An alpaca-derived nanobody that specifically binds to the trypsin analogue TrypLE and its applications

Abstract

The present invention belongs to the technical fields of molecular biology and immunology, and provides a camelid-derived nanobody that specifically binds to the trypsin analogue TrypLE and its application. It has a heavy chain variable region VHH, and the VHH contains the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO:1, CDR2 with the amino acid sequence shown in SEQ ID NO:2, CDR3 with the amino acid sequence shown in SEQ ID NO:3. The VHH includes 4 framework regions of FR1-4, and FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence. It is a nanobody with high neutralizing activity, has a strong binding ability to TrypLE protein, can effectively recognize TrypLE. This nanobody has a small molecular weight, low immunogenicity, better solubility and stability, and a longer CDR region, providing potential application value for the clinical detection of TrypLE residue content.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and immunology. More specifically, the present invention relates to a camelid-derived nanobody that specifically binds to the trypsin analogue TrypLE and its applications. Background Art

[0002] Trypsin (Parenzyme) is a protease and a serine proteolytic enzyme extracted from the pancreas of cattle, sheep, and pigs. In vertebrates, it functions as a digestive enzyme. After the precursor of trypsin, trypsinogen, is synthesized in the pancreas, it is secreted as a component of pancreatic juice and is limitedly decomposed by enterokinase or trypsin to become activated trypsin. It is an endopeptidase that can cut the carboxyl side of lysine and arginine residues in the polypeptide chain. It not only acts as a digestive enzyme but also activates other enzyme precursors such as chymotrypsinogen, carboxypeptidase, and phospholipase. Trypsin is the most specific protease and is an indispensable tool in determining the amino acid sequence of proteins. TrypLE produced by GIBCO is a trypsin analogue, a non-animal-derived recombinant enzyme prepared by bioengineering, which is widely used for the dissociation of tissues and monolayer cells and can be used to dissociate a series of adherent mammalian cells, including GHO, HEK 293, A529, primary human keratinocytes, and embryonic stem cells. In cell culture, it can replace porcine and bovine trypsin to dissociate adherent cells.

[0003] In 1993, scientists discovered in camels an immunoglobulin G that naturally lacks a part of the light chain and the constant region of the heavy chain but still has antigen-binding ability, called heavy chain antibody (HCAbs). By analyzing and identifying the structure and sequence of this heavy chain antibody, it was found that its antigen-binding region consists only of variable region fragments. Therefore, the antigen recognition region fragment of the heavy chain antibody is called VHH, and based on this, a single domain antibody (sdAb) containing only the VHH domain, also known as VHH or nanobody (Nb), was developed. In subsequent studies, this specific heavy chain antibody was also found in the blood of animals such as camels and sharks. Compared with other small molecule antibodies, nanobodies have unique properties such as small molecular mass (12 - 15 kDa), strong water solubility, etc., making them more advantageous in disease diagnosis and treatment. In recent years, a variety of nanobody drugs have entered the clinical stage.

[0004] There is a natural light-chain-deficient antibody in camelids, namely the heavy-chain antibody, whose variable region consists only of the heavy chain. The diameter of the variable region protein is less than 10 nanometers, so it is also called nanobody. Nanobodies have the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, and easy binding to multiple epitopes.

[0005] Nanobodies are currently the smallest antibody molecules. They were initially discovered by Belgian scientist Hamers in camel blood and are a class of highly concerned engineered antibody products. The main advantages of nanobodies are as follows: First, their volume is 1 / 10 of that of ordinary antibodies. Due to their small size, they have strong penetrability in animal tissues, can pass through the human brain tissue, can reach the inside of high-density tumors, and can be used to treat certain tumors or brain diseases through nanobodies; Second, they have good antigen specificity; Third, they are easy to genetically modify, facilitating artificial modification to obtain antibodies against different pathogens; Fourth, they have high stability. The time that nanobodies are not naturally decomposed in the body is longer than that of ordinary antibodies, and the drug effect time is more persistent. Nanobodies can even pass through the human stomach and remain effective.

