the suspension was resuspended in 10 mL of fresh 2×YT liquid medium. Amp (100 μg / mL) and Kana (50 μg / mL) were added in sequence and cultured overnight in a shaking incubator at 30°C and 200 rpm.

[0105] (10) The next day, centrifuge at 5000 rpm and 4°C for 30 min.

[0106] (11) Filter the supernatant and add 1 / 4 volume of PEG / NaCl, and let it stand on ice for 1 h.

[0107] (12) Centrifuge at 5000 rpm and 4°C for 30 min, discard the supernatant, and resuspend in 1 mL of PBS to prepare the phage antibody library obtained in the first round of panning.

[0108] (2) Second and third rounds of selection

[0109] The panning plan is similar to the first round of panning, with the following changes:

[0110] (1) The coating concentration of human HER2 recombinant protein in the ELISA plate was reduced to 4 μg / mL and 2 μg / mL in successive rounds.

[0111] (2) In step (6), the number of times the ELISA plate was washed was increased to 15 and 20 times in successive rounds.

[0112] (3) The blocking solution used in the second round of panning was replaced with 5% MPBS, and the blocking solution used in the third round of panning was still 3% BSA solution.

[0113] The titer of the selected phages increased in the third round compared with the second round, and the polyclonal ELISA test showed that specific phages were also enriched (see Table 7 and Figure 2 ).

[0114] Table 7: Titer results of the original and three rounds of panning phage nanobody libraries

[0115]

[0116] Soluble phage antibodies were prepared from 96 monoclonal colonies randomly selected from the solid agar plates obtained in the third and second rounds of panning and then tested by monoclonal phage ELISA. To ensure that the amount of phage antibodies amplified by each monoclonal colony was essentially the same, each colony was cultured in the same volume of culture medium under the same experimental conditions, and all other experimental procedures were kept consistent.

[0117] The 50 clones with the highest absorbance values ​​were selected and sent to a sequencing company for sequence analysis. The NCBI-BLAST tool was used to perform homology comparison of the sequencing results, the EXPASY-translate tool was used to translate the amino acid sequence, and the amino acid sequence diversity was analyzed in BioEdit. Finally, the anti-HER2 VNAR sequence was obtained and named VNAR-G4.

[0118] The amino acid sequence of VNAR-G4 is shown in SEQ ID NO.10, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.11; the VNAR-G4 sequence was compared with the NCBI database, and the results showed that this sequence was a shark-derived nanoantibody gene sequence.

[0119] The amino acid sequence and nucleotide sequence of the shark nanobody VNAR-G4 are as follows:

[0120] Amino acid sequence of shark nanobody VNAR-G4 (SEQ ID NO.10)

[0121] ARVEQTPTTTTKEAGESLTINCVLKGSNYAVCNTYWYFTKKGATKKESLTNGGRYSV TMNKASKSFSLRISDLRVDDSGTYHCKAFSWGDDGYCDLFTWVEGGGTPLTV

[0122] Nucleotide sequence of shark nanobody VNAR-G4 (SEQ ID NO.11)

[0123] AATGACTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTGAATTCAGGAGGAATTTAAAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCGCCGCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAACTATGCAGTGTGTAACACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTAACAAATGGCGGACGATACTCAGTCACAATGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGATGACAGTGGTACATATCACTGTAAAGCGTTTAGTTGGGGAGATGACGGGTACTGTGATTTGTTTACCTGGGTCGAAGGAGGCGGCACCCCTCTGACTGTGGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAGGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGTGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAACCATATGAATTTCCATTGATGGGACAAATAACTTATTCCGGGGGGCTTTGCGTTCTTTAATG

[0124] The shark nanobody VNAR-G4 includes a framework region FR, a complementary determining region CDR, and a hypervariable region HV, wherein the framework region FR includes the amino acid sequences of FR1, FR2, FR3a, FR3b, and FR4, which are as follows:

[0125] FR1:ARVEQTPTTTTK(SEQ ID No.14);

[0126] FR2: VLKGSNY (SEQ ID No. 15);

[0127] FR3a:AVCNTYWYFTKKGATKKESLTNGGRYSVTMNKAS(SEQ ID No. 16);

[0128] FR3b: KAFSWGDDGYC (SEQ ID No. 17);

[0129] FR4: DLFTWVEGGGTPLTV (SEQ ID No. 18);

[0130] The amino acid sequences of the hypervariable region HV include HV2 and HV4, which are as follows:

[0131] HV2:KESLTNGGRYS (SEQ ID No. 19)

