A method for preparing shark nanobodies targeting pancreatic lipase and its application

Abstract

The present invention belongs to the field of biotechnology and relates to a method for preparing shark nanobodies targeting pancreatic lipase and its application. The amino acid sequence of the shark nanobody VNAR-A4 is shown in SEQ ID No. 10. The shark nanobody VNAR-A4 prepared by the present invention has a molecular weight of about 15 kDa, which is smaller than traditional monoclonal antibodies, has an elongated CDR3 structure, and can better bind to the hidden epitopes of antigens; the shark nanobody VNAR-A4 has good stability, can maintain its activity under extreme pH conditions, and has good affinity and specificity for PL; the shark nanobody VNAR-A4 is expressed using an Escherichia coli prokaryotic expression system, is more easily genetically engineered, and can be obtained in large quantities. The shark nanobody VNAR-A4 has the potential to inhibit the activity of pancreatic lipase and provides a new lipolysis inhibitor for the treatment of obesity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to the preparation of antibodies, and particularly relates to a shark nanobody VNAR-A4 targeting pancreatic lipase and its application. Background Art

[0002] Obesity refers to the abnormal or excessive accumulation of fat that may have an adverse impact on physical health. As a complex chronic disease, it has currently become one of the global public health problems. Obesity is a complex metabolic disease, closely related to diseases such as non-alcoholic fatty liver, type 2 diabetes, rheumatoid arthritis, gastrointestinal system cancers, etc., and will 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. Pancreatic lipase (PL) plays a crucial role in the process of fat hydrolysis. After food reaches the gastrointestinal tract, the carboxyl group of dietary triglyceride is attacked by the nucleophilic serine, resulting in hydrolysis, releasing free fatty acids, monoglycerides, diglycerides, cholesterol and other substances, and being absorbed at the small intestine and re-synthesized into triglycerides. The excess fat is stored in the body, forming white adipose tissue and causing obesity. Orlistat is a gastric and pancreatic lipase inhibitor that can bind to the active site serine of gastric and pancreatic lipases to inhibit their activities, thereby inhibiting the hydrolysis of dietary triglycerides. The unhydrolyzed triglycerides are excreted from the body with feces, and play an anti-obesity role by reducing the absorption of dietary fat. Compared with other anti-obesity drugs, it has higher safety, but still has adverse reactions such as gastrointestinal discomfort, oily stools, loose stools, etc. Rare adverse reactions involve the kidneys and liver, and will also affect the absorption of fat-soluble vitamins and other drugs. With the improvement of people's quality of life, obesity has become a global public health problem. Therefore, it is necessary to find new PL inhibitors with low toxicity and low side effects.

[0003] Monoclonal antibodies have been widely used in a large number of biotechnological and biomedical applications for decades. However, they are limited by their relatively large size (about 150 kDa), which prevents them from accessing epitopes in the surface crevices of antigens (such as the active sites of enzymes). In addition, monoclonal antibodies may be affected by high temperature and high humidity conditions, which can reduce their sensitivity. To overcome these limitations, there is an urgent need to explore new antigen-binding agents that are smaller in size and have natural thermal stability. In the 1990s, foreign scholars discovered VNARs with only heavy chains in sharks. Since they only have heavy chains, the antigen-binding sites are formed by a single domain, and they are also known as nanobodies. VNARs are the smallest antibody fragments discovered so far (about 12 - 15 kDa). They have evolved by compensating for CDR2 and thus only have CDR1 and CDR3. Compared with conventional antibodies, VNARs have many advantages due to their smaller molecular weight and special structure, such as high antigen affinity, good water solubility and stability, strong tissue penetration, and the ability to recognize hidden antigen epitopes. These properties make them a good alternative source for therapeutic and diagnostic reagents. Shark nanobodies, as a novel type of antibody, have unique advantages. Searching for shark nanobodies that can bind and inhibit PL has important application value for the treatment of obesity. Summary of the Invention

[0004] The object of the present invention is to solve the above problems existing in the prior art, and propose a shark nanobody VNAR-A4 targeting PL and its application. The present invention has screened and obtained a brand-new shark nanobody VNAR-A4, which has high affinity and high specificity for PL.

