Anti-ouabain nanobody and use thereof

Abstract

The present invention belongs to the technical field of nanobodies, and specifically relates to an anti-ouabain nanobody and the use thereof. The amino acid sequence of the anti-ouabain nanobody is as shown in SEQ ID NO. 1. The nucleotide sequence thereof is as shown in SEQ ID NO. 2. The antibody has a good affinity to ouabain, can neutralize and antagonize the pharmacological activity of ouabain, and can be used to detect the content of ouabain in a solution in combination with an ELISA method. The present invention provides a method for constructing an ouabain antibody having a light weight, a high thermostability and a high detection sensitivity, and provides a research tool for deeply exploring the new biological functions of endogenous ouabain.

Description

Anti-ouabain nanoantibody and its application Technical Field

[0001] The present invention belongs to the technical field of nano antibodies, and in particular relates to an anti-ouabain nano antibody and its application. Background Art

[0002] Ouabain is a type of cardiotonic steroid compound, which is widely found in digitalis plants and has high medicinal value. In 1785, the British folk doctor Wellington used the steroidal plant medicine digitalis decoction to treat edema with remarkable results, which attracted widespread attention in the medical community. In the middle of the last century, European and American pharmaceutical companies competed to develop cardiotonic steroids. The most famous one is digoxin preparation, which has unique therapeutic effects on a variety of cardiovascular diseases. After decades, it is still an important drug for the clinical treatment of heart diseases such as congestive heart failure. As a cardiotonic steroid compound, ouabain can also be used to treat heart failure and arrhythmia. In terms of biological mechanism, ouabain mainly inhibits Na on the cell membrane. + / K + -ATPase plays a role. Ouabain inhibits Na + / K + -ATPase makes Na + Levels rise, Na + The increase in the level of Ca2+ inhibits the activity of the sodium-calcium exchanger, causing the intracellular Ca 2+ The concentration increases, activating Ca 2+ The related signaling pathways play a role in enhancing myocardial contractility and treating heart failure. Currently, many countries, including the United States, France, and Germany, have stopped the clinical use of ouabain. The main reason is that ouabain has a narrow therapeutic window, with therapeutic doses approaching toxic doses, making overdose extremely likely to cause toxicity. However, some scientists believe that ouabain can be taken orally to reduce its toxicity and used to treat angina pectoris and myocardial infarction. In the late 20th century, American Professor Hamlyn JM published a research paper in Nature demonstrating the existence of endogenous ouabain in the human body. Researchers detected and purified this unique substance by mass spectrometry from 300 liters of mixed human plasma. Professor Hamlyn JM also discovered that the chemical structure of endogenous ouabain is similar to that of ouabain derived from plants or toads, and that it is secreted into the metabolic circulation through the adrenal glands, playing a vital role in regulating arterial pressure, heart, and kidney function. Further research has revealed that ouabain is closely related to the development and progression of diseases such as immunity, tumors, and viral infections, suggesting that it may have new biological functions beyond the cardiovascular system. To confirm this hypothesis, we need a clearer understanding of the process of ouabain in the body, accurate quantification of ouabain, and the development of new antibodies that can block ouabain.

[0003] Since the advent of hybridoma technology in 1975, monoclonal antibody drugs have been widely used in the diagnosis and treatment of a wide range of diseases, becoming a major research hotspot. This technology has achieved significant progress in tumor therapy, diagnosis, and molecular detection. In tumor therapy, traditional monoclonal antibodies can target and kill tumor cells, even inducing programmed necrosis. In diagnostics, antibodies labeled with radioisotopes or fluorescein play an important role in PET and disease imaging. Monoclonal antibodies offer advantages such as target specificity, mature preparation processes, and ease of labeling. However, as their application expands, their shortcomings have become increasingly apparent. First, their large molecular weight makes them unstable in vitro and in vivo. Second, their strong immunogenicity can lead to severe rejection reactions. Furthermore, the high background signal of monoclonal antibodies in molecular imaging applications limits their clinical application. Consequently, a wave of new antibody development has emerged, with small molecule antibodies, including Fab fragments, single-chain antibodies, and nanobodies being the most representative.

[0004] Nanobodies (Nb) are a new type of small molecule antibody. They were originally derived from the variable region sequence of a heavy chain antibody discovered in camels in 1993 by Hamers-Casterman et al. Further research revealed that two antibody structures naturally occur in camel serum. One is a conventional tetrameric antibody with two heavy chains and two light chains, while the other is a heavy chain antibody with only the heavy chain missing both light chains. The variable region of the heavy chain antibody is a single-chain antibody, lacking both light chains and the typical constant heavy chain CH1 domain. Its molecular mass is approximately 15 kDa, one-tenth that of a conventional antibody, and its molecular volume is approximately 4.8 nm by 2.2 nm. This unique, naturally occurring small molecule antibody is the smallest biologically active functional fragment discovered to date. In camels and sharks, these heavy chain antibodies recognize antigens solely through a single variable region, thus possessing biological activity. Therefore, Nb represents the smallest complete antigen-binding domain. This new type of antibody, found in animals such as camels and cartilaginous fish, shares similarities in structure and physiological function with monoclonal antibodies, and holds great promise for future applications. Nb has unique advantages over monoclonal antibodies and even other antibodies. It has high affinity, good stability, and is easier to achieve soluble expression in microorganisms, which greatly reduces the production cycle and production costs. It has broad application space in the fields of precise diagnosis, targeted treatment, food safety monitoring and other fields of various diseases.

[0005] The present invention aims to provide materials for constructing lightweight, high-affinity ouabain Nb and for establishing a heat-stable and highly sensitive ouabain detection method; and to provide research tools for further exploring new biological functions of endogenous ouabain.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to develop a new type of ouabain Nb with small molecular weight, strong binding ability and stable properties by genetic engineering methods, establish an ELISA immunoassay based on ouabain Nb to detect ouabain content, and provide a tool for related research.

