Nanobodies specifically binding to pancreatic kallikrein and uses thereof

Abstract

The application provides a kind of nanobody specifically binding to pancreatic kallikrein, wherein the nucleotide sequence of the nanobody is as shown in any one of SEQ ID No.1, 3, 5.The application also provides corresponding applications.The application successfully screens and identifies anti-pancreatic kallikrein nanobody with good expression activity, uses pichia pastoris system to express and produce, can greatly reduce production cost, and is more conducive to the industrialization of the antibody;It also successfully constructs the purification filler using anti-pancreatic kallikrein nanobody as accessory, and lays a good foundation for subsequent purification of pancreatic kallikrein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical or biopharmaceutical technology, specifically relating to a nanobody that specifically binds to kallikrein and its applications. Background Technology

[0002] Pancreatic kallikrein is a proteolytic enzyme primarily extracted from the pancreas of pigs. It is mainly composed of four sugars and 18 amino acids. It is a kallikrein-releasing enzyme found in human tissues and mammals, and is an important member of the kallikrein-kinin system. In vivo, it exists as an inactive precursor, kallikreinogen, which degrades kallikreinogen into kinins to exert its pharmacological effects. These effects include dilating blood vessels and capillaries, increasing vascular permeability, improving microcirculation, and regulating blood pressure; reducing myocardial oxygen consumption; and stimulating... Active phospholipase A2 promotes the secretion of prostaglandin E2 from the renal medulla, inhibits oxidative stress, and increases blood flow; it also promotes the secretion of prostacyclin (PGI2), inhibits the formation of thromboxane (TXA2), avoids excessive platelet aggregation, reduces blood viscosity, prevents thrombosis, and avoids aggravating renal microcirculatory damage; by activating the kinin system, it dilates the glomerular arterial wall and capillaries, which can significantly improve microcirculation, restore renal capillary function, thereby inhibiting mesangial cell proliferation and basement membrane thickening, and delaying the progression of diabetic nephropathy.

[0003] Nanobodies are a class of monoclonal antibodies derived from camels, possessing only a variable region structure. Their molecular weight is approximately 15 kDa, about one-tenth that of conventional antibodies. Compared to conventional monoclonal antibodies, nanobodies offer advantages such as high tissue permeability, high stability, high solubility, low aggregation, and ease of cloning. While conventional monoclonal antibodies are mostly produced through mammalian cell culture, nanobodies can be produced using microbial fermentation systems, significantly shortening development time and reducing production costs. Therefore, nanobodies offer numerous advantages for downstream development and application.

[0004] Currently, the mature purification process for kallikrein mainly involves a multi-step organic solvent precipitation method, which has low production efficiency, consumes a lot of manpower, material resources and organic solvents, and there is no affinity purification method for kallikrein on the market. Summary of the Invention

[0005] The main objective of this invention is to address the problems in the prior art by providing a nanobody that specifically binds to pancreatic kallikrein and its applications.

[0006] To achieve the above objectives, the present invention provides a nanobody that specifically binds to kallikrein, characterized in that the nucleotide sequence of the nanobody is as shown in any one of SEQ ID No. 1, 3, or 5.

[0007] Another aspect of the present invention provides a nanobody that specifically binds to kallikrein, characterized in that the amino acid sequence of the nanobody is as shown in any one of SEQ ID No. 2, 4, or 6.

[0008] The present invention also provides a recombinant vector, the main feature of which is that it includes a nucleotide sequence encoding the nanobody described above.

[0009] The present invention also provides a recombinant bacterium, the main feature of which is that it includes the recombinant vector, such as yeast cells.

[0010] The present invention also provides the use of the nanobody that specifically binds to kallikrein, the main feature of which is for purifying kallikrein; or for detecting the level of kallikrein.

[0011] The present invention also provides a chromatography packing material, the main feature of which is that the chromatography packing material includes a phase-coupled base medium and a ligand, wherein the ligand is the nanoantibody.

[0012] Preferably, the base medium is agarose microspheres, polystyrene-divinylbenzene microspheres, or polymethacrylate microspheres.

[0013] The present invention also provides a method for purifying kallikrein, the main feature of which is that the kallikrein is subjected to immunoaffinity chromatography using the aforementioned chromatographic packing material.

