Bovine serum albumin combined nano antibody and application thereof

Abstract

The invention discloses a bovine serum albumin combined nano antibody and a preparation method and application thereof, the amino acid sequence of the bovine serum albumin combined nano antibody Nb1 is shown as SEQ.ID No.1, the nano antibody Nb not only has high affinity to bovine serum albumin, but also has good binding activity to both human serum albumin and mouse serum albumin, and the nano antibody Nb has high affinity to human serum albumin and mouse serum albumin. And the antibody has a relatively good broad-spectrum application range, and can be widely applied to the aspects of drug half-life period enhancement, tumor targeted therapy and imaging, immunodetection or diagnosis, recombinant albumin purification or enrichment and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody engineering, and particularly relates to a bovine serum albumin-binding nanobody and its application. Background Art

[0002] Serum albumin (such as human serum albumin HSA, bovine serum albumin BSA or murine serum albumin MSA) has become an ideal target for drug modification mediated by nanobodies (VHH) due to its highly conserved gene sequence and structure (the homology among the three is 70-80%). By designing nanobodies that specifically bind to serum albumin and conjugating them with therapeutic drugs, a "nanobody-serum albumin-drug" ternary complex system can be constructed, significantly optimizing pharmacokinetics and therapeutic effects. Modifying drugs with nanobodies that bind to serum albumin, especially nanobody-conjugated drugs, can extend the drug half-life, achieve targeted drug delivery, reduce the drug dosage, and can also enhance the biocompatibility of the drug to promote drug absorption, maximizing the efficacy of the drug.

[0003] Bovine serum albumin (BSA) is the main component in bovine serum, has good water solubility, high stability and abundant chemical modification sites, and is widely used in the biomedical field, such as as a protein standard, a drug carrier, etc. Nanobodies are variable regions of heavy-chain antibodies that are naturally lacking in light chains in camelids, and have unique biological characteristics such as small molecular weight, high affinity, strong stability, easy expression and modification. Its unique structure enables it to recognize epitopes that are difficult to reach by traditional antibodies, and shows great application potential in immunoassay, targeted therapy and other aspects.

[0004] At present, although certain progress has been made in the research on both BSA and nanobodies, the systematic research on the preparation, characterization and biological application of BSA nanobodies is still relatively scarce. Especially in how to efficiently prepare BSA nanobodies with high affinity and high stability and apply them to actual immunoassay and targeted therapy, there are obvious research gaps. Conducting in-depth research in this area is expected to provide new strategies and methods for the diagnosis and treatment of BSA-related diseases. Summary of the Invention

[0005] The core objective of the present invention is to prepare bovine serum albumin nanobodies with high affinity and stability, comprehensively characterize them, and explore their application potential in the biomedical field. The specific content of the present invention is as follows: In the first aspect of the present invention, a bovine serum albumin-binding nanobody is provided, and the amino acid sequence of the bovine serum albumin nanobody is as shown in SEQ ID NO.1.

[0006] Furthermore, the bovine serum albumin-binding nanobody can also bind to human serum albumin (HSA) and murine serum albumin (MSA).

[0007] In a second aspect of the present invention, there is provided a nucleic acid encoding the bovine serum albumin-binding nanobody, including the currently disclosed nucleotide sequence or a nucleotide sequence optimized according to actual needs. Preferably, the nucleotide sequence is a nucleotide sequence optimized according to the codon preference of Escherichia coli. More preferably, the nucleic acid sequence is as shown in SEQ ID NO.2 or has 95% homology with the sequence shown in SEQ ID NO.2.

[0008] In a third aspect of the present invention, there is provided a biological material selected from any one of the following: I. A vector containing the nucleic acid; II. A transformant containing the vector described in I; III. A recombinant cell capable of producing the bovine serum albumin-binding nanobody and / or containing the nucleic acid.

[0009] Furthermore, the vector includes at least one of pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, and pYES2 vectors.

[0010] Furthermore, the recombinant cell includes at least one of Chinese hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0011] In a fourth aspect of the present invention, there is provided the use of the bovine serum albumin-binding nanobody, the nucleic acid, or the biological material in any of the following aspects: A1. Use in the preparation of products for prolonging the half-life of drugs; A2. Use in the preparation of products for tumor-targeted therapy and imaging; A3. Use in the preparation of immunoassay or diagnostic products; A4. Use in the preparation of products for promoting the purification or enrichment of recombinant albumin.