[0008] In order to achieve the above purpose, the present invention is implemented through the following scheme:

[0009] The present invention provides an anti-ouabain nanobody, the amino acid sequence of which is shown in SEQ ID NO.1:

[0010] In SEQ ID NO.1, 1-23 are amino acids of the framework region FR1 of the nanobody: EVQLLQSGGLAQPGGSLRLTCTA (SEQ ID NO.3); 24-40 are amino acids of the framework region FR2 of the nanobody: MGWYRQPPGKQRELVAS (SEQ ID NO.4); 41-78 are amino acids of the framework region FR3 of the nanobody: NYTDSVKSRFIISRDNTKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO.5); 79-89 are amino acids of the framework region FR4 of the nanobody: WGQGTQVTVSS (SEQ ID NO.6); 90-96 are amino acids of the complementary determining region CDR1 of the nanobody: SIGAVYV (SEQ ID NO.7); 97-103 are amino acids of the complementary determining region CDR2 of the nanobody: ITSAGIT (SEQ ID NO.8); 104-117 are the amino acids of the complementarity determining region CDR3 of the nanobody, which is NAPLRGRDDYGGFD (SEQ ID NO.9).

[0011] The present invention also provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID NO.2:

[0012] The present invention provides a recombinant plasmid comprising the gene of the anti-ouabain nanobody.

[0013] The present invention provides an expression host cell comprising the recombinant plasmid.

[0014] The present invention also provides a method for preparing the anti-ouabain nanobody, characterized in that the recombinant plasmid is transformed into BL-21 genetically engineered bacteria, and after induction, it is preliminarily purified by His-tag Purification resin and then further purified by AKTA ion exchange.

[0015] The present invention provides the use of the anti-ouabain nanobody in preparing a preparation for detecting ouabain by an ELISA method.

[0016] The present invention provides a kit for detecting ouabain. The kit detects ouabain based on a competitive ELISA method, and the kit uses the nanobody shown in SEQ ID NO.1 as a detection antibody.

[0017] The present invention provides a method for detecting ouabain, comprising the following steps:

[0018] (1) Optimization of antibody concentration: Add 100 μL of 20 ng / ml ouabain-OVA to a 96-well ELISA plate and coat overnight; discard the liquid in the wells, wash 5 times with PBST, add 100 μL of OVA and block at room temperature for 1 hour; discard the liquid in the wells, wash 5 times with PBST, add a series of concentrations of biotinylated ouabain nanoantibodies and incubate at room temperature for 2 hours; discard the liquid in the wells, wash 5 times with PBST, add 100 μL of streptomycin-HRP and incubate at room temperature for 1 hour; discard the liquid in the wells, wash 5 times with PBST, add 100 μL of TMB color development liquid, incubate at 37°C for 10 minutes, and then add 50 μL of color stop solution. Detect absorbance at 450 nM using a microplate reader; select the optimal antibody concentration for subsequent detection experiments.

[0019] (2) Competitive ELISA assay: Add 100 μL of 20 ng / mL ouabain-OVA to a high-adsorption 96-well ELISA plate and coat overnight; discard the liquid in the wells, wash five times with PBST, add 100 μL of OVA and block at room temperature for 1 hour; discard the liquid in the wells, wash five times with PBST. For the standard group, add 100 μL of 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, and 0.6 ng / mL of ouabain. For the blank control group (B0), add 100 μL of PBS. For the test group, add 100 μL of the test serum sample. FITC-labeled ouabain nanobody was added to each sample well and incubated at room temperature for 2 hours. The wells were then discarded, washed five times with PBST, and 100 μL of streptomycin-HRP was added and incubated at room temperature for 1 hour. The wells were then discarded, washed five times with PBST, and 100 μL of TMB colorimetric solution was added. After incubation at 37°C for 10 minutes, 50 μL of colorimetric stop solution was added. The absorbance was measured at 450 nM using a microplate reader. The average absorbance value (B) of each standard solution and sample was divided by the absorbance value of the first blank (0 standard) (B0) and multiplied by 100% to obtain the percent absorbance. A standard curve was plotted with the logarithm of the ouabain concentration on the X-axis and the percent absorbance on the Y-axis. Based on the percent absorbance of the sample, the abscissa of the corresponding point on the curve was calculated as the logarithm of the ouabain concentration. The inverse value, C, was then calculated as the ouabain concentration in the sample.

[0020] The present invention provides an application of ouabain nanoantibodies capable of significantly neutralizing and antagonizing the pharmacological activity of ouabain in vitro.

[0021] The present invention discloses the following technical effects:

[0022] The present invention prepares antigens to immunize alpacas and monitors immune responses. Immunized alpaca lymphocytes are isolated and purified to extract total RNA. A large phage library is constructed based on phage display technology. DNA sequencing and database analysis confirm that the library exhibits good diversity and insertion rate, meeting the requirements for screening nanoantibodies. The library is enriched with the antigen ouabain-OVA, and after three rounds of solid-phase biopanning, positive clones are effectively enriched in the library. Multiple rounds of screening using an indirect ELISA method reveal multiple clone sequences exhibiting strong signal reactions with the ouabain antigen. The selected clone sequences are expressed in prokaryotes and purified to obtain highly pure antibody proteins. The binding ability of the antibody proteins to ouabain is then determined using MST. High-affinity ouabain nanoantibodies are screened and their in vitro biological activity is verified. The results demonstrate that the ouabain nanoantibodies described herein can neutralize and antagonize the pharmacological activity of ouabain.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The nanobodies obtained by the present invention have advantages such as small relative molecular mass, high affinity, low production cost, and the ability to achieve large-scale expression. They can be used to detect the content of ouabain in biological samples and as neutralizing antibodies to antagonize the pharmacological activity of ouabain. Furthermore, the method of preparing small-molecule nanobodies of the present invention is universal and can be used to screen and prepare nanobodies targeting other small molecules, thus possessing high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1: SDS-PAGE electrophoresis analysis of ouabain and OVA conjugated products

[0027] Figure 2: Immune alpaca serum titer test

[0028] Figure 3: Agarose gel analysis of nested PCR amplification products of VHH fragments. Left lane 1: First-round PCR amplification product; right lane 1: Second-round PCR amplification product.

[0029] Figure 4: Agarose gel analysis of phage library construction products. The left panel shows agarose gel analysis of vector digestion products at 50°C for 15, 30, 60, and 120 min. The right panel shows lanes 1-3: gel-recovered digestion products, vector digestion products, and negative control, respectively.

