Preparation method and application of shark nano antibody targeting pancreatic lipase

Abstract

The invention belongs to the technical field of biology, and relates to a preparation method and application of a shark nano antibody targeting pancreatic lipase. The amino acid sequence of the shark nano antibody VNAR-A4 is as shown in SEQ ID No. 10. The molecular weight of the shark nano antibody VNAR-A4 prepared by the invention is about 15 kDa, is smaller than that of a traditional monoclonal antibody, has a slender CDR3 structure, and can be better combined with hidden epitopes of antigens; the shark nano antibody VNAR-A4 is good in stability, can keep activity under an extreme pH condition, and has good affinity and specificity with PL; the shark nano antibody VNAR-A4 is expressed by using an escherichia coli prokaryotic expression system, is easier for genetic engineering modification, and can be prepared in a large scale. The shark nano antibody VNAR-A4 has the potential of inhibiting the activity of pancreatic lipase, and a novel fat hydrolysis inhibiting reagent is provided for treating obesity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the preparation of antibodies, in particular to a shark nano antibody VNAR-A4 targeting pancreatic lipase and an application thereof. Background Art

[0002] Obesity refers to abnormal or excessive fat accumulation that may have adverse effects on physical health. As a complex chronic disease, it has become one of the global public health issues. Obesity is a complex metabolic disease that is closely related to diseases such as non-alcoholic fatty liver disease, type 2 diabetes, rheumatoid arthritis, and gastrointestinal cancer, and it can have a negative impact on female fertility. Obesity is closely related to fat metabolism in the body. Dietary fat cannot be directly absorbed by the human body and needs to be hydrolyzed by lipase before it can be absorbed. In the process of fat hydrolysis, pancreatic lipase (PL) plays a vital role. After food reaches the gastrointestinal tract, the carboxyl group of dietary triglycerides is hydrolyzed by the nucleophilic attack of serine, releasing free fatty acids, monoglycerides, diglycerides, cholesterol and other substances, which are absorbed in the small intestine and resynthesized into triglycerides. Excess fat is stored in the body to form white adipose tissue, causing obesity. Orlistat is a gastric and pancreatic lipase inhibitor that can bind to the active site serine of gastric and pancreatic lipase to inhibit its activity, thereby inhibiting the hydrolysis of triglycerides in the diet. Unhydrolyzed triglycerides are excreted from the body with feces, playing an anti-obesity role by reducing the absorption of dietary fat. Compared with other anti-obesity drugs, it has higher safety, but there are still adverse reactions such as gastrointestinal discomfort, oily stools, and soft stools. Rare adverse reactions involve the kidneys and liver, and can affect the absorption of drugs such as fat-soluble vitamins. As people's quality of life improves, obesity has become a global public health problem, so there is a need to find new PL inhibitors with low toxicity and low side effects.

[0003] Monoclonal antibodies have been commonly used in a large number of biotechnology and biomedical applications for decades, but they are limited by their large size (about 150 kDa), which prevents them from entering epitopes in cracks on the surface of antigens (such as the active site of enzymes). In addition, monoclonal antibodies may affect sensitivity under high temperature and high humidity conditions. In order to overcome these situations, it is urgent to explore new antigen binders with small size and natural thermal stability. In the 1990s, foreign scholars discovered VNAR with only heavy chains in sharks. Because it only has heavy chains, the antigen binding site is formed by only a single domain, which is also called nanoantibody. VNAR is the smallest antibody fragment discovered so far (about 12-15 kDa). It has evolved by compensating CDR2 and therefore has only CDR1 and CDR3. Compared with conventional antibodies, VNAR's smaller molecular weight and special structure have many advantages, such as high antigen affinity, good water solubility and stability, strong tissue penetration, and the ability to recognize hidden antigen epitopes, making it a good alternative source of therapeutic and diagnostic reagents. Shark nanobodies, as a new type of antibody, have unique advantages. Finding shark nanobodies that can bind to and inhibit PL to treat obesity has important application value. Summary of the invention

[0004] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a shark nanoantibody VNAR-A4 targeting PL and its application. The present invention obtains a brand-new shark nanoantibody VNAR-A4 through screening, which has high affinity and high specificity for PL.

