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] The present application belongs to the field of biomedical or biopharmaceutical technology, and particularly relates to a nanobody specifically binding to pancreatic kallikrein and application thereof. BACKGROUND

[0002] Pancreatic kallikrein is a proteolytic enzyme drug, which is currently mainly extracted from the pancreas of pigs and mainly composed of 4 sugars and 18 amino acids. It is a kind of kallikrein in human tissues and mammals, and is an important member of the kallikrein-kinin system in the body. It exists in the form of inactive precursor-kallikrein in the body, can degrade kallikrein into kallikrein to exert pharmacological effects, can dilate blood vessels and capillaries, increase vascular permeability, improve microcirculation, regulate blood pressure, etc.; can reduce myocardial oxygen consumption; can activate phospholipase A2, promote the secretion of prostaglandin E2 in the renal medulla, inhibit oxidative stress, increase blood flow; promote the secretion of prostacyclin (PGI2), inhibit the generation of thromboxane (TXA2), avoid excessive aggregation of platelets, reduce blood viscosity, prevent thrombosis, and avoid aggravating the damage of renal microcirculation; by activating the kallikrein system, dilating the glomerular arteriolar wall and capillary, the microcirculation can be significantly improved, and the function of renal capillary can be restored, thereby inhibiting the proliferation of mesangial cells and the thickening of the basement membrane, and delaying the progression of diabetic nephropathy.

[0003] Nanobody is a kind of monoclonal antibody with only variable region structure derived from Camelidae, and the molecular weight is about 15KD, which is one tenth of the conventional antibody. Compared with the conventional monoclonal antibody, the nanobody has the advantages of high tissue permeability, high stability, high solubility, low aggregation and easy cloning. Most of the conventional monoclonal antibodies are cultured and produced by mammalian cells, while the nanobody can be produced by microbial fermentation, which can greatly shorten the development time and significantly reduce the production and development cost. Therefore, the nanobody has many advantages for downstream development and application.

[0004] At present, the mature purification process of pancreatic kallikrein mainly adopts the method of multi-step organic solvent precipitation, which has low production efficiency, consumes a large amount of manpower, material resources and organic solvents, and there is no affinity purification method for pancreatic kallikrein on the market. SUMMARY

[0005] The main purpose of the present application is to provide a nanobody specifically binding to pancreatic kallikrein and application thereof aiming at the problems in the prior art.

[0006] In order to achieve the above-mentioned purpose, the present application provides a nanobody specifically binding to pancreatic kallikrein, and the main feature is that the nucleotide sequence of the nanobody is shown in any one of SEQ ID No. 1, 3, 5.

[0007] The present application provides a Nanobody specifically binding to pancreatic kallikrein, wherein the amino acid sequence of the Nanobody is shown in any one of SEQ ID No. 2, 4, 6.

[0008] The present application also provides a recombinant vector, wherein the nucleotide sequence encoding the Nanobody is comprised.

[0009] The present application also provides a recombinant bacterium, wherein the recombinant vector is comprised, such as a yeast cell.

[0010] The present application also provides the use of the Nanobody specifically binding to pancreatic kallikrein, wherein the Nanobody is used for purifying pancreatic kallikrein; or, detecting the level of pancreatic kallikrein.

[0011] The present application also provides a chromatography packing material, wherein the chromatography packing material comprises a base medium and a ligand coupled to the base medium, and the ligand is the Nanobody.

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

[0013] The present application also provides a method for purifying pancreatic kallikrein, wherein the chromatography packing material is used for immunoaffinity chromatography of pancreatic kallikrein.

[0014] The present application also provides a kit, wherein the kit comprises the Nanobody, and is used for detecting the level of pancreatic kallikrein. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is the enrichment result of the Nanobody library in Example 1.

[0016] Figure 2 Figure 2 is the result graph of the Nanobody EC50 binding activity.

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

[0018] Figure 4 Figure 4 is the protein expression of the sampling in the fermentation process.