[0012] Furthermore, the drugs in the prolonging of the half-life of drugs include protein drugs, antibody fragments, small molecule chemotherapeutic drugs, and other biological agents; preferably, the protein drugs include at least one of IL-6R-targeted drugs, TNF-α-targeted drugs, and IL-17A / IL-17F-targeted drugs; the small molecule chemotherapeutic drugs include at least one of doxorubicin, sorafenib, and sirolimus. In a specific embodiment of the present invention, the drug is interferon.

[0013] Furthermore, the products for prolonging the drug half-life described in A1 include at least one of a fusion protein composed of a bovine serum albumin-binding nanobody and a protein drug, a conjugate formed by linking a bovine serum albumin-binding nanobody and a therapeutic agent, and a pharmaceutical composition of the bovine serum albumin-binding nanobody.

[0014] Furthermore, the protein drugs include at least one of a polypeptide, an antibody, an antibody fragment, a cytokine, and a tumor marker molecule that can play a therapeutic role.

[0015] Furthermore, the pharmaceutical composition of the bovine serum albumin-binding nanobody further includes additives acceptable in the pharmaceutical field.

[0016] The fifth aspect of the present invention provides a method for preparing the bovine serum albumin-binding nanobody described above. The method includes the step of introducing the nucleic acid into a recombinant cell to express the bovine serum albumin-binding nanobody.

[0017] The recombinant cell is selected from at least one of Chinese hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0018] In a specific embodiment of the present invention, the bovine serum albumin nanobody gene (nucleotide sequence shown in SEQ ID NO.2) was cloned into the expression vector pET-22b (containing His tag), and transformed into BL21(DE3) Escherichia coli to express the bovine serum albumin nanobody, achieving the purpose of preparing the bovine serum albumin nanobody.

[0019] The beneficial effects of the present invention include but are not limited to: The bovine serum albumin nanobody provided by the present invention not only has a high affinity for bovine serum albumin, but also has good binding activity to human serum albumin and mouse serum albumin. It has a good broad-spectrum usage range and can be widely applied in aspects such as enhancing drug half-life, tumor targeted therapy and imaging, immunoassay or diagnosis, recombinant albumin purification or enrichment, etc., and has a wide application prospect in the pharmaceutical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 is a schematic diagram of the SDS-PAGE detection result in the embodiment of the present invention; Figure 2Schematic diagram of ELISA test results of Nb1 with BSA, HSA and MSA in the embodiments of the present invention; Figure 3 Blood drug activity-time curve of IFNα-2b in the embodiments of the present invention; Figure 4 Blood drug activity-time curve of the fusion protein Nb1-IFNα-2b in the embodiments of the present invention. Detailed implementation manners

[0021] The present invention will be described in detail below in conjunction with embodiments. However, the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0022] Phage display library: natural alpaca / camel VHH library, purchased from Pujian Biotechnology (Wuhan) Co., Ltd.; Bovine serum albumin: self-produced by Shanghai Xinruite Biopharmaceutical Technology Co., Ltd.; Coating buffer: PBS (pH 7.4); Blocking solution: 3% skim milk in PBST (PBS + 0.05% Tween-20); Acidic elution: 0.1 M Glycine-HCl (pH 2.2), immediately neutralized with 1 M Tris-HCl (pH 9.0); Competitive elution: 1-5 mg / mL soluble BSA (dissolved in PBS); Host bacteria: XL1-Blue Escherichia coli, BL21(DE3) Escherichia coli, purchased from Beijing Biovector Science Lab Co., Ltd.; Helper phage: M13KO7, purchased from Thermo Fisher Scientific; Transfer membrane buffer (25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3); PVDF membrane (0.45 μm) or nitrocellulose membrane (NC membrane).