[0005] The technical solution of the present invention is: The present invention protects a shark nanobody VNAR-A4 targeting PL, and its amino acid sequence is shown in SEQ ID No.10.

[0006] Furthermore, the shark nanobody VNAR-A4 includes a framework region, a complementary 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.

[0007] The present invention also protects the gene encoding the shark nanobody VNAR-A4, and the nucleotide sequence of the gene is shown in SEQ ID No.11.

[0008] The present invention further protects a recombinant vector, which contains the above-mentioned coding gene.

[0009] The present invention further protects a recombinant strain, which contains the above-mentioned encoding gene or the above-mentioned recombinant vector.

[0010] The present invention also protects the use of the shark nano antibody VNAR-A4 in the preparation of PL protein inhibitors.

[0011] The present invention also protects the use of the shark nano antibody VNAR-A4 in the preparation of anti-obesity drugs.

[0012] Beneficial effects of the present invention: (1) The shark nanoantibody VNAR-A4 prepared by the present invention has the ability to recognize PL, and its sequence composition is FR1, FR2, FR3a, FR3b and FR4, HV2, HV4, CDR1 and CDR3; due to the special variable domain of VNAR-A4, it is the antibody structure with the smallest molecular weight, only about 15 kDa, 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 the cracks on the antigen surface (such as the active site of the enzyme).

[0013] (2) The shark nanoantibody VNAR-A4 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-A4 has high stability and good tissue permeability, is easier to store and transport than conventional antibodies, and has good affinity and specificity for PL.

[0014] (3) The shark nanobody VNAR-A4 provided by the present invention has the potential to inhibit PL protein and can be used to prepare PL protein inhibitors, providing a new PL protein inhibitory drug for the treatment of obesity.

[0015] The gene or recombinant plasmid encoding VNAR-A4 or the recombinant cell containing the recombinant plasmid of the gene can be used for preparing PL protein inhibitors and treating obesity.

[0016] (4) The present invention selects the striped bamboo shark as a model animal for antibody preparation to prepare VNAR. The bamboo shark is not an endangered shark species, is small in size, and is easy to breed artificially, making it suitable for antibody development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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; Figure 2 The results are for phage polyclonal ELISA; Figure 3 The results of VNAR-A4 purification are shown in Figure 1. Lane description: M: Protein Marker, 1-3: 200 mM imidazole eluent, 4-6: 250 mM imidazole eluent.

[0018] Figure 4 ELISA was used to detect the binding of shark nanobodies to recombinant human PL protein; Figure 5 The results show that the antibody VNAR-A4 inhibits the activity of recombinant human PL protein; DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0020] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0021] The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art and are 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.

[0022] Example 1 Construction of a shark phage antibody library targeting PL 1. Construction of VNAR immune library (1) Obtaining cDNA Peripheral blood lymphocytes (PBMCs) and spleen tissues of immunized striped bamboo sharks were collected, RNA was extracted using the TRIZOL method, and then reverse transcribed into cDNA.

[0023] (2) Cloning of target gene cDNA was amplified by PCR using primer combination 1 (upstream primers NewVF1+NewVF2, downstream primers NewVR1+NewVR2), primer combination 2 (upstream primers NewVF2+BamVF3, downstream primers BamVR3+BamVR4), and primer combination 3 (upstream primer BamVF4, downstream primer BamVR5). The resulting PCR products were digested with SfiI and ligated to the phagemid vector pComb3xss.