[0006] Specific nanobodies are obtained by screening a nanobody phage library. Nanobody phage libraries are divided into immune libraries and non-immune libraries. Immune libraries are prepared by immunizing animals such as alpacas and camels with proteins. Non-immune libraries are prepared by randomly editing the variable region while retaining a certain constant region according to the structure of the constant region and variable region of nanobodies. When the library capacity of the nanobody library reaches 10 7 or more, specific nanobodies against antigens can be obtained. The use of non-immune libraries saves time and avoids the harm caused to animals by immunizing animals and collecting animal blood.

[0007] As a currently commonly used trypsin analogue, the importance of detecting the residual amount of TrypLE has become increasingly significant. At present, there is no enzyme-linked immunosorbent assay (ELISA) kit product for quantitatively detecting TrypLE with nanobody as the detection antibody at home and abroad, and the market potential is huge.

[0008] Due to its preparation method, TrypLE does not contain animal and human-derived components and generally has a high purity, which also results in its stronger specificity than natural trypsin preparations. In addition, TrypLE acts more gently on cells, and when terminating digestion, it does not require a Trypsin enzyme inhibitor. Only dilution with a diluent is needed to stop the reaction, which greatly reduces the interference of future substances on cultured cells. TrypLE has been proven to be able to decompose cells from media with or without serum supply.

[0009] As an enzyme extracted from animal bodies, trypsin inevitably has problems of large batch - to - batch differences and low purity. TrypLE, as a non - animal - sourced product through bioreactors, has high purity and small batch - to - batch differences, which also means its specificity is more significant. By comparing trypsin and TrypLE, it can be found that TrypLE has more superior performance in many aspects. Trypsin must be stored at - 20°C. After multiple freeze - thaw cycles or long - term storage at 4°C, its stability is lost. TrypLE can be stably stored at room temperature for 24 months, which not only saves freezer space but also provides the convenience of immediate availability. Some research shows that compared with trypsin, TrypLE improves the cloning survival and growth of human embryonic stem cells by 3 times, which makes it more conducive to cell growth and passage in cell culture. Through the above comparison, it can be seen that TrypLE is a very promising substitute for trypsin inhibitors. Summary of the Invention

[0010] The object of the present invention is to provide a llama - derived nanobody, a kit and an application that specifically bind to the trypsin analogue TrypLE. Specifically, it provides a llama - derived nanobody or its antigen - binding fragment that binds to TrypLE, a polynucleotide encoding it, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a preparation method thereof, a transformed cell, and a pharmaceutical composition containing the above.

[0011] The llama - derived nanobody or its antigen - binding fragment of the present invention is a nanobody with high neutralizing activity, has a strong binding ability to the TrypLE protein, can effectively recognize TrypLE. This nanobody has the advantages of small molecular weight, low immunogenicity, better solubility and stability, and a longer CDR region, providing potential application value for the detection of TrypLE.

[0012] To achieve the above object, the present invention provides the following technical solutions:

[0013] A llama - derived nanobody or its antigen - binding fragment that specifically binds to the trypsin analogue TrypLE, having a heavy - chain variable region VHH, wherein the VHH contains the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, CDR3 with an amino acid sequence as shown in SEQ ID NO:3. The VHH includes 4 framework regions of FR1 - 4, and the FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0014] The amino acid sequence of the FR1 framework region is shown in SEQ ID NO:4, the amino acid sequence of the FR2 framework region is shown in SEQ ID NO:5, the amino acid sequence of the FR3 framework region is shown in SEQ ID NO:6, and the amino acid sequence of the FR4 framework region is shown in SEQ ID NO:7.

[0015] The amino acid sequence of the heavy chain variable region is shown in the following SEQ ID NO:8, where: positions 1-25 are the FR1 framework region, positions 30-43 are the FR2 framework region, positions 60-91 are the FR3 framework region, positions 105-115 are the FR4 framework region, CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

[0016] A polynucleotide encoding a llama-derived nanobody or an antigen-binding fragment thereof that specifically binds to TrypLE as described above, and the sequence of the polynucleotide is shown in SEQ ID NO:9.

[0017] A nucleic acid construct comprising the polynucleotide.