[0030] Figure 5: Library colony capacity estimation of the original library diluted 10 4 , 10 5 , 10 6 Pick up 100 μL of colonies (colonies represent independent clones).

[0031] Figure 6: Library diversity and sequence feature analysis. Amino acid sequence alignment analysis of random clones from the phage library. Library diversity and feature sequence analysis in the IMGT database.

[0032] Figure 7: ELISA detection of phage library enrichment effect.

[0033] Figure 8: ELISA analysis of monoclonal expression products to screen positive clones. Monoclonal expression products were analyzed by indirect ELISA, with a value of 0.75, 5 times greater than the blank control, used as the dividing line between positive and negative clones.

[0034] Figure 9: Nanobody prokaryotic protein expression and purification. FT refers to the flow-through, and lanes 1-8 correspond to fraction batches collected from the ion exchange column.

[0035] Figure 10: MST detection of affinity between ouabain and nanobody.

[0036] Figure 11: Ouabain nanobody reduces ouabain-induced cytotoxicity. Different concentrations of ouabain nanobody protect cells from viability after treatment with 1 μM and 2 μM ouabain for 24 and 48 hours.

[0037] Figure 12: Ouabain nanobody antagonizes ouabain-induced Ca 2+ The concentration of cellular Ca increases. 2+ The results of flow cytometry and quantification showed that compared with the NC group and the ouabain group, the Ca2+ level in the ouabain nanobody group and the ouabain incubation group was significantly increased. 2+ The experiment was repeated three times independently, *P<0.05; #P<0.05; statistically significant.

[0038] FIG13 shows the standard curve of competitive binding ELISA established based on ouabain nanobody. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the examples, but the description of the examples does not limit the scope of protection of the present invention in any way.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0041] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0042] Example 1 Antigen preparation and alpaca immunization

[0043] 1.1 Experimental Materials and Animals

[0044] Ouabain and were purchased from MCE; BSA protein was purchased from Beyotime Biotechnology and Sigma-Aldrich, USA, respectively; Coomassie Brilliant Blue dye was purchased from Beyotime Biotechnology; carbodiimide and OVA protein were purchased from Sigma-Aldrich, USA; lymphocyte separation medium was purchased from Yisheng Biotechnology; dialysis bags were purchased from Shanghai Shenggong Bioengineering Co., Ltd.; alpacas were bred by Shenzhen Kangti Life Science Co., Ltd., number 1607.

[0045] 1.2 Experimental methods

[0046] 1.2.1 Conjugation of ouabain to carrier protein OVA

[0047] The present invention couples an ouabain compound with an OVA protein carrier by a carbodiimide method. 5 mg of ouabain and 5 mg of BSA are dissolved in 0.5 mL of TE buffer. The ouabain and BSA are added dropwise to the carbodiimide solution while shaking at 37° C., and the mixture is reacted on a shaker for 2 hours. The solution is placed in a dialysis bag and dialyzed for 3 days. A small amount of the dialyzed product is collected and the coupled product is detected by SDS-PAGE protein electrophoresis. The remaining product is frozen in a -20° C. refrigerator for subsequent use.

[0048] 1.2.2 SDS-PAGE and Coomassie Brilliant Blue Staining

[0049] Prepare SDS-PAGE gel according to the proportions in the recipe table. Prepare a protein separation gel of appropriate concentration (8% to 15%) based on the molecular weight of the target protein. After the lower gel is completely solidified, prepare a 5% concentration of stacking gel and insert it into a 10- or 15-well gel comb for fixation. After protein electrophoresis, cut the stacking gel, rinse lightly with deionized water, carefully discard the liquid, add approximately 20mL of rapid Coomassie Brilliant Blue staining solution, and shake at a constant speed on a horizontal shaker for 30 minutes. Finally, recover the Coomassie Brilliant Blue staining solution and shake the gel pieces on a shaker with deionized water to decolorize and rinse. Rinse three times with fresh deionized water every 10 minutes, and decolorize for 2 hours.

[0050] 1.2.3 Antigen immunization of alpacas

[0051] In the present invention, the antigen is coupled with the BSA carrier protein to prepare an antigen-immunized blank alpaca. The animal immunization scheme is shown in the following table.

[0052] Blood was collected after each immunization to monitor the immune response. Six rounds of immunization were performed, and 50 mL of peripheral blood was collected from the immunized alpacas 10 days after the last antigen injection.

[0053] 1.2.4 Isolation of alpaca peripheral blood mononuclear lymphocyte B cells

[0054] Mix the collected peripheral blood with PBS in a volume ratio of 1:2, and mix thoroughly by pipetting with a Pasteur pipette. Take an appropriate amount of lymphocyte separation solution and add it to a 50mL centrifuge tube. Slowly add the peripheral blood diluent to the centrifuge tube at 25°C and pipette evenly, and centrifuge at 1000g for 10 minutes. Aspirate the lymphocytes, add the cell suspension to 1 50mL centrifuge tube for every 5 tubes, and fill it up to 50mL with PBS. Invert the centrifuge tube to mix thoroughly, centrifuge at 1000g for 10 minutes at 25°C, and discard the supernatant. Add pre-cooled PBS to the centrifuge tube, pipette to resuspend the lymphocytes, centrifuge at 2500g for 10 minutes at 4°C, and discard the supernatant. Resuspend the lymphocytes by pipetting with PBS, count the cells, and adjust the concentration of the suspension to 5×10 6 The cell suspension was aliquoted into 1.5 mL RNase-free centrifuge tubes at a volume of 1 mL per tube. The tubes were centrifuged at 1000 g for 10 min at 4°C, and the supernatant was removed. The peripheral blood mononuclear cells obtained were used for subsequent experiments.

[0055] 1.3 Experimental Results

[0056] Ouabain was conjugated to OVA using the carbodiimide method. Subsequently, OVA and the ouabain-OVA conjugate were analyzed by SDS-PAGE electrophoresis. As shown in Figure 1, the control OVA sample exhibited a distinct band near 44 kDa, consistent with the theoretical molecular weight of OVA of 44 kDa. Compared to the control OVA sample, the ouabain-OVA conjugate exhibited a distinct delayed band near 44 kDa. Furthermore, multiple bands were evident above 44 kDa, indicating that the ouabain-OVA conjugate was successfully prepared.