[0023] Example 1: Bovine serum albumin-binding nanobody and its preparation method The natural alpaca-derived phage display nanobody library was screened using the immunotube method. The screening steps are as follows: (1) BSA coating and blocking: BSA was completely dissolved in PBS solution to prepare a BSA solution with a final concentration of 25 μg / mL. The BSA solution was coated on the immunotubes and incubated at room temperature for 2 hours. After discarding the coating solution, it was washed 3 times with PBST (1 min each time), and then blocked with the blocking solution at room temperature for 1 h and washed 3 times with PBST; (2) Pre-clearing of the phage library: The phage library was incubated with immunotubes not coated with BSA for 30 min (at room temperature) to remove non-specifically bound phages. The supernatant of the unbound phages was collected for subsequent screening; (3) Three rounds of affinity enrichment screening: The specifically bound phages were collected, and the screening process is shown in Table 1; (4) Amplification: The eluted phages were used to infect XL1-Blue Escherichia coli, and helper phage M13KO7 was added. It was cultured with shaking at 37°C for 12 - 16 h, and the phages were purified by NaCl precipitation for the next round of screening. (5) Identification of positive clones: The monoclonal clones screened in step (4) were picked and inoculated into 96-well plates for culture. Helper phage M13KO7 was added. After culture, the supernatant (containing phages) was collected. An ELISA plate was coated with BSA (concentration 10 μg / mL) overnight at 4°C, the phage supernatant was added, and it was incubated at room temperature for 1 h. The binding signal was detected to obtain ELISA positive clones; (6) Amplification and sequencing: After amplifying the positive clones, a biological company was commissioned to sequence the obtained nanobodies.

[0024] Table 1

[0025] As a result, a bovine serum albumin-binding nanobody Nb1 with the amino acid sequence shown in SEQ ID NO.1 was obtained.

[0026] Example 2: Expression and purification of bovine serum albumin-binding nanobody LB medium formula: Tryptone 10 g / L, Yeast extract powder 5 g / L, Sodium chloride 10 g / L, Ampicillin 100 mg / L, pH 7.4.

[0027] Fermentation medium: Tryptone 12 g / L, Yeast extract 24 g / L, Glycerol 4 mL / L, K2HPO4 12.54 g / L, KH2PO4 2.31 g / L, pH 7.0.

[0028] Feed medium formula: Trace elements 100 mL / L, Glucose 400 g / L, Yeast powder 100 g / L, Magnesium sulfate 15.5 g / L.

[0029] The nucleotide sequence encoding the nanobody was codon-optimized according to the preference of Escherichia coli to obtain the corresponding nucleotide sequence, and the nucleotide sequence encoding the nanobody Nb1 of bovine serum albumin is shown in SEQ ID NO.2.

[0030] A biological company was commissioned to clone the bovine serum albumin nanobody gene (the nucleotide sequences are respectively as SEQ ID NO.2) into the expression vector pET-22b (containing His tag, His6-TEV site-nanobody), and the expression vector pET-22b was used to transform BL21(DE3) Escherichia coli, and the transformed bacteria were spread on an LB agar plate containing ampicillin and cultured at 37 °C for 12 hours.

[0031] Single colonies were picked and inoculated into 5 mL of LB medium (containing Amp) and cultured with shaking at 37 °C for 12 hours. Then, the culture was transferred to 500 mL of LB medium at a ratio of 1:100 and cultured at 37 °C until OD 600 = 1.2 to obtain a seed culture; the seed culture was transferred to a 5 L fermenter containing 2.5 L of fermentation medium at an inoculation amount of 5%, and the temperature was set at 37 °C, the aeration rate was 25 L / min, the pH was 7.0, the pressure was 0.050 Mpa, and the rotation speed was 200 rpm. The control mode was automatic, and the dissolved oxygen was controlled above 30% by adjusting the rotation speed and aeration rate during fermentation. When OD 600 no longer increased, fed-batch was started to ensure that the glucose concentration > 5 g / L; when OD600 ≥ 30, the fed-batch was stopped, the tank temperature was reduced to 20 °C within 1 hour, IPTG was added to a final concentration of 0.5 mM, and the cells were induced at 25 °C for 16 hours. Then, the cells were collected and centrifuged at 4 °C and 8000×g for 10 minutes, and the supernatant was discarded. The cell pellet was washed with PBS buffer (pH 7.4). The cells were resuspended in lysis buffer (containing 1 mM PMSF and 10 mM imidazole) and sonicated on ice (power 300 W, working for 3 seconds / interval 5 seconds, for a total of 20 minutes). Then, the mixture was centrifuged at 4 °C and 12000×g for 30 minutes, and the supernatant was collected.