[0018] An expression vector comprising the nucleic acid construct.

[0019] A transformed cell comprising the polynucleotide, nucleic acid construct or expression vector.

[0020] The nucleic acid construct further comprises at least one expression regulatory element operably linked to the polynucleotide, such as a histidine tag, a stop codon, etc.

[0021] A pharmaceutical composition containing the llama-derived nanobody or an antigen-binding fragment thereof that specifically binds to TrypLE and a pharmaceutical carrier.

[0022] Use of a llama-derived nanobody or an antigen-binding fragment thereof that specifically binds to TrypLE in the preparation of a kit for detecting TrypLE.

[0023] The present invention establishes an ELISA method for detecting TrypLE using a nanobody as a detection antibody, develops a kit, and realizes an effective, rapid and convenient detection of the content of TrypLE in a sample, so as to accumulate experience for establishing a detection method for other impurity proteins in biological products.

[0024] The present invention is directed to the development of nanobody drugs against TrypLE. After immunizing alpacas with the antigen, peripheral blood is collected, an antibody library is constructed, and high-affinity nanobodies that specifically bind to TrypLE are obtained by phage display technology. The obtained alpaca-derived nanobodies or their antigen-binding fragments are nanobodies with high neutralizing activity, have a strong binding ability to the TrypLE protein, can effectively detect TrypLE, and have the advantages of small molecular weight, low immunogenicity, better solubility and stability, and a longer CDR region, providing potential application value for TrypLE detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] One or more embodiments are illustrated by way of example in the corresponding drawings, and these exemplary illustrations do not limit the embodiments. The special word "exemplary" here means "serving as an example, embodiment, or illustrative". Any embodiment illustrated as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0026] Figure 1 It is a schematic diagram of the TrypLE-SDS-PAGE identification result of Example 1 of the present invention; in the figure: Line 1: Marker, Line 2-4: TrypLE;

[0027] Figure 2 It is a schematic diagram of the VHH fragment amplification result of Example 2 of the present invention; in the figure: A is the result of the first round of PCR; B is the result of the second round of PCR; Line 1-12: VHH nucleic acid fragments amplified from lymphocytes, Line 13: Marker;

[0028] Figure 3 It is a graph showing the library capacity of the TrypLE-VHH nanolibrary detected on a plate in Example 3 of the present invention;

[0029] Figure 4 It is a graph showing the library abundance of the TrypLE-VHH nanolibrary detected on a plate in Example 3 of the present invention;

[0030] Figure 5 It is a schematic diagram of the prokaryotic expression and purification results of TrypLE-VHH in Example 4 of the present invention; in the figure: Line 1: Marker, Line 2: Bacterial lysate of Escherichia coli expressed at 16°C, Line 3: Supernatant of Escherichia coli expressed at 16°C, Line 4: Precipitate of Escherichia coli expressed at 16°C, Line 5: Flow-through of Escherichia coli expression and purification, Line 6: Washing of Escherichia coli purification, Line 7-10: Elution of Escherichia coli purification;

[0031] Figure 6It is a schematic diagram of the purification result after the renaturation of TrypLE-VHH; in the figure: A is the antibody purified by nickel ion affinity chromatography; B is the antibody purified by gel filtration chromatography and the SDS-PAGE diagram;

[0032] Figure 7 It is the binding kinetic curve of TrypLE-VHH and TrypLE. Specific implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the protection scope of the present invention. In addition, to better illustrate the present invention, numerous specific details are given in the following specific implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, details of raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail to highlight the gist of the present invention. The present invention is described in detail below.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The materials cited herein and the materials they cite will be incorporated by reference.

[0035] Equivalent technologies of the specific implementation manners described that can be understood by those skilled in the art through conventional experiments will be included in this application.

[0036] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0037] I. Materials: Male alpacas, 2 years old, in good physical condition, are raised in the Alpaca Breeding Center of Shanxi Agricultural University. 293F cells, EcoRI and XhoI restriction endonucleases, lymphocyte separation medium, total RNA extraction kit, cDNA reverse transcription kit, Taq Green PCR Mix, PstⅠ and BstEⅡ restriction endonucleases.