[0057] The titer of immunized alpaca serum was detected by indirect ELISA. As shown in Figure 2, the titer of alpaca antigen serum increased to 10 after the first immunization compared with that of unimmunized alpaca. 3 times, and the serum titer level increased to 10 after the third immunization 4 times, and the serum titer level reached 10 after the fifth immunization 5 This result shows that the antigen produces a strong immune response in alpacas.

[0058] Example 2 Construction and screening of nanobody phage display library

[0059] 2.1 Experimental Materials

[0060] Helper phage VCSM13 was purchased from Beijing Baokewei Food Safety Biological Company; SifⅠ enzyme was purchased from Vicente Biotechnology Company; T4 DNA ligase and restriction endonucleases were purchased from Thermo Fisher Scientific; DNA gel recovery kit was purchased from Shanghai Shenggong Bioengineering Company; Fast pfu high-fidelity amplification enzyme was purchased from Beijing Quanshijin Biotechnology Company; PCR amplification premix was purchased from Qingke Biotechnology Company; DNA marker was purchased from General Biotechnology Company; and cDNA reverse transcription kit was purchased from Toyobo Company, Japan.

[0061] 2.2 Experimental methods

[0062] 2.2.1 cDNA synthesis and nested PCR amplification

[0063] RNA was used as a template to generate a cDNA library through reverse transcription. The amount of RNA used for reverse transcription in 10 μL was 500 ng. The reverse transcription system was prepared as shown in the following table:

[0064] Using the first round cDNA as a template, nested PCR was performed to amplify the heavy chain variable region VHH gene in large quantities. The PCR system was prepared as shown in the following table:

[0065] After the system is prepared, place it in a PCR instrument for amplification. The reaction procedure is as follows:

[0066] Specific amplification primers are as follows:

[0067] In order to increase the capacity of the library, the present invention uses the first round of gel recovery products as PCR templates to perform nested PCR amplification, and performs the second round of PCR reaction according to the conditions shown in the following table:

[0068] After the system is prepared, place it in a PCR instrument for amplification. The second round of PCR reaction procedure is as follows:

[0069] Specific amplification primers are as follows:

[0070] 2.2.2 Phage library construction

[0071] The present invention uses pComb3xss phagemid vector as the vector for phage library construction. First, the vector and nested PCR products are simultaneously digested with restriction endonuclease Sfi I (the digestion sites are located at 282 bp and 1954 bp of the vector, respectively).

[0072] Prepare 20 μL of reaction system as follows:

[0073] The vector and fragment were simultaneously digested at 50°C for 60 minutes, followed by agarose electrophoresis, and the digested vector and fragment were recovered for ligation. The present invention uses T4 ligase for ligation, taking 20 ng of the digested vector and 8 ng of the digested insert (molar ratio of 1:4) for ligation. The ligation system was prepared according to the following table:

[0074] The enzyme ligation system was then connected at 22°C for 30 minutes. After the connection was completed, the cells were transformed into TG1 competent cells and cultured in a 37°C constant temperature incubator overnight.

[0075] 2.2.3 Phage superinfection amplification and titer determination

[0076] The present invention uses VCSM13 helper phage for superinfection, scrapes all the library plate clones, dilutes with 10mL 2×TY medium and freezes, takes 100μL bacterial solution and adds it to a conical flask containing 2×TY liquid culture, shakes in a constant temperature shaker until the bacteria grows in the logarithmic phase OD 600 = 0.3-0.5. Add helper phage VCSM13 at a volume ratio of 1:100 and incubate in a 37°C incubator for 30 minutes. Transfer the bacterial solution to a 50mL centrifuge tube and centrifuge at 2700 rpm for 10 minutes at 4°C. Resuspend the pellet and add it to 250mL of 2×TY liquid culture medium supplemented with 100mg / mL ampicillin and 50mg / mL kanamycin. Incubate overnight at 37°C, 225 rpm on a horizontal shaker for 16 hours. Harvest the culture and centrifuge at 20,000g for 30 minutes at 4°C. Add 1 / 4 volume of PEG8000 / NaCl to the supernatant, mix thoroughly by inversion, and incubate on ice for at least 30 minutes. Centrifuge at 4000 rpm for 30 minutes at 4°C and discard the supernatant. The phage precipitate was resuspended in 1 mL of PBS solution and placed in a 1.5 mL Eppendorf tube. The tube was centrifuged at 20,000 g for 1 min at 4°C. The supernatant was collected and a small amount was used for phage titer determination. The rest was frozen and stored in a -80°C refrigerator.

[0077] The present invention adopts the double-layer agar method to determine the phage titer, and the phage is diluted to 10 -1 to 10 -10 pfu, 1 μL of phage of each dilution was taken and mixed with 100 μL of TG1 bacterial solution. After standing at 37°C for 20 min, 3 mL of top agar was added respectively, mixed and poured onto LB solid medium plates, cultured at 37°C overnight, and the plaques were counted and the phage titer was calculated.

[0078] 2.2.4 Phage Library Biopanning

[0079] The library was biopanned using a binding, washing, elution, and amplification cycle. 20× ELISA coating buffer from a biotechnology company was pre-mixed with ouabain-OVA and 100 μL was added to each well of a 96-well microtiter plate. Three rounds of coating were performed at concentrations of 30 μg / mL, 15 μg / mL, and 7 μg / mL, respectively. Coating was incubated overnight at 4°C and washed five times with PBST. 100 μL of 5% BSA was prepared in advance and added to each well for blocking. The plates were then washed five times with PBST. Phage titers of 10-11 pfu (phage:bacteria ratio = 50:1) were prepared and 100 μL of phage was added to each well. The plates were incubated on a horizontal shaker for 2 hours and washed five times with PBST. The plate was then discarded and 100 μL of glycine was added to each well for 15 minutes. Elution was then completed by adding Tris-HCl to neutralize the pH. The eluate was recovered and prepared to a phage titer of 10-11 pfu. This was then used to infect TG1 E. coli. The titer was measured and the enrichment factor was calculated. The infected bacteria were plated and all colonies were collected. The enrichment titer was used to determine whether to proceed to the next round of screening.