[0014] The present invention also provides a kit, the main feature of which is that the kit contains the nanobody described above, for detecting the level of kallikrein. Attached Figure Description

[0015] Figure 1 The enrichment results are shown for the nanobody library in Example 1.

[0016] Figure 2 The image shows the binding activity results of the EC50 nanobody.

[0017] Figure 3 This is a schematic diagram of the fermentation process conditions in Example 2.

[0018] Figure 4 This shows the protein expression status during the fermentation process.

[0019] Figure 5 SDS-PAGE electrophoresis image of anti-kallikrein nanobody purified by nickel column.

[0020] Figure 6 SDS-PAGE electrophoresis image of trypsinogenase eluted for affinity purification. Detailed Implementation

[0021] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0022] This invention provides an anti-kallikrein nanobody that can specifically bind to kallikrein and can be used as an affinity ligand in chromatography packing materials; or, in ELISA kits, etc.

[0023] The anti-kallikrein nanobody provided by this invention can be combined with various microsphere carriers to form a chromatography packing material, which can be used to purify kallikrein, capture kallikrein in one step, effectively save costs, and reduce the loss of activity in multiple steps.

[0024] The chromatographic packing material provided by this invention includes a phase-coupled base medium and a ligand, wherein the ligand is the anti-kallikrein nanobody. The specific synthesis method includes the following steps:

[0025] 1) Prepare microspheres with hydroxyl-rich surfaces as the base medium. You can use any one of the following: agarose microspheres, hydrophilically treated polystyrene-divinylbenzene microspheres (PS-DVB), or acid-treated polymethyl methacrylate microspheres (PMMA).

[0026] 2) Activation: The surface of the basic microspheres is modified to obtain an activation medium rich in epoxy groups;

[0027] 3) Couple the anti-kallikrein nanobody to the activation medium;

[0028] 4) The excess epoxy groups are blocked to obtain anti-kaloplasm nanobody chromatography packing material.

[0029] Example 1

[0030] Antibody library preparation

[0031] Purchase the national standard of kallikrein as the antigen. Mix the high-purity antigen with an equal volume of immune adjuvant and immunize one Bactrian camel from Xinjiang. Immunize the camel according to the strategy in Table 1. After 6 immunizations, collect peripheral blood from the camel, separate peripheral blood mononuclear cells from the peripheral blood, and take approximately 1×10⁻⁶ cells. 7 Cells were transferred to 1.5 mL centrifuge tubes, 1 mL of TRIZOL Reagent was added, total RNA was extracted, and cDNA was synthesized by reverse transcription using the Reverse Transcription System kit. The gene of the nanobody was amplified by reverse transcription and PCR, and then cloned into the phage vector pMECS. The gene was then transformed into TG1 host cells to construct a phage display library.

[0032] Table 1

[0033]

[0034] Subsequently, phage display technology was used for library screening. After four rounds of "adsorption-washing-enrichment" screening, 400 single clones were selected for positive clone identification after the first screening, resulting in 70 sequence-differential positive clones. The results are as follows: Figure 1 As shown.

[0035] Seventy phage clones were randomly selected from the enriched phage clones for two batches of antigen identification. Twelve double-positive clones were purified through prokaryotic expression and EC50 binding activity verification. Figure 2 As shown.

[0036] Depend on Figure 2 It was found that YTM Nb 3-11 did not bind; the remaining antibodies all bound to the kallikrein antigen to varying degrees. Among them, YTM Nb 1-30, YTM Nb 1-70, YTM Nb 1-77, YTM Nb 2-13, YTM Nb 2-34, YTM Nb 2-41, YTM Nb 3-52 (nucleotide sequence as shown in SEQ ID No. 1, amino acid sequence as shown in SEQ ID No. 2), YTM Nb 1-96 (nucleotide sequence as shown in SEQ ID No. 3, amino acid sequence as shown in SEQ ID No. 4), and YTM Nb 3-17 (nucleotide sequence as shown in SEQ ID No. 5, amino acid sequence as shown in SEQ ID No. 6) showed better binding activity, while YTM Nb 2-06 and YTM Nb 3-71 showed weaker binding activity. Based on the EC50 value, nine sequences with better activity were selected for subsequent experiments, including sequencing and gene synthesis.