[0038] II. Statistical method: In the immune effect detection experiment, the absorbance values of the sera of alpacas in each group are expressed as mean ± standard error (Means ± SE), and the data are tested by one-way analysis of variance using SPSS 16.0 statistical software.

[0039] III. Expression and purification of target protein, preparation of TrypLE antigen: The TRYPLE antigen was purchased from the Thermo Fisher website (https: / / www.thermofisher.cn / cn / zh / home. html) (LOT. A1217702) and stored at -80 °C for later use. The size of the TrypLE-his protein identified by SDS-PAGE was about 24 KD, and the results are as Figure 1 shown.

[0040] IV. Alpaca immunization and antibody library construction:

[0041] 1. Alpaca immunization and identification of immunization effect: Select adult male alpacas in good physical condition. Before immunization, 10 ml of blood was collected from the experimental alpacas, and the serum was separated and stored at -20 °C for later use. 100 μg of the purchased TrypLE protein was diluted with PBS to a final volume of 1 ml and subcutaneously injected into the alpacas at multiple points. For the first immunization, 1 ml of complete Freund's adjuvant was used for emulsification for 5 min. After that, immunization was carried out every two weeks with incomplete Freund's adjuvant. After the 3rd immunization, alpaca blood was collected and the serum was separated. The diluted TrypLE protein was added to the ELISA plate for coating. Blank wells (without adding any liquid) and negative wells (only adding coating solution) were set, and they were left standing overnight at 4 °C. After that, the coating solution was discarded, and after washing with PBS, 5% calf serum was added and incubated at 37 °C for 40 min. After the blocking was completed, the coated plate was washed, and the washing solution was blotted dry with filter paper for later use. The serially diluted serum was used as the primary antibody, and the HRP-labeled goat anti-alpaca antibody was used as the secondary antibody and added to the ELISA plate pre-coated with TrypLE protein in sequence for reaction. The pre-immunization serum was used as a blank control. After the reaction, TMB-hydrogen peroxide urea solution chromogenic solution was added, and after color development in the dark at 37 °C for 15 min, the termination solution was added to terminate the reaction. The absorbance was measured at a wavelength of 450 nm by an enzyme-linked immunosorbent assay reader within 15 min to detect the immunization effect of the alpacas.

[0042] The detection results of the TrypLE antigen protein activity are shown in Table 1: The antibody against TrypLE was not contained in the pre-immune serum, while the positive reaction in the post-immune whole serum was very strong and still had a positive reaction after dilution 100-fold. The results showed that after immunization with TrypLE protein, heavy-chain antibodies against TrypLE protein were produced in the alpacas and could be used for subsequent library construction.

[0043] Table 1: ELISA detection results of alpaca serum immunological activity

[0044]

[0045] 2. Isolation and library construction of alpaca peripheral blood lymphocytes (PBMC): On the 12th day after the 4th immunization, 50 - 60 mL of anticoagulated venous blood was collected from the jugular vein for the isolation of peripheral blood lymphocytes (PBMC). After mixing with PBS at a ratio of 1:1, it was slowly added to the lymphocyte separation medium and centrifuged at 2000 rpm and 20 °C for 20 minutes. The liquid of the PBMC layer was aspirated. Total RNA was extracted from the isolated PBMCs according to the instructions of the kit. Using the extracted total RNA as a template, cDNA was synthesized with random primer oligo-dT using the Superscript II First-Strand Synthesis System for RT-PCR kit. Using the cDNA as a template, the first-round PCR experiment was carried out with specific primers CALL001 and CALL002. The primer sequences and amplification temperatures of the first-round PCR reaction are shown in Table 2.

[0046] Table 2: Primer sequences and amplification temperatures of the first-round PCR reaction

[0047]

[0048] Among them, Call001-F corresponds to the Leader region of the alpaca antibody, and Call002-R corresponds to the second constant region (CH2) of the alpaca antibody. The 700bp nucleic acid fragment in the PCR reaction product was recovered as the template for the second-round PCR. The reverse transcription product was divided into 30 reactions, and each PCR reaction system was 50 ul. The continued PCR reaction system is shown in Table 3, and the continued PCR amplification reaction conditions are shown in Table 4.