[0080] 2.2.5 Phage library screening of positive clones

[0081] The present invention adopts the indirect ELISA method to screen positive clones in the enrichment library, select the super-infected bacterial plate clones after three rounds of enrichment and add them to a 96-well plate, use VCSM13 to assist phage infection for 14 hours, and transfer 100 μL of the supernatant to a 96-well plate pre-coated with 5 μg / mL ouabain-OVA after centrifugation, and incubate at 37°C for 45 minutes. Wash the plate 6 times with PBST, add (5000:1) HRP-labeled anti-M13 monoclonal antibody, 100 μL / well, and incubate at room temperature for 45 minutes on a horizontal shaker. Wash it again with PBST 3 times, add TMB color development solution, 100 μL / well, and incubate at 37°C for 10 minutes. When the solution gradually turns blue and shows obvious changes in depth, add 2mM dilute sulfuric acid, 50 μL / well, stop the color reaction, and immediately read the OD with an enzyme reader. 450 The wells with a 5-fold higher expression than the negative control were sequenced to obtain the final Nanobody sequence, which encodes a Nanobody against ouabain.

[0082] 2.3 Experimental Results

[0083] 2.3.1 Nested PCR amplification of VHH fragments

[0084] The present invention uses nested PCR to amplify the VHH gene. As shown in Figure 3, the total RNA extracted above is reverse transcribed to obtain cDNA. Two pairs of specific primers are used to amplify the VHH gene fragment in two rounds of PCR. The first round of PCR amplification products are analyzed by agarose gel electrophoresis. A DNA band of approximately 700 bp in size can be seen in lane 1. The target band is recovered by gel electrophoresis and used as a template for the next round of PCR. The second round of PCR products are analyzed by agarose gel electrophoresis to obtain a DNA band of approximately 400 bp in size. The target band product is recovered by gel for subsequent construction of a recombinant plasmid. The band sizes of the two rounds of PCR amplification products are consistent with the expected DNA fragment size and can be used for phage library construction.

[0085] 2.3.2 Phage library construction

[0086] As shown in Figure 4, to construct the library, the amplified VHH fragment product and the phagemid vector pComb3xss were digested with enzymes, both using Sfi I enzyme for single digestion. First, the conditions for vector digestion efficiency were optimized, and the digestion products of the vector under different conditions were analyzed by agarose gel electrophoresis. It can be seen that digestion at 50 ° C for 60 minutes showed clear vector digestion bands of 1700bp and 3200bp respectively, which are consistent with the expected vector digestion band size, achieving a good digestion effect. According to the aforementioned digestion reaction conditions, the vector and VHH fragment were digested simultaneously, and clear digested vector and VHH fragment bands were displayed on the agarose gel, which are consistent with the expected band size.

[0087] 2.3.3 Phage library quality analysis

[0088] As shown in Figure 5, the library was estimated to contain approximately 1×10 8 Therefore, the colony capacity of the library constructed in the present invention is 1×10 8 cfu, which meets the standards for screening nanoantibody library capacity and can be used for subsequent nanoantibody screening.

[0089] 20 monoclonal colonies were randomly selected on the gradient dilution plate and compared by DNA sequencing and amino acid sequence translation with biological software. As shown in the upper figure of Figure 6, the 20 amino acid sequences are significantly different, especially in the CDR3 region of different sequences, which is highly different. As an antigenic determining region, the high degree of difference of CDR3 indicates that they may recognize different antigenic epitopes. Therefore, the present invention preliminarily judges that the diversity of the constructed library is 100%. At the same time, the present invention randomly selects 1 amino acid sequence and submits it to the IGMT database for analysis. As shown in the lower figure of Figure 6, the sequence shows a high degree of homology with the sequence from Vigucna IGHV3S53*1, indicating that the sequence meets the basic characteristics of the existing nano antibody sequences in the database. In summary, the nano antibody library constructed by the present invention has a high insertion rate and good diversity. The nano antibody library is successfully constructed and of good quality, and can be used for subsequent biological screening.

[0090] Example 3 Nanobody Biopanning and Expression Application

[0091] 3.1 Experimental Materials

[0092] Glycerol was purchased from Sigma-Aldrich, USA; DMEM medium, trypsin cell digestion solution, and penicillin-streptomycin were purchased from Vicente Biotechnology; serum-free cell freezing medium was purchased from New Cyme Biotechnology; HRP-labeled rabbit secondary antibody and HRP-labeled mouse secondary antibody were purchased from Bioworld, China; Trizol reagent was purchased from Shanghai Sangon Biotechnology Co., Ltd.; T4 DNA ligase was purchased from Thermo Fisher Scientific, USA; DNA gel recovery kit was purchased from Shanghai Sangon Biotechnology Co., Ltd.; Fast Pfu high-fidelity amplification enzyme was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction endonucleases were purchased from Thermo Fisher Scientific, USA; RIPA lysis buffer (strong) and RIPA lysis buffer (weak) were purchased from Beyotime Biotechnology Co., Ltd.; PCR amplification premix, DL5000 DNA Marker, and agarose were purchased from Qingke Biotechnology Co., Ltd.; and cDNA reverse transcription kit was purchased from Toyobo, Japan.

[0093] 3.2 Experimental methods

[0094] 3.2.1 Phage Library Biopanning

[0095] The library was bio-panned in the manner of binding, washing, elution, and amplification cycles. 20× ELISA coating buffer from a bioengineering company was mixed with ouabain-OVA in advance and 100 μL was added to each well of a 96-well enzyme-labeled plate. The three rounds of coating concentrations were 30 μg / mL, 15 μg / mL, and 7 μg / mL, respectively. The plates were coated overnight at 4°C and washed 5 times with PBST. 5% BSA was prepared in advance and 100 μL was added to each well for blocking. The plates were washed 5 times with PBST. The phage titer was prepared to 10 -11 Add 100 μL of phage to each well (phage: bacteria volume = 50:1), incubate on a horizontal shaker for 2 hours, and wash 5 times with PBST. Discard the liquid in the plate, add 100 μL of glycine to each well, elute for 15 minutes, and then add Tris-HCl to neutralize the pH value to end the elution. Recover the eluate to prepare a phage titer of 10 -11 pfu were used to infect TG1 E. coli, and the titer was determined and the enrichment coefficient was calculated. The infected bacteria were spread on plates, and all colonies were collected. The enrichment titer was used to determine whether to proceed to the next round of screening.