[0132] HV4:KASKS (SEQ ID No. 20)

[0133] The complementary determining regions (CDRs) include the amino acid sequences of CDR1 and CDR3. Corresponding to the above FRs, the CDR amino acid sequences of the shark nanobody VNAR-G4 are:

[0134] CDR1:EAGESLTINC(SEQ ID No.21)

[0135] CDR3: KSFSLRISDLRVDDSGTYHC(SEQ ID No.22)

[0136] Example 3 In vitro recombinant expression and purification of nanobodies

[0137] The positive clone phage plasmid was used as a template. According to the sequence information of the sequencing results, the forward primer: 5'-TAAGAAGGAGATATACCATGGGCGGTTGGGTGGACCAAACACC-3' (SEQ ID NO.12) and the reverse primer: 5'-TGGTGGTGCTCGAGTGCGGCCGCAGCGTAGTCCGGAACGTCG-3' (SEQ ID NO.13) were designed. The VNAR-G4 sequence was amplified by PCR. The pET28a vector was double-digested with Nco I and Nco I. The shark nanobody gene VNAR-G4 fragment was connected to the pET28a vector. The above connection product was transformed into DH5a competent cells. After the sequencing alignment was correct, the plasmid was transformed into E. coli BL21 (DE3) competent cells. The his tag was purified using nickel ion chelating filler. Each purified protein was aliquoted and stored at -80 ° C. The results are shown in the figure. Figure 3 As shown, the position of the target band is consistent with the theoretical molecular weight of VNAR-G4, proving that VNAR-G4 was successfully expressed and purified, and VNAR-G4 could be eluted using 200 mM imidazole eluent.

[0138] Example 4 ELISA detection of the binding of shark nanobodies to recombinant human HER2 protein

[0139] 100 μL of 1 μg / mL recombinant human HER2 protein was added to each well of the ELISA plate and incubated at 4°C overnight. The next day, the supernatant was discarded and each well was washed three times with 300 μL of PBST. 200 μL of PBS containing 3% BSA was added to each well and incubated at 37°C for 1.5 h. Each well was washed three times with 300 μL of PBST. 100 μL of different concentrations (5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 μg / mL) of 1% BSA was added to each well. M) of VNAR-G4, incubated at 37°C for 1.5 h, and washed three times with 300 μL PBST per well; 100 μL of HRP-anti-HAtag antibody (1:10,000 diluted in PBS containing 3% BSA) was added to each well, incubated at 37°C for 1.5 h, and washed three times with 300 μL PBST per well; 100 μL of TMB substrate was added to each well, incubated at 37°C in the dark for 10 min, and after color development, 50 μL of 1 M H2SO4 was added to each well to terminate the reaction, and the OD was measured. 450 value.

[0140] like Figure 4 As shown, based on the ELISA results, the EC of VNAR-G4 was calculated. 50 The value is 298.9nM, and VNAR-G4 can specifically bind to recombinant human HER2 protein.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A shark nanobody VNAR-G4 targeting HER2, the amino acid sequence of which is shown in SEQ ID No.

10.

2. The shark nanobody VNAR-G4 according to claim 1, characterized in that The shark nanobody VNAR-G4 includes a framework region, a complementarity determining region and a hypervariable region; The framework regions are FR1, FR2, FR3a, FR3b and FR4; The amino acid sequence of FR1 is shown in SEQ ID No. 14; The amino acid sequence of FR2 is shown in SEQ ID No. 15; The amino acid sequence of FR3a is shown in SEQ ID No. 16; The amino acid sequence of FR3b is shown in SEQ ID No. 17; The amino acid sequence of FR4 is shown in SEQ ID No. 18; The complementary determining regions are CDR1 and CDR3; The amino acid sequence of the CDR1 is shown in SEQ ID No 21; The amino acid sequence of the CDR3 is shown in SEQ ID No 22; The hypervariable regions are HV2 and HV4; The amino acid sequence of HV2 is shown in SEQ ID No. 19; The amino acid sequence of HV4 is shown in SEQ ID No.

20.

3. The shark nanobody VNAR-G4 according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

11.

4. A recombinant vector, characterized in that The recombinant vector contains the gene according to claim 3.

5. A recombinant strain, characterized in that The recombinant strain contains the gene according to claim 3 or the recombinant vector according to claim 4.

6. Use of the shark nanobody VNAR-G4 according to claim 1 in the preparation of HER2 protein inhibitors.

7. Use of the shark nanobody VNAR-G4 according to claim 1 in the preparation of anti-cancer drugs.

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