[0049] Table 3: Continued PCR reaction system

[0050]

[0051] Table 4: Continued PCR amplification reaction conditions

[0052]

[0053] The DNA fragment was cut and recovered, and the 700bp nucleic acid fragment was purified using a gel recovery kit. 1 ul of the gel recovery product was ligated to the pMD19-T simple vector, and the reaction system is shown in Table 5.

[0054] Table 5: Reaction system

[0055]

[0056] The recycled nucleic acid fragments were ligated to the pMD19-T simple vector. The ligation products were transferred into DH5α competent cells, and monoclonal colonies were picked for determination of the base sequence. The specific method was as follows: The reaction system was vortexed instantaneously and reacted overnight at 4°C. The ligation products and DH5α competent cells were mixed evenly, incubated on ice for 25 min, heat shocked at 42°C for 90 s, incubated on ice for 4 min, 400 μl of LB medium was added and incubated at 37°C and 200 rpm for 40 min. 50 μl of the transformed bacterial solution was evenly spread on the surface of the LB solid medium containing AMP and cultured overnight at 37°C in an inverted position. The next day, 20 monoclonal colonies were picked and inoculated into 5 ml of LB liquid medium containing AMP and cultured overnight, and then their base sequences were determined.

[0057] The sequencing results were analyzed by Vector NTI software, and the second-round PCR primers VHH2-F and VHH2-R for library construction were designed. Using the 700 bp recycled fragment generated by the first-round PCR as a template, the VHH fragment was amplified with the primers VHH2-F and VHH2-R designed by sequencing.

[0058] The isolated lymphocytes after alpaca immunization were detected by a cell counter to be 3.6×10 7 cells, and the two PCR products were detected by agarose gel electrophoresis. The results of the first-round PCR were as Figure 2 shown in A. The VHH fragments of the conventional antibody (900 bp) and the heavy-chain antibody (700 bp) were amplified by PCR. The results of the second-round PCR were as Figure 2 shown in B: The product size was about 400 bp, which was consistent with the expectation.

[0059] The (VHHs) sequences of the nano-amplified heavy-chain antibody were amplified by the second-round PCR, and the VHHs sequences with a size of about 400 bp were recovered and purified. A 50 μl reaction system was used, and a total of 24 PCR reactions were performed. The specific method was as follows: The second-round PCR primers are shown in Table 2, and the reaction conditions for the second-round PCR amplification are shown in Table 6. The PCR reaction system was 2 μl of cDNA, 1 μl of each primer, 25 μl of Taq Green PCRMix, and 21 μl of deionized water.

[0060] Table 6: Reaction conditions for the second-round PCR amplification

[0061]

[0062] The VHHs fragment was ligated into plasmid pMES4 through the restriction enzyme sites PstⅠ and BstEⅡ, and then transferred into electrocompetent E. coli TG1 cells. After culturing, the bacterial liquid was centrifuged to discard the supernatant, and the precipitate was resuspended with fresh medium. 100 μl of the bacterial liquid was taken for calculating the library capacity, and the remaining bacterial liquid was spread on 20 2×YTAG solid culture plates. After overnight culture, the colonies were collected, which was the single-domain antibody library of TrypLE protein.

[0063] 3. Detection of the library capacity and abundance of antibodies

[0064] (1)The method for measuring the library capacity was as follows: The electrotransformed bacterial liquid was diluted in gradients of 10 -1 ~10 -8 ; 100 μl of each dilution was spread on a solid culture plate, and two plates were used for each gradient. The culture was carried out overnight at 25 °C. The next day, the colonies on the gradient plates were counted to calculate the library capacity, and the results were as Figure 3 shown. The library capacity was (3 + 4) ÷ 2 ÷ (100 × 10 -6 ) × 30 × 10 3 = 1.05 × 10 9 colonies.