[0096] 3.2.2 Phage library screening of positive clones

[0097] The present invention adopts the indirect ELISA method to screen positive clones in the enrichment library, select the super-infected bacterial plate clones after three rounds of enrichment and add them to a 96-well plate, use VCSM13 to assist phage infection for 14 hours, and transfer 100 μL of the supernatant to a 96-well plate pre-coated with 5 μg / mL ouabain-OVA after centrifugation, and incubate at 37°C for 45 minutes. Wash the plate 6 times with PBST, add (5000:1) HRP-labeled anti-M13 monoclonal antibody, 100 μL / well, and incubate at room temperature for 45 minutes on a horizontal shaker. Wash it again with PBST 3 times, add TMB color development solution, 100 μL / well, and incubate at 37°C for 10 minutes. When the solution gradually turns blue and shows obvious changes in depth, add 2mM dilute sulfuric acid, 50 μL / well, stop the color reaction, and immediately read the OD with an enzyme reader. 450 The wells with a 5-fold higher expression than the negative control were sequenced to obtain the final Nanobody sequence, which encodes a Nanobody against ouabain.

[0098] 3.2.3 Comparative analysis of positive sequences

[0099] Positive clones were imported into MEGA software for sequence alignment, and molecular evolutionary tree analysis was performed after alignment. After analysis, the candidate nanobody sequences were uploaded to the IMGT database for sequence analysis.

[0100] 3.2.4 Construction of Nanobody Expression Vector

[0101] The present invention uses pET-32a as an expression vector, selects restriction sites as BamH I and Xho I, and performs agarose electrophoresis on the PCR products amplified from the positive clones. Specific primers are used to amplify the VHH fragment using the positive clones as templates.

[0102] The PCR amplification system is shown in the following table:

[0103] The reaction system is as follows:

[0104] The specific amplification primer sequences are as follows:

[0105] The pET-32a plasmid and the above gel-recovered product were digested with BamH I and Xho I. After digestion at 37°C for 30 min, the fragments were detected by agarose electrophoresis. The digested fragments were recovered from the gel and their concentrations were determined. The digestion system is shown in the following table:

[0106] The digested vector and fragment were ligated at a molar ratio of 1:4. The ligation reaction system is shown in the following table.

[0107] After ligation at 22°C for 30 min, the cells were transformed into BL21(DE3) competent cells, and positive clones were obtained for sequencing the next day.

[0108] 3.2.5 Prokaryotic Expression of Nanobodies

[0109] The bacterial solution containing the recombinant plasmid was added to 100 mL of LB + kanamycin liquid medium at a volume ratio of 1:50, and cultured in a shaking incubator at 37°C until the OD 600 ≈0.6. IPTG was added to the culture at concentrations of 0, 0.1, and 10 mM at different temperatures and times for induction. Protein expression was analyzed by SDS-PAGE electrophoresis to optimize the induction conditions. Centrifuge at 8000 rpm for 15 min at 4°C and discard the supernatant. Resuspend the bacterial pellet in PBS solution and disrupt the bacterial lysis protein with ultrasonication. The specific program of the ultrasonic disruptor is ultrasonication for 3 seconds, pause for 9 seconds, and a total of 45 minutes. The disrupted solution was centrifuged at 13000 rpm for 30 minutes at 4°C, the supernatant was collected, and stored at 4°C. The pellet was frozen at -20°C.

[0110] 3.2.6 Protein Purification of Nanobodies

[0111] Add 1 mL of mixed 50% His-tag BeyoGold to every 4 mL of bacterial lysate supernatant, and mix the bacterial lysate supernatant and 50% His-tag BeyoGold. Incubate slowly with shaking at 4°C for 60 minutes. Add a mixture of lysate and His-tag Purification resin to an empty column tube. Allow the liquid in the column to flow out and be collected under gravity. Add 1-2 column volumes of non-denaturing lysis buffer for a total of 5 times, and then add 1-2 column volumes of non-denaturing wash buffer for a total of 5 times. Elute the target protein 3-5 times with one column volume of non-denaturing elution buffer each time. Collect each eluate into different Eppendorf tubes. Use Milipore protein concentrator tubes as needed, and select 3kDa specification concentrator tubes to concentrate the sample to an appropriate volume. Add the sample to the concentrator tube and centrifuge at 3000g for 30 minutes at 25°C to collect the supernatant and measure the concentration. Depending on the protein purity requirement, the protein was further purified using an AKTA pure protein purifier, using a Superdex 75 gel chromatography column or an ion exchange column for separation and purification. The buffer system was 0.02M Tris-HCl, 1M NaCl, pH 8.0 (the equilibration buffer was adjusted according to the isoelectric point). The system flow rate was set at 0.5-1 mL / min depending on the instrument pressure. After pre-equilibration of the column, the sample was loaded using a sample loop, and the effluent fractions were collected at different times using an automated fraction collector. The different protein fractions were analyzed by SDS-PAGE electrophoresis, and the protein fractions were frozen and stored at -80°C.

[0112] 3.2.7 Fluorescent labeling of proteins

[0113] The present invention uses microthermophoresis (MST) technology to detect the affinity of antibody proteins and antigens. This method requires that at least one of the ligand molecule or receptor protein is fluorescently labeled. The present invention uses the Monolith RED-NHS second-generation protein labeling kit to label the target antibody.

[0114] (1) Protein labeling

[0115] In a 1.5mL Eppendorf tube, add 7μL of RED-NHS II dye and 7μL of NHS labeling buffer. Pipet and mix thoroughly to obtain a dye solution with a final concentration of 300μM. In a new 1.5mL Eppendorf tube, add 90μL of protein sample (concentration: 10μM). Add 10μL of the dye solution to the protein sample and pipette and mix thoroughly to obtain 100μL of dye-protein solution. Incubate at room temperature in the dark for 30 minutes. For protein purification, refer to Experimental Methods 4.3.3.