[0024] The sequences of the primer combinations are as follows: Upstream primer NewVF1 (SEQ ID NO.1) CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC Upstream primer NewVF2 (SEQ ID NO.2) CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAAAAACACCG Downstream primer NewVR1 (SEQ ID NO.3) CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC Downstream primer NewVR2 (SEQ ID NO.4) CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC Upstream primer BamVF3 (SEQ ID NO.5) CAATTTGATTGGGCCCAGGCGGCCCAATGGGTTGAACAAACACCGA Upstream primer BamVF4 (SEQ ID NO.6) CAATTTGATTGGGCCCAGGCGGCCGCATGGGTTGACCAAACAC Downstream primer BamVR3 (SEQ ID NO.7) CAATTTGATTGGGCCGGCCTGCCAGGTTTCACAGTCAGAATGGTG Downstream primer BamVR4 (SEQ ID NO.8) CAATTTGATTGGGCCGGCCTGGCCGGGTTTTACACTCAGAATGGTG Downstream primer BamVR5 (SEQ ID NO.9) CAATTTGATTGGGCCGGCCTGCCAGGTTTCACTGCCAGAAAAGTGC The primer combinations were used to perform PCR reactions as shown in Table 1 PCR reaction system and Table 2 PCR reaction program.

[0025] Table 1 PCR reaction system

[0026] Table 2 PCR reaction procedure

[0027] The results of nucleic acid electrophoresis of the VNAR region of the bamboo shark amplified by PCR are as follows: Figure 1 .

[0028] (3) Construction of phagemid The PCR product digestion system is as follows: Table 3 PCR product digestion system

[0029] Prepare 13 tubes of enzyme digestion reaction system as shown in Table 3 on ice and place them in a PCR instrument at 50℃ for 2 h. Perform 1% agarose gel electrophoresis on the digestion products, use the kit to recover all the digestion products, and store them at -20℃ for later use.

[0030] pComb3XSS plasmid restriction enzyme digestion system is as follows: Table 4 pComb3XSS plasmid restriction enzyme digestion system

[0031] Prepare 8 tubes of pComb3XSS plasmid digestion reaction system as shown in Table 4 on ice and place them in a PCR instrument at 50℃ for 2 h. Perform 1% agarose gel electrophoresis on the digestion products, and use a kit to recover the digested pComb3XSS plasmid and store it at -20℃ for later use in the construction of pComb3XSS recombinant plasmid.

[0032] The T4 DNA ligase ligation system is as follows: Table 5 T4 DNA ligase ligation system

[0033] Prepare 20 tubes of the ligation system shown in Table 5 on ice and place them in a PCR instrument at 16°C for ligation overnight. The next day, inactivate the ligation system in a PCR instrument at 65°C for 10 min. Collect all ligation products, i.e., pComb3XSS recombinant plasmids, and store them at 4°C for later use.

[0034] 2. Library construction The above ligation products were transformed into E. coli In TG1 competent cells, the original phage library is formed. The original library capacity is calculated by limiting dilution coating method. The original library capacity of shark nanoantibody (cfu) = colony number × 10 × dilution gradient × library volume. The colony number is counted according to the colony growth, and the constructed shark nanoantibody library capacity is calculated. The quality evaluation of VNAR library is shown in Table 6, including library capacity, gene insertion rate and gene diversity.

[0035] Table 6: Evaluation of the quality of phage libraries Library capacity Insertion rate Diversity <![CDATA[1×10 8 ]]> 100% 91.7% Example 2 Selection of Nanobody Phage Targeting Human PL Shark The original phage library of Example 1 was amplified by adding helper phage M13KO7 to prepare phage for panning.

[0036] 1. The first round of selection: (1) Antigen coating: Dilute the human PL recombinant protein stock solution with PBS solution to 10 μg / mL, and add 100 μL per well to a 96-well ELISA 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. Place the ELISA plate at 4°C overnight.

[0037] (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.

[0038] (3) Blocking: Add 200 μL / well of 3% BSA solution to the antigen wells and incubate at 37°C for 1 h. Add 100 μL / well of phage antibody library (pre-diluted to 1000 times the library capacity with 3% BSA) to the BSA wells and incubate at 37°C for 1 h.

[0039] (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 times. Then, 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 h.

[0040] (5) Activation of TG1 strain: Take an appropriate amount of overnight cultured TG1 bacterial liquid and transfer it to fresh 2×YT liquid culture medium at a ratio of 1:100. Place it in a shaker incubator at 37°C and 180 rpm for about 2 h to reach the logarithmic growth phase.