[0019] Figure 5 Figure 5 is the SDS-PAGE electrophoresis diagram of the anti-pancreatic kallikrein Nanobody purified by the nickel column.

[0020] Figure 6 Figure 6 is the SDS-PAGE electrophoresis diagram of the pancreatic kallikrein eluted by affinity purification. DETAILED DESCRIPTION

[0021] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further described below in combination with specific embodiments.

[0022] The present application provides an anti-pancreatic kallikrein nanobody, which can specifically bind to pancreatic kallikrein and can be used as an affinity ligand for chromatography filler.

[0023] The anti-pancreatic kallikrein nanobody provided by the present application is combined with various microsphere carriers to form a chromatography filler, which can be used for purifying pancreatic kallikrein, one-step capturing pancreatic kallikrein, effectively saving cost, and reducing the loss of activity in multiple steps.

[0024] The chromatography filler provided by the present application comprises a basic medium and a ligand coupled thereto, and the ligand is the anti-pancreatic kallikrein nanobody. The specific synthesis method comprises the following steps:

[0025] 1) Prepare a microsphere with a surface rich in hydroxyl groups as a basic medium, which can be any one of agarose microspheres, hydrophilic treated polystyrene-divinylbenzene microspheres (PS-DVB), and acid treated polymethacrylate microspheres (PMMA);

[0026] 2) Activation: surface modification of the basic microspheres to obtain an activated medium rich in epoxy groups;

[0027] 3) Coupling of the anti-pancreatic kallikrein nanobody to the activated medium;

[0028] 4) Excess epoxy group blocking treatment to obtain an anti-pancreatic kallikrein nanobody chromatography filler.

[0029] Example 1

[0030] Antibody library preparation

[0031] A national standard pancreatic kallikrein was purchased as an antigen, and a high-purity antigen was mixed with an equal volume of immunoadjuvant to immunize one Xinjiang two-humped camel. The camel was immunized according to the strategy in Table 1, and peripheral blood was extracted from the camel after 6 immunizations. Peripheral blood mononuclear cells were separated from the peripheral blood of the camel, and about 1×10 7 Cells were added to a 1.5 mL centrifuge tube, 1 mL of TRIZOL Reagent was added, total RNA extraction was performed, Reverse Transcription System kit was used for reverse transcription to synthesize cDNA, and the gene of the nanobody was amplified by reverse transcription and PCR, and then cloned into the phage vector pMECS, and transformed into TG1 host cells to construct a phage display library.

[0032] Table 1

[0033] A national standard pancreatic kallikrein was purchased as an antigen, and a high-purity antigen was mixed with an equal volume of immunoadjuvant to immunize one Xinjiang two-humped camel. The camel was immunized according to the strategy in Table 1, and peripheral blood was extracted from the camel after 6 immunizations. Peripheral blood mononuclear cells were separated from the peripheral blood of the camel, and about 1×10

[0034] Subsequently, the library screening was performed by using the phage display technology, and after 4 rounds of screening process of "adsorption-washing-enrichment", 400 single clones were selected for positive clone identification after the first screening, and 70 sequence difference positive clones were obtained in total, and the results are shown in Figure 1 .

[0035] From the above enriched phage clones, 70 strains were randomly selected for 2 batches of antigen identification, 12 double positive clones were subjected to prokaryotic expression and purification, and EC50 binding activity verification was performed as shown in Figure 2 .

[0036] As shown in Figure 2 , YTM Nb 3-11 does not bind; the rest of the antibodies bind to the tryptase antigen to varying degrees, among which YTM Nb 1-30 (nucleotide sequence as shown in SEQ ID No. 1, amino acid sequence as shown in SEQ ID No. 2), YTM Nb 1-70 (nucleotide sequence as shown in SEQ ID No. 3, amino acid sequence as shown in SEQ ID No. 4), YTM Nb 1-77 (nucleotide sequence as shown in SEQ ID No. 5, amino acid sequence as shown in SEQ ID No. 6), YTM Nb 2-13, YTM Nb 2-34, YTM Nb 2-41, YTM Nb 3-52, YTM Nb 1-96, YTM Nb 3-17 have relatively high binding activity, and YTM Nb 2-06, YTM Nb 3-71 have relatively weak binding activity. According to the value of EC50, nine sequences with relatively high activity were selected for subsequent experiments including sequencing and gene synthesis.