[0037] Example 2

[0038] Pichia pastoris expression

[0039] Nine highly active kallikrein nanobody sequences were linked together to form the following bivalent structure.

[0040] HHHHHH-YTM-GGGGSGGGS-YTM-CC

[0041] The N-terminal six histidine sequence is used for nickel column affinity purification of the tag, the middle is the GGGGSGGGS flexible linker sequence, and the C-terminus is the 2-cysteine ​​CC sequence. MTY is the kallikrein nanobody sequence.

[0042] (1) Construct the above sequences into the pPICZaA vector respectively;

[0043] (2) Linearized with Sac I restriction endonuclease and then electroporated into X-33 competent cells;

[0044] (3) Spread the electroporated samples onto YPD plates containing different concentrations of bleomycin resistance and incubate them in a 30°C incubator for 3-4 days.

[0045] (4) After single colonies grow on the plate culture medium, pick single colonies from plates of different concentrations and place them in BMGY medium. When the OD value of BMGY culture medium reaches about 20, collect the cells and replace them in BMMY medium. Incubate at 28℃ and 250rpm.

[0046] (5) Samples were taken at intervals thereafter and methanol was added to a final volume of 1%; the samples were centrifuged at 12,000 rpm for 5 min, the supernatant was collected and stored at -20℃; the induction was continued for 5 days and the culture was then terminated.

[0047] (6) Perform SDS-PAGE analysis on the obtained supernatant sample.

[0048] Nine highly active trypsinogenase nanobody sequences were expressed and produced using a Pichia pastoris system. The protein expression level increased continuously over time, significantly reducing production costs and facilitating the industrialization of this antibody. The results for YTM Nb 1-30 are provided as an illustration; specifically, the fermentation conditions are as follows... Figure 3 As shown, the intermediate sampling expression (sampling times 23h, 38h, 45h, 62h, 71h, 86h) is as follows: Figure 4 As shown in the electrophoresis diagram of the sampled expression, there were no protein bands at the 23h sampling point. After the addition of the inducing agent, the amount of protein expression increased continuously with time.

[0049] Example 3

[0050] Nickel column purification of anti-kaloplasm nanobodies

[0051] The nine fermentation supernatants from Example 2 were filtered through a 0.8 μm filter membrane, and the filtrates were purified by nickel column chromatography (the nanobody construction contained the His tag).

[0052] After mixing the homemade nickel column packing material, add it to the chromatography column and let it stand at room temperature for 10 minutes (the thickness of the packing material is about 2 ml). After the gel and solution separate into layers, open the outlet at the bottom and let the ethanol flow out slowly by gravity.

[0053] After rinsing the packed column with 10 ml of deionized water to remove ethanol, equilibrate the column with 20 ml of 10 mM Binding Buffer. Once equilibrated, the sample can be loaded.

[0054] Purification of soluble proteins

[0055] 1) Load 5 ml of sample at a flow rate of 1 ml / 3 min and collect the flow-through solution.

[0056] 2) Rinse the column with 30ml of 10mM Binding Buffer to remove impurities and proteins.

[0057] 3) Elute with an appropriate amount of Elution Buffer for 1 ml / 3 min and collect the elution peak.

[0058] 4) After elution, wash the column with 20 ml of deionized water, then equilibrate with 6 ml of 20% ethanol (the ethanol should completely submerge the packing material), seal the column, and store at 2-8℃.

[0059] The anti-kallikrein nanobody purified by nickel column was subjected to SDS-PAGE electrophoresis. The results for YTM Nb 1-30 are provided as an illustration, specifically as follows: Figure 5 As shown.

[0060] Example 4

[0061] Synthesis of affinity chromatography packing materials

[0062] 1) Prepare microspheres with hydroxyl-rich surfaces as the base medium. You can use any one of the following: agarose microspheres, hydrophilically treated polystyrene-divinylbenzene microspheres (PS-DVB), or acid-treated polymethyl methacrylate microspheres (PMMA).

[0063] 2) Activation: The surface of the basic microspheres is modified to obtain an activation medium rich in epoxy groups;

[0064] The method for epoxy modification of a base medium rich in hydroxyl groups is as follows: Mix the base medium with an equal volume of 0.1-1 mol / L sodium hydroxide solution, add an equal volume of epoxy reagent to the medium under stirring, stir and react at 20-40℃ for 16-24 h, and wash away excess reagent with ethanol and water in sequence to obtain an activated medium rich in epoxy groups on the surface.