[0041] (6) Washing: Wash the antigen wells with 0.1% PBST solution at a volume of 300 μL / well and wash ten times in total.

[0042] (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 2×YT agar plate (Amp+) and culture inverted at 37°C overnight.

[0043] (8) Helper phage superinfection: Add 20 times more helper phage M13KO7 to the remaining TG1 bacterial solution and incubate at 37°C for 1 h. Mix once every 30 min.

[0044] (9) Progeny phage amplification: After incubation, the phage solution was placed in a centrifuge at 8000 rpm for 5 min. After centrifugation, the supernatant was discarded and resuspended in 10 mL of fresh 2×YT liquid culture medium. Amp (100 μg / mL) and Kana (50 μg / mL) were added in sequence and placed in a shaker incubator at 30°C and 200 rpm for overnight culture.

[0045] (10) The next day, centrifuge at 5000 rpm and 4 °C for 30 min. (11) Filter the supernatant, add 1 / 4 volume of PEG / NaCl, and let it stand on ice for 1 h.

[0046] (12) Centrifuge at 5000 rpm and 4 °C for 30 min, discard the supernatant, and resuspend in 1 mL PBS to prepare the phage antibody library obtained in the first round of panning.

[0047] (II) Second and third rounds of selection The panning scheme is similar to the first round of panning, with the following changes: (1) The coating concentration of human PL recombinant protein in the ELISA plate was reduced to 4 μg / mL and 2 μg / mL in successive rounds.

[0048] (2) In step (6), the number of times the ELISA plate is washed is increased to 15 and 20 times in succession.

[0049] (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.

[0050] The titer of the selected phages in the third round was higher than that in the second round, and the polyclonal ELISA test showed that specific phages were also enriched (see Table 7 and Figure 2 ).

[0051] Table 7: The titer results of the original and three rounds of panning phage nanobody libraries

[0052] 96 monoclonal colonies were randomly selected from the solid agar plates obtained from the third and second rounds of panning to prepare soluble phage antibodies and perform monoclonal phage ELISA tests. To ensure that the amount of phage antibodies amplified by each monoclonal colony was basically the same, each colony was cultured in the same volume of culture medium under the same experimental conditions, and other experimental operations were kept consistent.

[0053] The 53 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-PL VNAR sequence was obtained and named VNAR-A4.

[0054] The amino acid sequence of VNAR-A4 is shown in SEQ ID NO.10, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.11; the VNAR-A4 sequence was compared with the NCBI database, and the results showed that this sequence was a shark-derived nanoantibody gene sequence.

[0055] The amino acid sequence and nucleotide sequence of shark nanobody VNAR-A4 are as follows: Amino acid sequence of shark nanobody VNAR-A4 (SEQ ID NO.10) GWVDQTPTTTTKETGESLTINCVLRDSVRALDRTYWYFTKKGATKKESLSTGGRYAETVNKASKSFSLRISDLRVEDSGTYDCKAYAGGKFLIGGSYEEGGGTILTVKP Nucleotide sequence of shark nanobody VNAR-A4 (SEQ ID NO.11) GGTTGGGTGGACCAAACACCGACAACGACAACAAAGGAGACAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCGTTCGTGCATTGGATAGGACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAACTGGCGGACGAT ACGCGGAAACAGTGAACAAGGCATCAAAGTCTTTCTCATTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATGACTGTAAAGCGTATGCAGGGGGGAAATTCCTTATAGGTGGAAGCTATGAAGAAGGAGGCGGCACCATTCTGACTGTGAAACCT The shark nanobody VNAR-A4 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: FR1: GWVDQTPTTTTKETGESLTINCVLR (SEQ ID No. 14); FR2: TYWYFT (SEQ ID No.15); FR3a: GRYAETV (SEQ ID No. 16); FR3b:FSLRISDLRVEDSGTYDC (SEQ ID No. 17); FR4:EGGGTILTVKP (SEQ ID No.18); The amino acid sequences of the hypervariable region HV include HV2 and HV4, which are as follows: HV2:;KKGATKKESLSTG (SEQ ID No.19) HV4:;NKASKS (SEQ ID No.20) The complementary determining region CDR includes the amino acid sequences of CDR1 and CDR3. Corresponding to the above FRs, the CDR amino acid sequences of the shark nanobody VNAR-A4 are: CDR1:DSVRALDR (SEQ ID No.21) CDR3: KAYAGGKFLIGGSYE (SEQ ID No. 22) Example 3 In vitro recombinant expression and purification of nanobodies 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-A4 sequence was amplified by PCR. The pET28a vector was double-digested with Nco I and Nco I. The shark nanobody gene VNAR-A4 fragment was connected to the pET28a vector. The above connection product was transformed into DH5a competent cells. After the sequencing was correct, the plasmid was transformed into E.coliBL21 (DE3) competent cells. The his tag was purified with 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-A4, proving that VNAR-A4 is successfully expressed and purified, and VNAR-A4 can be eluted using 200 mM imidazole elution buffer.