[0116] (II) Determination of labeling efficiency

[0117] The labeling efficiency (DOL) is calculated as follows:

[0118] 3.2.8 MST Detection of the Affinity of Antibody Protein to Ouabain

[0119] First, prepare a serial dilution of the ouabain solution in PBS containing 0.05% Tween-80. Use this solution to dilute ouabain to 1 μM. Pipette 10 μL into a 0.2 mL PCR tube, labeling each tube as PCR tube 1 through 16. Add 10 μL of PBS containing 0.05% Tween-80 to PCR tubes 2 through 16, and serially dilute the ouabain ligand in each tube to half the concentration of the previous tube. Dilute the labeled Nb protein to be tested to a concentration of 10 nM. Add 10 μL of the labeled Nb protein solution to each PCR tube and mix thoroughly by pipetting. Ensure that the concentration of the antibody protein to be tested in each PCR tube is 5 nM. Add an equal volume of the labeled antibody protein to each PCR tube and pipette into a capillary tube. The affinity between the antibody and ligand is determined by MST. Use a dedicated capillary tube to aspirate the contents of the PCR tubes, taking care to avoid introducing air bubbles. Set the parameters on the machine and detect the affinity between the nanobody and ouabain.

[0120] 3.3 Experimental Results

[0121] 3.3.1 Phage library biopanning enrichment

[0122] After the original library was constructed, the present invention subjected the phage library to three rounds of biopanning. In the first round, the coating antigen ouabain-OVA concentration was 30 μg / mL; in the second round, the coating antigen concentration was 15 μg / mL; and in the third round, the coating antigen concentration was 7 μg / mL. The results, as shown in Table 1, show that the third round of panning enriched the number of positive clones by 1000-fold compared to the first round, with a significantly improved recovery rate. Furthermore, the input-to-elution ratio of phages remained nearly stable between the second and third rounds, further demonstrating significant enrichment of positive clones.

[0123] Table 1: Phage enrichment parameters

[0124] To further evaluate the library enrichment efficiency, indirect ELISA was used to monitor changes in antigen-antibody binding signals during each round of panning. As shown in Figure 7, the binding signal for OVA and the antigen ouabain-OVA was low in the original library before panning. After three rounds of enrichment, the binding signal for OVA was significantly enhanced, increasing eightfold compared to the OVA binding signal intensity, indicating that positive clones specifically binding to Ouabain-OVA were effectively enriched in the library. Combined analysis of the panning recovery rate and indirect ELISA test results demonstrates that the Nb library was well enriched, providing a guarantee for subsequent screening to obtain ouabain Nbs.

[0125] 3.3.2 Screening and identification of positive clones

[0126] The present invention screens positive clones by indirect ELISA. Monoclonal clones are selected from the enriched library for multiple rounds of screening, and the periplasmic expression products of the monoclonal clones are detected by indirect ELISA. As shown in Figure 8, the blank control OD 450 The index with a value of 5 times or more was used as the dividing line, and finally multiple positive clones were selected for DNA sequencing and database analysis.

[0127] 3.3.3 Prokaryotic Expression of Nanobodies

[0128] The present invention constructs a prokaryotic expression recombinant plasmid. Once constructed and verified by sequencing, the recombinant plasmid is transformed into a host bacterium and induced with 100 mM IPTG for 4 hours at 37°C. The bacterial broth is disrupted by ultrasound, and the supernatant is collected and purified using a nickel column and ion exchange. The purified product is then analyzed by SDS-PAGE electrophoresis. As shown in Figure 9, the recombinant protein products all exhibit a distinct band around 14 kDa, which is consistent with the theoretical molecular weight of the recombinant protein, indicating successful expression of the recombinant protein.

[0129] 3.3.4 MST determination of the affinity of nanobodies to ouabain

[0130] The present invention expresses an antibody protein and purifies it via nickel affinity chromatography. The antibody protein is labeled with a nucleic acid dye using the Monolith RED-NHS second-generation protein labeling kit. The affinity between the candidate Nb protein and ouabain is determined using MST. As shown in Figure 10, MST measures the fluorescence changes produced by the reaction between the protein and the small molecule, enabling affinity determination in a relatively short time. The results show uniform protein fluorescence distribution, indicating effective fluorescent protein labeling. The binding ability of Nb to ouabain is also confirmed. Its amino acid sequence is shown in SEQ ID NO. 1 and is designated G2-Nb.

[0131] Example 4: Nanobodies neutralize the pharmacological activity of ouabain in vitro

[0132] 4.1 Experimental Materials

[0133] See 3.1 for experimental materials

[0134] 4.2 Experimental methods

[0135] 4.2.1 Cell culture and passaging

[0136] Cells were cultured in DMEM supplemented with 10% serum and penicillin in a 37°C, 5% CO2 incubator. When the cell density reached 80%-90% of the plate, the cells were washed with sterile PBS and digested with an appropriate volume of trypsin. The cells were incubated in a cell incubator for 2 minutes. Culture medium was added to terminate the digestion, and the cells were centrifuged at 800 g for 3 minutes in a 15 mL centrifuge tube. The supernatant was removed and fresh culture medium was added. The cells were resuspended by pipetting, and 1 / 3 of the suspension was evenly distributed across the culture dish containing fresh culture medium. The cells were then incubated in a 37°C, 5% CO2 incubator.

[0137] 4.2.2 Extraction of cell proteins

[0138] Select cells in good growth condition and, after drug addition or transfection, place them into a centrifuge tube. Wash twice with sterile PBS and add an appropriate amount of RIPA lysis buffer. Vortex thoroughly to mix, lyse on ice, and vortex three times every 10 minutes. Centrifuge at 12,000 rpm for 10 minutes and save the supernatant.

[0139] 4.2.3 Protein concentration determination by BCA method

[0140] Mix BCA working solution with solutions A and B in a 50:1 volume ratio to prepare 200 μL of working solution for each sample. Take 2 μL of each sample, fill it up to 10 μL with PBS, and then mix it thoroughly with 200 μL of BCA working solution. Incubate this solution at 37°C for 30 minutes and measure the OD value by colorimetry. 560 The absorbance was measured and the content was calculated from the standard curve.