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

[0006] The present invention protects a shark nanobody VNAR-A4 targeting PL, and its amino acid sequence is shown in SEQ ID No.10.

[0007] Further, the shark nanobody VNAR-A4 includes a framework region, a complementarity-determining region, and a hypervariable region;

[0008] The framework region is FR1, FR2, FR3a, FR3b, and FR4;

[0009] The amino acid sequence of FR1 is shown in SEQ ID No.14;

[0010] The amino acid sequence of FR2 is shown in SEQ ID No.15;

[0011] The amino acid sequence of FR3a is shown in SEQ ID No.16;

[0012] The amino acid sequence of FR3b is shown in SEQ ID No.17;

[0013] The amino acid sequence of the FR4 is shown in SEQ ID No. 18;

[0014] The complementarity-determining regions are CDR1 and CDR3;

[0015] The amino acid sequence of the CDR1 is shown in SEQ ID No. 21;

[0016] The amino acid sequence of the CDR3 is shown in SEQ ID No. 22;

[0017] The hypervariable regions are HV2 and HV4;

[0018] The amino acid sequence of the HV2 is shown in SEQ ID No. 19;

[0019] The amino acid sequence of the HV4 is shown in SEQ ID No. 20.

[0020] 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.

[0021] The present invention also protects a recombinant vector, and the recombinant vector contains the above-mentioned encoding gene.

[0022] The present invention also protects a recombinant strain, and the recombinant strain contains the above-mentioned encoding gene or the above-mentioned recombinant vector.

[0023] The present invention also protects the application of the shark nanobody VNAR-A4 in the preparation of a PL protein inhibitor.

[0024] The present invention also protects the application of the shark nanobody VNAR-A4 in the preparation of an anti-obesity drug.

[0025] Advantages of the present invention:

[0026] (1) The shark nanobody VNAR-A4 prepared by the present invention has the ability to recognize PL, and its sequence consists of 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, smaller than the traditional monoclonal antibody. Therefore, it overcomes the disadvantages and deficiencies of traditional antibodies in application and can better bind to the epitope (such as the active site of an enzyme) in the surface crack of the antigen.

[0027] (2) The shark nanobody VNAR-A4 of the present invention is expressed using the Escherichia coli prokaryotic expression system, which is easier to express and genetically engineered, and a large amount of shark nanobodies can be obtained. The prepared shark nanobody VNAR-A4 has high stability, good tissue permeability, is easier to store and transport than conventional antibodies, and has good affinity and specificity for PL.

[0028] (3) The shark nanobody VNAR-A4 provided by the present invention has the potential to inhibit PL protein and can be applied to the preparation of PL protein inhibitors, providing a new PL protein inhibitory drug for the treatment of obesity.

[0029] The gene encoding VNAR-A4, or the recombinant plasmid, or the recombinant cell containing the recombinant plasmid of this gene can be applied to the preparation of PL protein inhibitors and the treatment of obesity.

[0030] (4) The present invention selects the bamboo shark 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. Description of the Drawings

[0031] Figure 1 For PCR amplification of the shark nanobody gene; Lane description: M: DNA Marker, 1: PCR product of primer combination one, 2: PCR product of primer combination two, 3: PCR product of primer combination three;

[0032] Figure 2 For the results of phage polyclonal ELISA;

[0033] Figure 3 For the purification results of VNAR-A4. Lane description: M: Protein Marker, 1-3: 200 mM imidazole eluate, 4-6: 250 mM imidazole eluate.