[0056] Example 4 ELISA detection of the binding of shark nanoantibodies to recombinant human PL protein 100 mL / well of 1 mg / mL recombinant human PL protein was added to the ELISA plate and incubated overnight at 4°C. The next day, the supernatant was discarded and the plate was washed three times with 300 mL / well containing 0.1% PBST. 200 mL / well of PBS containing 3% BSA was added, incubated at 37°C for 1.5 h, and washed three times with 300 mL / well 0.1% PBST. 100 mL / well of different concentrations of VNAR-A4 (5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 μM) was added, incubated at 37°C for 1.5 h, and washed three times with 300 mL / well 0.1% PBST. 100 mL / well of HRP-anti-HA tag antibody (1:10000 diluted in PBS containing 3% BSA) was added, incubated at 37°C for 1.5 h, and washed three times with 300 mL / well 0.1% PBST. 100 mL / well of mL of TMB substrate, incubate at 37°C in the dark for 10-15 min, add 50 mL of 1 MH2SO4 to each well after color development to terminate the reaction, and detect OD 450 value.

[0057] like Figure 4 As shown, based on the ELISA results, the EC of VNAR-A4 was calculated. 50 The value is 307.9 nM, and VNAR-A4 can specifically bind to recombinant human PL protein.

[0058] Example 5 Detection of the Inhibition of Shark Nanobodies on the Activity of Recombinant Human PL Protein by p-Nitrophenol Method Add 3.75 mL of recombinant human PL, 175 mL of Tris-HCl solution (100 mM Tris, 5 mM CaCl2, pH=7.0) and 66.25 mL of VNAR-A4 dilutions of different concentrations and mix well, so that the final concentration of A4 is 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 mg / ml, and incubate at 37°C for 15 min to allow VNAR-A4 to fully bind to recombinant human PL. Add 5 mL of 50 mM p-nitrophenylbutyrate (dissolved in DMSO, prepared immediately before use) and mix well, and react at 37°C for 20 min. Pipette 200 mL into a 96-well plate and incubate at OD 405 Readings in nm.

[0059] like Figure 5 As shown, according to OD 405 As a result, the IC of VNAR-A4 was calculated. 50 The value was 0.04481 mg / mL, and VNAR-A4 was able to inhibit the activity of recombinant human PL protein.

[0060] The above description is only 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 above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A shark nanobody VNAR-A4 targeting pancreatic lipase, whose amino acid sequence is shown in SEQ ID No.

10.

2. The shark nanobody VNAR-A4 according to claim 1, characterized in that The shark nanobody VNAR-A4 includes a framework region, a complementary 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 coding gene of the shark nanobody VNAR-A4 according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown as 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-A4 according to claim 1 in the preparation of a pancreatic lipase protein inhibitor.

7. Use of the shark nanobody VNAR-A4 according to claim 1 in the preparation of anti-obesity drugs.

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