[0065] Epoxy reagents can be epichlorohydrin, allyl glycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, or glycerol triglycidyl ether, etc.

[0066] 3) Anti-kallikrein nanobodies were coupled to the activation medium respectively;

[0067] The method for conjugating nanobodies is as follows: The purified nanobodies from Example 3 are dissolved in 0.05-0.2 mol / L phosphate buffer containing 0.5-1.5 mol / L sodium sulfate at pH 6-8, with a nanobodies concentration of 0.5-20 mg / ml; the epoxy-modified activation medium is mixed with the nanobodies solution at a ratio of 1:5, and the mixture is stirred at 18-40°C for 8-24 hours. After the reaction is completed, the medium is filtered and washed 5 times with pure water.

[0068] 4) The excess epoxy groups are blocked to obtain anti-kallikrein nanobody chromatography packing material;

[0069] The method for blocking excess epoxy groups is as follows: Mix the medium for conjugating nanobodies with an equal volume of aqueous ethanolamine solution (concentration of 0.1-1.0 mol / L, pH 8.5-11.5), stir and react at 20-40℃ for 4-16 hours, filter the medium after reaction, wash it 5 times with pure water, and finally store it in 20% ethanol at 2-8℃.

[0070] Affinity packing material using kallikrein nanobodies as ligands can be made into analytical columns or preparative columns for the analysis and purification of kallikrein, or into ELISA kits for detecting kallikrein levels.

[0071] Example 5

[0072] Purification of pancreatic kallikrein

[0073] 1) Preparation: Pack the anti-kaloplasm nanobody affinity chromatography packing material prepared in Example 4 into 5 mL columns;

[0074] 2) Equilibration: Equilibrate the column with 10 times its volume of loading buffer to make the column conditions suitable for loading.

[0075] 3) Sample loading: The kallikrein solution is fed into the packing column at a rate of 1 ml / min using a sample pump. The kallikrein protein will bind to the nanobodies in the packing.

[0076] 4) Washing: Wash with 5 times the volume of equilibration buffer to remove impurities and proteins until the UV280 baseline is leveled.

[0077] 5) Elution: Elute using elution buffer, collect the solution at which the UV280 peak occurs, and stop collecting when the UV280 baseline flattens.

[0078] 6) Regeneration: Clean the column with 5 times its volume of equilibration fluid and store at 4 degrees Celsius.

[0079] Results: Analysis of the purification chromatogram.

[0080] The purified and eluted kallikrein was subjected to SDS-PAGE electrophoresis, demonstrating that each affinity chromatography packing material could adsorb kallikrein in one step, yielding a certain amount of purified kallikrein protein. The results for YTM Nb 1-30 are provided as an illustration, specifically as follows: Figure 6 As shown.

[0081] Therefore, the nanobody that specifically binds to kallikrein provided by the present invention, and the anti-kallikrein nanobody affinity chromatography packing material formed by the combination of the antibody and microspheres, can effectively bind kallikrein in one step, solving the problem of low efficiency in current purification processes.

[0082] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. A nanobody that specifically binds to kallikrein, characterized in that, The amino acid sequence of the nanobody is shown in any one of SEQ ID No. 2, 4, or 6.

2. A recombinant vector, characterized in that, Includes nucleotides encoding the nanobody of claim 1.

3. A recombinant bacterium, characterized in that, Includes the recombinant vector as described in claim 2.

4. The use of the nanobody that specifically binds to kallikrein according to claim 1, characterized in that, Used to purify pancreatic kallikrein.

5. A chromatography packing material, characterized in that, The chromatography packing material comprises a phase-coupled base medium and a ligand, wherein the ligand is the nanobody described in claim 1.

6. The chromatography packing material according to claim 5, characterized in that, The base medium is agarose microspheres, polystyrene-divinylbenzene microspheres, or polymethacrylate microspheres.

7. A method for purifying kallikrein, characterized in that, Immunoaffinity chromatography of kallikrein was performed using the chromatographic packing material described in claim 5.

8. A reagent kit, characterized in that, The kit contains the nanobody as described in claim 1.

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