[0034] Figure 4 For ELISA detection of the binding of shark nanobody to recombinant human PL protein;

[0035] Figure 5 For the results of the antibody VNAR-A4 inhibiting the activity of recombinant human PL protein; Detailed Embodiments

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] To further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0038] The experimental methods in the following embodiments are all conventional methods in the art unless otherwise specified, and are carried out according to the techniques described in the literature in the art or according to the product specifications; various biological materials, reagents, etc. used in the embodiments can be obtained from commercial channels unless otherwise specified.

[0039] Example 1 Construction of a Shark Phage Antibody Library Targeting PL

[0040] 1. Construction of VNAR Immune Library

[0041] (1) Obtaining cDNA

[0042] Collect the peripheral blood lymphocytes PBMC and spleen tissues of immunized Chiloscyllium plagiosum, extract RNA by the TRIZOL method, and then reverse transcribe it into cDNA.

[0043] (2) Cloning of the Target Gene

[0044] Use primer combination one (upstream primers NewVF1 + NewVF2, downstream primers NewVR1 + NewVR2), primer combination two (upstream primers NewVF2 + BamVF3, downstream primers BamVR3 + BamVR4), and primer combination three (upstream primer BamVF4, downstream primer BamVR5) to perform PCR amplification on the cDNA. The obtained PCR product is digested with SfiI enzyme and then ligated to the phagemid vector pComb3xss.

[0045] The sequences of the primer combinations are as follows:

[0046] Upstream primer NewVF1 (SEQ ID NO.1)

[0047] CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC

[0048] Upstream primer NewVF2 (SEQ ID NO.2)

[0049] CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAACAAACACCG

[0050] Downstream primer NewVR1 (SEQ ID NO.3)

[0051] CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC

[0052] Downstream primer NewVR2 (SEQ ID NO.4)

[0053] CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC

[0054] Upstream primer BamVF3 (SEQ ID NO.5)

[0055] CAATTTGATTGGGCCCAGGCGGCCCAATGGGTTGAACAAACACCGA

[0056] Upstream primer BamVF4 (SEQ ID NO.6)

[0057] CAATTTGATTGGGCCCAGGCGGCCGCATGGGTTGACCAAACAC

[0058] Downstream primer BamVR3 (SEQ ID NO.7)

[0059] CAATTTGATTGGGCCGGCCTGGCCAGGTTTCACAGTCAGAATGGTG

[0060] Downstream primer BamVR4 (SEQ ID NO.8)

[0061] CAATTTGATTGGGCCGGCCTGGCCGGGTTTTACACTCAGAATGGTG

[0062] Downstream primer BamVR5 (SEQ ID NO.9)

[0063] CAATTTGATTGGGCCGGCCTGGCCAGGTTTCACTGCCAGAAAAGTGC

[0064] The primer combinations are used for PCR reactions according to the PCR reaction system in Table 1 and the PCR reaction program in Table 2 as follows.

[0065] Table 1 PCR reaction system

[0066]

[0067]

[0068] Table 2 PCR reaction program

[0069]

[0070] The nucleic acid electrophoresis results of the VNAR region of Chiloscyllium plagiosum obtained by PCR amplification are as follows Figure 1 .

[0071] (3) Construction of phagemid

[0072] The enzyme digestion system of the PCR product is as follows

[0073] Table 3 Enzyme digestion system of PCR product

[0074]

[0075] Prepare 13 tubes of the enzyme digestion reaction system shown in Table 3 on ice, place them in a PCR instrument and digest at 50 °C for 2 h. Perform 1% agarose gel electrophoresis on the enzyme digestion products, use a kit to recover all the enzyme digestion products, and store them at -20 °C for later use

[0076] The enzyme digestion system of pComb3XSS plasmid is as follows

[0077] Table 4 Enzyme digestion system of pComb3XSS plasmid

[0078]

[0079] Prepare 8 tubes of the pComb3XSS plasmid enzyme digestion reaction system shown in Table 4 on ice, place them in a PCR instrument and digest at 50 °C for 2 h. Perform 1% agarose gel electrophoresis on the enzyme digestion products, use a kit to recover the digested pComb3XSS plasmid, and store it at -20 °C for later use, for subsequent construction of pComb3XSS recombinant plasmid