[0065] (2)The method for measuring the library abundance was as follows: The primary library bacterial liquid was diluted in gradients from 10 -4 ~10 -10 ; 100 μl of each dilution of the bacterial liquid was spread on a solid culture plate, and the culture was carried out overnight at 25 °C. The next day, the colonies on the gradient plates were counted to calculate the library abundance. The results were as Figure 4 shown. The abundance of the library was 11 ÷ (100 × 10 -8 ) × 10 3 = 1.1 × 10 10 colonies / ml.

[0066] (3)The method for measuring the insertion rate and insertion diversity of the VHH fragment in the library was as follows: After the library capacity was determined, 34 monoclonal colonies were randomly selected from the solid culture plate for measuring the library capacity, and cultured overnight with shaking at 37 °C. A part of the collected bacterial liquid was used for PCR identification. By analyzing the PCR identification results and sequencing results, the fragment lengths of 34 bacterial liquid PCR samples were all consistent with the VHH fragment, and the insertion rate of the VHH fragment in the library was calculated to be 100%.

[0067] V. Screening, identification and expression of nanobodies: The coding sequence of 6 histidine tags and the translation termination codon TGA were ligated after the nucleotide sequence of the alpaca-derived nanobody shown in SEQ ID NO:9, and it was constructed into the pET23a plasmid through the restriction enzyme sites EcoRI and XhoI to construct an expression vector. The specific method was as follows:

[0068] Add the colonies collected in the previous step to the VCSM13 helper phage at a multiplicity of infection of 1:20. After overnight culture, centrifuge and collect the supernatant. Add PEG6000 / NaCl at a volume ratio of 1:4, place it at 4 °C for at least 1 hour, and then centrifuge for 30 min. Resuspend the precipitate after centrifugation with PBS, and the collected phage particles are obtained.

[0069] Mix the collected phages (1×10 10 pieces) with an equal volume of 5% (w / v) skim milk, add it to a 96-well plate coated with TrypLE antigen, incubate at room temperature for 1 hour, elute the specific phages with 0.2 M glycine, and then neutralize the eluted phages with Tris-HCl (pH 9.1). Infect E. coli TG1 cells with the eluted phages to amplify the phage single-chain antibody library. Perform the next round of panning in the same way, and a total of 3 rounds of phage panning are carried out.

[0070] Mix the screened eluate with the Escherichia coli culture in the early logarithmic growth phase, culture it in 2×YTAG liquid medium for 2 h, add the helper phage at a ratio of cell:phage = 1:20, culture overnight, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG-Nacl, mix well and place it at 4 °C for 3 h, centrifuge to remove the supernatant, resuspend the precipitate in 1 ml of PBS, centrifuge for 1 min. Randomly select 50 single colonies from the plate of the titer of the eluate from the last round of screening and inoculate them into 1 ml of 2×YTAG, shake and culture for 12 h, inoculate at an inoculation amount of 1% into 2×YTAG, culture until the early logarithmic growth phase, add the helper phage at a ratio of cell:phage = 1:1, culture overnight, centrifuge for 1 min, resuspend the precipitate in an equal volume of 2×YTAG, shake vigorously and culture for 12 h, centrifuge and collect the supernatant for ELISA identification. Positive clone judgment standard: The ratio (S / N) of the absorbance value (S) of the test sample to the absorbance value (N) of the negative control ≥ 2.1. Use specific primers MP57 and GⅢ to sequence the plasmid of the positive clone (the primers are shown in Table 7) to obtain the sequence encoding VHHs in its plasmid. Through sequence determination, the core coding sequence of the nanobody is obtained.

[0071] Table 7: Sequencing primer sequences

[0072]

[0073] Construct the obtained core coding sequence of the nanobody (NB) into the pcoldI plasmid, ligate the coding sequence of 6 histidine tags and the translation termination codon TGA after the nucleotide sequence of the alpaca-derived nanobody shown in SEQ ID NO:9, and construct it into the pET23a plasmid through the restriction enzyme sites EcoRI and XhoI to construct an expression vector.