[0141] 4.2.4 Determination of the effect of ouabain on cell viability by MTT assay

[0142] Cell viability was determined by MTT (thiazolyl blue). When the cell density in the 6-well plate reached 80%-90%, the cells were digested and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in 1 mL of culture medium and counted using a cell counting plate. 100 μL of cell suspension was added to a 96-well plate and 1×10 4Cells were cultured on a 96-well plate for 12-24 hours. When the cell growth density reached 60%-80%, drugs could be added to the cells. MTT was prepared to a concentration of 5 mg / mL under light-shielding conditions, and 15 μL was added to the cell wells. The cells were placed in a cell culture incubator for 4 hours. The solution in the 96-well plate was discarded, 150 μL of DMSO was added to each well, and the 96-well plate was placed on a horizontal shaker and shaken at room temperature for 10 minutes. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader to calculate the cell survival rate.

[0143] The formula for calculating cell viability is: Cell viability = [(OD 实验组 -OD 零处理组 ) / (OD 对照组 -OD 零处理组 )]*100%.

[0144] 4.2.5 Flow cytometry detection of intracellular Ca 2+ level

[0145] Cells were cultured in 12-well culture dishes. When the cell density reached 60%-80%, the cells were treated with drugs. The treated cells were deprived of the culture medium and rinsed three times with prewarmed HBSS to eliminate esterases and other substances in the culture medium, which could potentially degrade the ion probe. A 1-5 μM Fluo4-AM working solution was added to completely cover the cells and incubated in a cell culture incubator at a constant temperature for 40 minutes. The Fluo4-AM working solution was discarded, and the cells were washed three times with HBSS to completely remove the Fluo4-AM working solution from the dish. 1 mL of HBSS was then added and incubated at room temperature for 20-30 minutes to ensure complete deesterification of the AM bodies. The cells were then completely digested with trypsin, centrifuged at 2500 rpm for 5 minutes, and washed three times with HBSS. Finally, the cells were resuspended in 400 μL of HBSS and transferred to a flow cytometer. The BL1 / FITC channel was selected for analysis.

[0146] 4.2.6 Competitive Binding ELISA for Endogenous Ouabain Detection

[0147] For specific steps, please refer to the content of the invention.

[0148] 4.3 Experimental Results

[0149] 4.3.1 Ouabain Nanobodies Reduce Ouabain-Induced Cytotoxicity

[0150] High concentrations of ouabain can cause cell apoptosis. To investigate the pharmacological effects of ouabain Nb, this study treated Hela cells with ouabain and ouabain Nb. The effects of ouabain and antibody protein on cell survival were examined using the MTT assay. As shown in Figure 11, when Hela cells were treated with 1 μM and 2 μM ouabain for 48 and 24 hours, respectively, the cell survival rate was reduced to half of its original level after ouabain treatment. Therefore, this ouabain concentration was selected, and cells were pre-incubated with three concentrations of ouabain Nb (0.5, 1, and 2 μM) for 30 minutes before treatment. MTT results showed that different concentrations of ouabain Nb had a certain neutralizing effect compared to the ouabain control group, reducing the toxicity of ouabain on cells and increasing the survival rate of Hela cells. These results demonstrate that the ouabain Nb prepared in this study can neutralize a certain concentration of ouabain in vitro, reducing its toxicity.

[0151] 4.3.2 Ouabain Nanobodies Antagonize Ouabain-Induced Ca 2+ Increased concentration

[0152] Ouabain can inhibit Na + / K + -ATPase induces intracellular K + Reduced Na + Aggregation, depolarization of the cell membrane, activation of Ca 2+ channel and form Na + -Ca 2+ exchange, causing Ca 2+ The concentration increases, eventually leading to the Ca 2+ To further verify the biological activity of ouabain Nb, ouabain Nb was used to neutralize ouabain monomer and detect the Ca release in Hela cells. 2+ As shown in Figure 12, the 250nM ouabain group had a significantly higher Ca 2+ After incubation of ouabain Nb (5 μM) with ouabain, cells were treated and Ca 2+ concentration, and it was found that Ca 2+ The above results show that ouabain Nb plays a significant role in cells and can antagonize the Ca 2+ Concentration increased.

[0153] 4.3.3 Competitive Binding ELISA for Endogenous Ouabain Detection

[0154] The antigen was immobilized and the working concentration of the antibody was determined to be around 1 μg / mL. The optimal antibody concentration was selected for subsequent detection experiments. The competitive ELISA standard curve established based on the amino acid sequence of the antibody G2-Nb shown in SEQ ID N0.1 is shown in Figure 13 and Table 2. It can be observed that the curve has a good linear correlation. The detection range is 10-500 ng / mL based on the recovery rate and other parameters. The blank OD value of the standard was 0.05. 450 The average value of the values ​​was recorded as B0, and the OD values ​​at different drug concentrations were recorded as 450 The average absorbance of the sample wells is recorded as B. Calculate the B / B0 ratio for each drug concentration or sample well, as well as the standard deviation for each replicate. Plot a standard curve with the logarithm of the standard concentration as the abscissa and the B / B0 ratio as the ordinate. Based on the average absorbance of the sample wells, the abscissa of the corresponding point on the curve is the logarithm of the ouabain concentration. The antilogarithm of the logarithm represents the ouabain concentration in the sample being measured.

[0155] Table 2 Ouabain detection recovery data

Claims

1. An anti-ouabain nanobody, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1, 2. A gene encoding the anti-ouabain nanobody according to claim 1, whose nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that: A gene encoding the anti-ouabain nanobody according to claim 1.

4. The method for preparing the anti-ouabain nanobody according to claim 1, characterized in that: The recombinant plasmid as claimed in claim 3 was transformed into BL-21 genetically engineered bacteria, and after induction, it was preliminarily purified by His-tag Purification resin, and then further purified by AKTA ion exchange.

5. Use of the anti-ouabain nanobody according to claim 1 in preparing a preparation for detecting ouabain by ELISA method.

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