[0080] The ligation system of T4 DNA ligase is as follows

[0081] Table 5 Ligation system of T4 DNA ligase

[0082]

[0083] Prepare 20 tubes of the ligation system shown in Table 5 on ice, place them in a PCR instrument and ligate overnight at 16 °C. The next day, inactivate the ligation system in a PCR instrument at 65 °C for 10 min. Collect all the ligation products, that is, pComb3XSS recombinant plasmid, and store it at 4 °C for later use

[0084] 2. Library construction

[0085] The above ligation product was electrotransformed into E. coli TG1 competent cells to form the original phage library. The method of limited dilution plating was used to calculate the library capacity of the original library. The library capacity (cfu) of the shark nanobody original library = the number of colonies × 10 × dilution factor × library volume. The number of colonies was counted according to the colony growth situation, and the library capacity of the constructed shark nanobody library could be calculated. The quality evaluation of the VNAR library is shown in Table 6, including library capacity, gene insertion rate, and gene diversity.

[0086] Table 6: Quality evaluation of the phage library

[0087]

[0088] Example 2 Panning of shark nanobody phages targeting human PL

[0089] The phages were prepared by amplifying the original phage library of Example 1 with helper phage M13KO7 and used for panning.

[0090] (I) First-round panning:

[0091] (1) Coating antigen: The mother liquor of human PL recombinant protein was diluted to 10 μg / mL with PBS solution and added to a 96-well ELISA plate at 100 μL / well, with a total of 8 wells. At the same time, 200 μL of 3% BSA solution was added to the wells without coated antigen, with a total of 8 wells, for negative panning of the antibody library. The ELISA plate was placed at 4 °C overnight.

[0092] (2) Washing the plate: The next day, the coating solution was discarded, and the ELISA plate was washed with 0.1% PBST solution at 300 μL / well, and washed three times in total.

[0093] (3) Blocking: 200 μL / well of 3% BSA solution was added to the antigen wells and incubated at 37 °C for 1 h. 100 μL / well of phage antibody library (pre-diluted to 1000 times the library capacity with 3% BSA) was added to the BSA wells and incubated at 37 °C for 1 h.

[0094] (4) Discard the blocking solution in the antigen wells, wash the ELISA plate with 0.1% PBST solution at 300 μL / well, and wash three times in total. Subsequently, the pre-incubated phage antibody library in the BSA wells was transferred to the antigen wells at 100 μL / well and incubated at 37 °C for 1 h.

[0095] (5) Activating TG1 strain: An appropriate amount of overnight cultured TG1 bacterial solution was transferred to fresh 2×YT liquid medium at a ratio of 1:100 and cultured in a shaker incubator at 37 °C and 180 rpm for about 2 h to reach the logarithmic growth phase.

[0096] (6) Plate washing: Wash the antigen wells with 0.1% PBST solution at a volume of 300 μL / well, and wash a total of ten times.

[0097] (7) Infection: Add the TG1 bacterial solution in the logarithmic growth phase to the antigen wells at 100 μL / well, and incubate statically at 37 °C for 30 min. After the infection, collect all the TG1 bacterial solution. Repeat the above infection step twice. Combine the bacteria collected three times, and take 50 μL for limited dilution coating. The dilution factors are 10 2 、10 3 、10 4 、10 5 , take 100 μL and coat it on a 2×YT agar plate (Amp+), and incubate it upside down at 37 °C overnight.

[0098] (8) Helper phage superinfection: Add helper phage M13KO7 with a volume more than 20 times the bacterial volume to the remaining TG1 bacterial solution, and incubate statically at 37 °C for 1 h. During this period, it can be mixed once at 30 min.