[0037] Example 2

[0038] Pichia pastoris expression

[0039] Nine sequences of tryptase nanobodies with relatively high activity were connected into the following bivalent structure,

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

[0041] The N-terminal 6 histidine sequences are used as a nickel column affinity purification tag, the middle is a GGGGSGGGS flexible linker sequence, and the C-terminal is a 2 cysteine CC sequence. MTY is a tryptase nanobody sequence.

[0042] (1) The above sequences were respectively constructed into pPICZaA vectors;

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

[0044] (3) The electrotransformed samples were spread on YPD plates containing different concentrations of zeocin, and incubated in a 30°C incubator for 3-4 days;

[0045] (4) After single colonies grew on the plates, the single colonies on plates with different concentrations were picked and placed in BMGY medium. When the OD value of the BMGY culture reached about 20, the bacterial cells were collected and replaced in BMMY medium, and incubated at 28°C and 250 rpm;

[0046] (5) Then samples were taken at intervals, and 1% methanol was added to the final volume. The samples were centrifuged at 12000 rpm for 5 min, and the supernatant was taken and stored at -20°C. The induction was continued for 5 days, and the culture was ended;

[0047] (6) The supernatant samples taken were subjected to SDS-PAGE detection.

[0048] Nine active and optimal pancreatic kallikrein nanobody sequences were expressed and produced by using the Pichia pastoris system. With the increase of time, the amount of protein expression increased, which greatly reduced the production cost and was more conducive to the industrialization of the antibody. Among them, the results of YTM Nb 1-30 are provided as an illustration, and specifically, the fermentation process conditions are as shown in Figure 3 , and the expression conditions of the intermediate sampling (sampling time 23h, 38h, 45h, 62h, 71h, 86h) are as shown in Figure 4 . From the electrophoretogram of the sampling expression condition, it can be seen that there is no protein band at the 23h sampling point, and then the inducer is added. With the increase of time, the amount of protein expression increases.

[0049] Example 3

[0050] Nickel column purification of anti-pancreatic kallikrein nanobody

[0051] The nine fermentation supernatants in Example 2 were filtered with 0.8um filter membrane, and the filtrate was purified by nickel column (the nanobody contained his tag when constructed).

[0052] After the self-made nickel column filler was mixed, it was added to the chromatography column and placed at room temperature for 10 minutes (the thickness of the filler was about 2ml). After the gel and the solution were layered, the outlet at the bottom was opened, and ethanol slowly flowed out by gravity;

[0053] After the column was filled, 10ml of deionized water was added to wash away the ethanol, and then 20ml of 10mM Binding Buffer was used to balance the column. After the balance was completed, the sample was loaded.

[0054] Purification of soluble protein

[0055] 1) Load 5 ml at a flow rate of 1 ml / 3 min, collect flow through.

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

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

[0058] 4) After elution, wash the column with 20 ml of deionized water, and equilibrate with 6 ml of 20% ethanol (the ethanol should immerse the filler), and store the column at 2-8 °C after sealing.

[0059] SDS-PAGE electrophoresis of the anti-tryptase nanobodies purified by nickel column. The results of YTM Nb 1-30 are provided as an illustration, specifically, as shown in Figure 5 .

[0060] Example 4

[0061] Synthesis of affinity chromatography filler

[0062] 1) Prepare the microspheres rich in hydroxyl groups as the base medium, any one of agarose microspheres, hydrophilic treated polystyrene-divinylbenzene microspheres (PS-DVB), acid treated polymethacrylate microspheres (PMMA) can be used;

[0063] 2) Activation: modify the surface of the base microspheres to obtain activated medium rich in epoxy groups;

[0064] The method for modifying the base medium rich in hydroxyl groups with epoxy groups is as follows: mix the base medium with 0.1-1 mol / L sodium hydroxide solution in equal volume, add equal volume of epoxy reagent to the medium under stirring at 20-40 °C for 16-24 h, and then wash the excess reagent with ethanol and water in sequence, to obtain the activated medium rich in epoxy groups.