[0074] Identification: Take 1 μL of the constructed pET23a plasmid containing the target gene and add it to 50 μL of Escherichia coli BL21(DE3) competent cells. After placing it on ice for 30 minutes, heat shock it in a 42°C water bath for 60 seconds. After placing it on ice for 5 minutes, add 450 μL of LB culture medium to the bacterial solution. After mixing, place it on a shaker at 37°C and shake it at 200 rpm for 1 hour for recovery. Then, pipette 200 μL of the bacterial solution and spread it on an LB+Amp solid plate. Invert the plate and place it in a 37°C incubator overnight. Pick monoclonal colonies from the overnight culture plate and inoculate them into 5 mL of LB+Amp culture medium. After shaking and culturing at 37°C and 200 rpm for 8 hours, transfer all the bacterial solution to 4 L of LB+Amp culture medium. When it grows to the logarithmic phase, add 1 mM IPTG for induction and culture it overnight at 16°C. The next day, centrifuge to collect the bacterial cell precipitate, resuspend it with an appropriate amount of 1×PBS, and perform ultrasonic disruption on it. The conditions are 112.5 w, disrupt for 3 seconds, and intermittent for 5 seconds. Then, centrifuge at 4°C and 12,000 rpm for 20 minutes, and collect the supernatant and precipitate after disruption respectively. Figure 5 As shown in

[0075] Purification: Construct the obtained core coding sequence of the nanobody (NB) into the pcoldI plasmid and transfect it into 293F cells for eukaryotic expression. Collect the supernatant after cell culture and purify it by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM75 Increase Hiload column (GE Healthcare)), and identify the purified protein by SDS-PAGE. The specific method is as follows:

[0076] Collect the inclusion bodies in the precipitate and dissolve them with 10 mmol / L Tris-HCl (pH 7.0, containing 8 mol / L urea, 1 mmol / L DTT), centrifuge to remove insoluble substances, and then prepare a renaturation buffer (100 mmol / L Tris-HCl pH 8.0, 400 mmol / L arginine, 5 mmol / L reduced glutathione, 0.5 mmol / L oxidized glutathione, 0.1 mmol / L PMSF) for dilution and renaturation. Analyze the renatured product by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM75 Increase Hiload column (GE Healthcare)). The results are as Figure 6 shown. The target peak is determined by SDS-PAGE to obtain a relatively pure nanobody. Nickel ion affinity chromatography ( Figure 6 A) and gel filtration chromatography ( Figure 6B) The results showed that a relatively pure target protein was obtained. The target peak was determined by SDS-PAGE, and the position of the protein band was consistent with the size of the target protein.

[0077] This antibody: has a heavy chain variable region VHH, and the VHH contains the following CDRs:

[0078] The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:8 below (ESGGGLVQAGGSLRLSCTASQAFGR NAIN WYRQAPGEQREWVA TITSGGVTSYAPAVKG RFAISRDSAKNTGYLQMNSLKVEDTAVYYCNS LWGGGSGHIPTDY WGQGTQVTVSS); among them: positions 1-25 are the FR1 framework region, positions 30-43 are the FR2 framework region, positions 60-91 are the FR3 framework region, positions 105-115 are the FR4 framework region, CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

[0079] CDR1 with the amino acid sequence shown as SEQ ID NO:1 (NAIN), CDR2 with the amino acid sequence shown as SEQ ID NO:2 (TITSGGVTSYAPAVKG), CDR3 with the amino acid sequence shown as SEQ ID NO:3 (LWGGGSGHIPTDY), the VHH includes 4 framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0080] The amino acid sequence of the FR1 framework region is shown as SEQ ID NO:4 (ESGGGLVQAGGSLRLSCTASQAFGR), the amino acid sequence of the FR2 framework region is shown as SEQ ID NO:5 (WYRQAPGEQREWVA), the amino acid sequence of the FR3 framework region is shown as SEQ ID NO:6 (RFAISRDSAKNTGYLQMNSLKVEDTAVYYCNS), and the amino acid sequence of the FR4 framework region is shown as SEQ ID NO:7 (WGQGTQVTVSS).

[0081] The sequence of the polynucleotide encoding the alpaca-derived nanobody that specifically binds to TrypLE is shown as SEQ ID NO:9.