[0099] (9) Progeny phage amplification: Place the phage bacterial solution after incubation in a centrifuge and centrifuge at 8000 rpm for 5 min. After centrifugation, discard the supernatant, resuspend it in 10 mL of fresh 2×YT liquid medium, add Amp (100 μg / mL) and Kana (50 μg / mL) in sequence, and culture it overnight at 30 °C and 200 rpm in a shaking incubator.

[0100] (10) The next day, centrifuge at 5000 rpm and 4 °C for 30 min.

[0101] (11) Filter the supernatant and add 1 / 4 volume of PEG / NaCl, and let it stand on ice for 1 h.

[0102] (12) Centrifuge at 5000 rpm and 4 °C for 30 min, discard the supernatant, and resuspend it with 1 mL of PBS to prepare the phage antibody library obtained from the first round of panning.

[0103] (II) Second and third rounds of panning

[0104] The panning scheme is similar to that of the first round of panning, with only the following changes:

[0105] (1) The coating concentration of human PL recombinant protein on the enzyme-linked immunosorbent assay (ELISA) plate is gradually reduced to 4 μg / mL and 2 μg / mL round by round.

[0106] (2) The number of times of washing the ELISA plate in step (6) is gradually increased to 15 times and 20 times round by round.

[0107] (3) Replace the blocking solution used in the second round of panning with 5% MPBS, and still use 3% BSA solution as the blocking solution in the third round of panning.

[0108] The titer of the panned phages increased in the third round compared to the second round, and specific phages were also enriched as detected by polyclonal ELISA (see Table 7 and Figure 2 ).

[0109] Table 7: Titre results of the original and three-round panned phage nanobody libraries

[0110]

[0111]

[0112] Ninety-six monoclonal colonies were randomly picked from the solid agar plates obtained from the third-round and second-round panning to prepare soluble phage antibodies, and monoclonal phage ELISA was performed. To ensure that the amount of phage antibodies amplified from each monoclonal colony was basically the same, each colony was cultured in the same volume of medium under the same experimental conditions, and other experimental operations were kept consistent.

[0113] Fifty-three clones with the highest absorbance values were selected and sent to a sequencing company for sequence analysis. The homology comparison of the sequencing results was performed using the NCBI-BLAST tool, the amino acid sequence was translated using the EXPASY-translate tool, and the amino acid sequence diversity was analyzed in BioEdit. Finally, the VNAR sequence against PL, named VNAR-A4, was obtained.

[0114] 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 result indicated that this sequence was a nanobody gene sequence derived from sharks.

[0115] The amino acid sequence and nucleotide sequence of the shark nanobody VNAR-A4 are as follows:

[0116] Amino acid sequence of the shark nanobody VNAR-A4 (SEQ ID NO.10)

[0117] GWVDQTPTTTTKETGESLTINCVLRDSVRALDRTYWYFTKKGATKKESLSTGGRYA ETVNKASKSFSLRISDLRVEDSGTYDCKAYAGGKFLIGGSYEEGGGTILTVKP

[0118] Nucleotide sequence of the shark nanobody VNAR-A4 (SEQ ID NO.11)

[0119] GGTTGGGTGGACCAAACACCGACAACGACAACAAAGGAGACAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCGTTCGTGCATTGGATAGGACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAACTGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCTTTCTCATTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATGACTGTAAAGCGTATGCAGGGGGGAAATTCCTTATAGGTGGAAGCTATGAAGAAGGAGGCGGCACCATTCTGACTGTGAAACCT

[0120] The shark nanobody VNAR-A4 includes a framework region FR, a complementarity-determining region CDR, and a hypervariable region HV. The framework region FR includes the amino acid sequences of FR1, FR2, FR3a, FR3b, and FR4, which are as follows:

[0121] FR1: GWVDQTPTTTTKETGESLTINCVLR (SEQ ID No.14);

[0122] FR2: TYWYFT (SEQ ID No.15);

[0123] FR3a: GRYAETV (SEQ ID No.16);