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

[0066] 3) Coupling the anti-tryptase nanobodies to the activated medium, respectively;

[0067] The method for coupling the nanobodies is as follows: the purified nanobodies in Example 3 are dissolved in 0.05-0.2 mol / L phosphate buffer containing 0.5-1.5 mol / L sodium sulfate, pH 6-8, and the concentration of the nanobodies is 0.5-20 mg / ml; the activated medium with epoxy modification is mixed with 1:5 volume of the nanobody solution, and the mixture is stirred at 18-40°C for 8-24 h, after which the medium is filtered, and the filter is washed with pure water 5 times.

[0068] 4) The excess epoxy groups are blocked to obtain the nanobody chromatography filler for anti-pancreatic kallikrein;

[0069] The method for blocking the excess epoxy groups is as follows: the medium with coupled nanobodies is mixed with an equal volume of an aqueous ethanolamine solution (concentration: 0.1-1.0 mol / L, pH 8.5-11.5) uniformly, and the mixture is stirred at 20-40°C for 4-16 h, after which the medium is filtered, and the filter is washed with pure water 5 times, and finally the product is stored in 20% ethanol at 2-8°C.

[0070] The affinity filler with nanobodies for pancreatic kallikrein as ligand can be made into an analytical column or a preparative column, which is used for the analysis and purification preparation of pancreatic kallikrein, or can be made into an ELISA kit, which is used for detecting the level of pancreatic kallikrein.

[0071] Example 5

[0072] Purification of pancreatic kallikrein

[0073] 1) Preparation: the nanobody chromatography filler for anti-pancreatic kallikrein prepared in Example 4 is loaded into a 5 mL column;

[0074] 2) Equilibration: the column is equilibrated with 10 volumes of the loading buffer, so that the column conditions meet the loading requirements;

[0075] 3) Loading: the pancreatic kallikrein solution is fed into the filler column at a speed of 1 ml / min using a feeding pump, and the pancreatic kallikrein protein is combined with the nanobodies in the filler;

[0076] 4) Washing: 5 volumes of the equilibration buffer are used for washing to remove impurities, until the UV280 baseline is flat;

[0077] 5) Elution: the elution buffer is used for elution, and the solution with UV280 peak is collected, until the UV280 baseline is flat, and the collection is stopped;

[0078] 6) Regeneration: the column is washed with 5 volumes of the equilibration buffer, and is stored at 4°C.

[0079] Results: analysis of the purification profile.

[0080] The purified eluted pancreatic kallikrein is subjected to SDS-PAGE electrophoresis, proving that each affinity chromatography filler can adsorb pancreatic kallikrein in one step to obtain a certain purified pancreatic kallikrein protein. Among them, the results of YTM Nb 1-30 are provided as an illustration, specifically, as shown in Figure 6

[0081] Therefore, the nanobody specifically binding to pancreatic kallikrein provided by the application and the antibody combined with the microspheres to form the anti-pancreatic kallikrein nanobody affinity chromatography filler can effectively bind the pancreatic kallikrein in one step, solving the problem of low efficiency of the current purification process.

[0082] In this specification, the application has been described with reference to its specific embodiments. However, it is obvious that various modifications and changes can be made without departing from the spirit and scope of the application. Therefore, the specification and drawings should be considered as illustrative rather than limiting.​

Claims

1. A nanobody that specifically binds to kallikrein, characterized in that, The nucleotide sequence of the nanobody is shown in any one of SEQ ID No. 1, 3, and 5, and the amino acid sequence of the nanobody is shown in any one of SEQ ID No. 2, 4, and 6.

2. A recombinant vector, characterized in that, Includes the nucleotide sequence 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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