[0082] 6. ELISA detection of nanobody activity: Use ELISA plates pre-coated with TrypLE protein, dilute purified NB as the primary antibody, and use HRP-labeled llama as the secondary antibody to perform ELISA reaction to detect nanobody activity. After the reaction is completed, add TMB-hydrogen peroxide urea solution colorimetric solution, color at 37°C in the dark for 15 minutes, and then add stop solution to terminate the reaction. Measure the absorbance value within 15 minutes at a wavelength of 450nm using an enzyme reader.

[0083] The results of ELISA detection of specific binding of nanoantibodies to TrypLE protein are shown in Table 8. The NB nanoantibody has a positive reaction with the TrypLE antigen protein, indicating that the obtained NB nanoantibody has good immune activity.

[0084] Table 8: Neutralization effect of nanobodies on TrypLE antigen

[0085]

[0086] VII. Antigen and nanobody affinity experiment: NTA chip was used to fix the TrypLE protein on the chip. The fixed amount was about 100 RU. TrypLE-VHH protein was diluted with PBST buffer in multiple ratios and loaded one by one from low concentration to high concentration. The kinetic curve of antibody binding to TrypLE protein is shown in Figure 7 shown.

[0087] The equilibrium dissociation constant (KD) between TrypLE-VHH and TrypLE is less than 0.1 nM, which proves that TrypLE-VHH nanobody can bind to TrypLE with high affinity.

[0088] Since its discovery, nanobodies have become a research hotspot in the field of infectious diseases, tumors and immune diseases due to their unique properties compared to ordinary antibodies, such as high water solubility and structural stability, strong antigen affinity, and easy in vitro expression and humanization modification, etc., combined with phage display technology. Nanobodies can achieve the purpose of preventing diseases by binding to key proteins of pathogenic microorganisms, controlling the source of infection or cutting off the transmission route.

[0089] The present invention uses TrypLE protein to immunize alpacas and constructs a reservoir with a capacity of 1.05× 10 9 CFU / ml phage display antibody library, after three rounds of immune selection, identified a high-affinity anti-TrypLE protein nanoantibody NB1, which can effectively bind to TrypLE protein. Subsequently, multivalent nanoantibodies can be constructed based on NB1 to enhance the binding effect on TrypLE. The present invention lays a foundation for the detection and application of TrypLE by screening high-affinity anti-TrypLE nanoantibodies.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alpaca-derived nanobody or its antigen-binding fragment that specifically binds to the trypsin analogue TrypLE, having a heavy chain variable region VHH, characterized in that: The VHH comprises the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, CDR3 with an amino acid sequence as shown in SEQ ID NO:

3. The VHH includes four framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

2. The alpaca-derived nanobody or its antigen-binding fragment that specifically binds to the trypsin analogue TrypLE according to claim 1, characterized in that: The amino acid sequence of the FR1 framework region is as shown in SEQ ID NO:4, the amino acid sequence of the FR2 framework region is as shown in SEQ ID NO:5, the amino acid sequence of the FR3 framework region is as shown in SEQ ID NO:6, and the amino acid sequence of the FR4 framework region is as shown in SEQ ID NO:

7.

3. The alpaca-derived nanobody or its antigen-binding fragment that specifically binds to the trypsin analogue TrypLE according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is as shown in the following SEQ ID NO:8, wherein: positions 1-25 are the FR1 framework region, positions 30-43 are the FR2 framework region, positions 60-91 are the FR3 framework region, positions 105-115 are the FR4 framework region. CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

4. A polynucleotide encoding an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to TrypLE as described in any one of claims 1-3, and the sequence of the polynucleotide is as shown in SEQ ID NO:

9.

5. A nucleic acid construct comprising the polynucleotide described in claim 4.

6. An expression vector comprising the nucleic acid construct described in claim 5.

7. A transformed cell comprising the polynucleotide described in claim 4, the nucleic acid construct described in claim 5 or the expression vector described in claim 6.

8. The nucleic acid construct according to claim 5, characterized in that: The nucleic acid construct further comprises at least one expression regulatory element operably linked to the polynucleotide.

9. Use of an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to TrypLE as described in any one of claims 1-3 in the preparation of a kit for detecting TrypLE.

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