[0124] FR3b: FSLRISDLRVEDSGTYDC (SEQ ID No.17);

[0125] FR4: EGGGTILTVKP (SEQ ID No.18);

[0126] The hypervariable region HV includes the amino acid sequences of HV2 and HV4, which are as follows:

[0127] HV2: ;KKGATKKESLSTG (SEQ ID No.19)

[0128] HV4: ;NKASKS (SEQ ID No.20)

[0129] The complementarity-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 respectively:

[0130] CDR1: DSVRALDR (SEQ ID No.21)

[0131] CDR3: KAYAGGKFLIGGSYE (SEQ ID No.22)

[0132] Example 3 In Vitro Recombinant Expression and Purification of Nanobody

[0133] Using the positive clone phage plasmid as a template, according to the sequence information of the sequencing results, forward primer: 5'-TAAGAAGGAGATATACCATGGGCGGTTGGGTGGACCAAACACC-3' (SEQ ID NO.12) and 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 ligated with the pET28a vector. The above ligation product was transformed into DH5a competent cells. After correct sequencing, the plasmid was transformed into E. coli BL21(DE3) competent cells. Purification was carried out using nickel ion chelating filler with his tag. Each purified protein was aliquoted and stored at -80°C. The results are as Figure 3 shown. The position of the target band corresponded to the theoretical molecular weight of VNAR-A4, proving that VNAR-A4 was successfully expressed and purified. VNAR-A4 could be eluted using 200 mM imidazole eluent.

[0134] Example 4 ELISA Detection of the Binding of Shark Nanobody to Recombinant Human PL Protein

[0135] 100 μL / well of 1 μg / mL recombinant human PL protein was added to the enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4°C. The next day, the supernatant was discarded, and it was washed 3 times with 300 μL / well of 0.1% PBST. 200 μL / well of PBS containing 3% BSA was added and incubated at 37°C for 1.5 h, then washed 3 times with 300 μL / well of 0.1% PBST. 100 μL / well of VNAR-A4 at different concentrations (5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 μM) was added and incubated at 37°C for 1.5 h, then washed 3 times with 300 μL / well of 0.1% PBST. 100 μL / well of HRP-anti-HA tag antibody (diluted 1:10000 in PBS containing 3% BSA) was added and incubated at 37°C for 1.5 h, then washed 3 times with 300 μL / well of 0.1% PBST. 100 μL of TMB substrate was added to each well and incubated at 37°C in the dark for 10 - 15 min. After color development, 50 μL of 1 M H2SO4 was added to each well to terminate the reaction, and OD was detected450 Value

[0136] As Figure 4 shown, according to the ELISA results, the EC 50 value of VNAR-A4 was calculated to be 307.9 nM, and VNAR-A4 could specifically bind to recombinant human PL protein.

[0137] Example 5 Detection of the Inhibition of the Activity of Recombinant Human PL Protein by Shark Nanobody Using p-Nitrophenol Method

[0138] Add 3.75 μL of recombinant human PL, 175 μL of Tris-HCl solution (100 mM Tris, 5 mM CaCl2, pH = 7.0), and 66.25 μL of different concentration dilutions of VNAR-A4 and mix well to make the final concentration of A4 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 mg / ml. Incubate at 37 °C for 15 min to allow VNAR-A4 to fully bind to recombinant human PL. Add 5 μL of 50 mM p-nitrophenyl butyrate (dissolved in DMSO, freshly prepared) and mix well, and react at 37 °C for 20 min. Pipette 200 μL into a 96-well plate and read the value at OD 405 nm.

[0139] As Figure 5 shown, according to the OD 405 results, the IC 50 value of VNAR-A4 was calculated to be 0.04481 mg / mL, and VNAR-A4 could inhibit the activity of recombinant human PL protein.

[0140] The above description is only the preferred embodiment of the present invention and is not a limitation of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, modifications, etc. made within the spirit and principle of the present invention shall be included within 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 anti-obesity drugs.

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