[0032] Equilibrate the Ni-NTA column with binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). Load the crude extract onto the column at a flow rate of 1 mL / min to bind the His-tagged protein. Wash away non-specifically bound proteins successively with buffers containing 20 mM and 50 mM imidazole. Elute the nanobody with a buffer containing 250 mM imidazole and collect the elution peak. Load the eluate into a dialysis bag (molecular weight cut-off 7 kDa) and dialyze against PBS for 24 hours (4 °C, with buffer change 3 times) to remove imidazole and obtain purified bovine serum albumin-binding nanobody. Add TEV protease (1 U / mg protein) and incubate in a buffer of 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 8.0 at room temperature for 4 h. Then pass through the Ni column twice to remove the His-tag and protease, and obtain purified bovine serum albumin-binding nanobody.

[0033] Example 3: Characterization of Bovine Serum Albumin-Binding Nanobody (1)SDS-PAGE detection Perform SDS-PAGE detection on the purified sample: Mix the purified sample with Loading Buffer, boil for 5 minutes and then load the sample. Electrophorese on a 12% separating gel (constant voltage 120 V, 1 hour), stain with Coomassie Brilliant Blue to verify the molecular weight. The results are as Figure 1 shown. A band of approximately 14 kDa is obtained.

[0034] (2)ELISA binding verification of bovine serum albumin-binding nanobody Fuse the His-tag into the nanobody gene coding sequence, express the nanobody with His-tag. ELISA plates are respectively coated with bovine serum albumin (5 μM / mL, 100 μL / well) overnight at 4 °C. Block with 5% skim milk / PBST at 37 °C for 1 h. Then serially dilute the serum albumin-Nb complex, with the positive control being free nanobody and the negative control including only BSA. Incubate at 37 °C for 1 h, wash 3 times with PBST. Add TMB for color development, terminate with 2 M H2SO4, and measure OD 450 , and calculate the IC 50 (the concentration of BSA that inhibits 50% of Nb binding).

[0035] Replace bovine serum albumin with human serum albumin (BSA) and mouse serum albumin (MSA), and repeat the above steps.

[0036] The results are as Figure 2 shown.

[0037] As can be seen from Figure 2 it, the IC of Nb1-BSA50 is 9.08×10 -4 μM / mL; The IC 50 of Nb1-HSA is 1.56×10 -3 μM / mL; The IC 50 of Nb1-MSA is 3.42×10 -3 μM / mL.

[0038] According to the IC 50 values, the equilibrium dissociation constants were estimated, and the results are shown in Table 2.

[0039] Table 2

[0040] The equilibrium dissociation constants of Nb1 with BSA, HSA, and MSA are all less than 10 -8 , indicating that Nb1 has good binding affinity with BSA, HSA, and MSA. The nanobody Nb1 of bovine serum albumin of this application has a good broad-spectrum application range.

[0041] Example 4 Verification of drug half-life The fusion protein Nb1-IFNα-2b and IFNα-2b were prepared using Escherichia coli. The nanobody Nb1 was linked to the N-terminus of IFN through a flexible linker peptide ((GGGGS)3) to form the fusion protein Nb1-IFNα-2b (the nucleotide sequence is shown in SEQ ID NO.3). The nucleotide sequence of IFNα-2b is shown in SEQ ID NO.4. After protein purification, subsequent experiments were carried out.

[0042] Steps for culturing Escherichia coli: Pick a single colony and inoculate it into 5 mL of LB medium (containing Amp), and culture it with shaking at 37°C for 12 hours. Transfer it to 500 mL of LB medium at a ratio of 1:100 and culture it at 37°C until OD 600 = 1.2. Obtain the seed solution; transfer the seed solution to a 5 L fermenter containing 2.5 L of fermentation medium at an inoculation amount of 5%, set the temperature at 37°C, the ventilation volume at 25 L / min, the pH at 7.0, the pressure at 0.050 Mpa, and the rotation speed at 200 rpm. The control mode is automatic, and the dissolved oxygen is controlled above 30% by adjusting the rotation speed and ventilation volume during fermentation. When OD 600 no longer increases, start feeding to ensure that the glucose concentration > 5 g / L; when OD 600 ≥ 30, stop feeding, add IPTG to a final concentration of 1 mM, induce at 37°C for 16 hours, collect the bacteria, centrifuge at 8000×g for 10 minutes at 4°C, discard the supernatant, and wash the bacterial precipitate with PBS buffer (pH 7.4).

[0043] The purification steps are as follows: Add binding buffer (50 mM Tris-HCl, 300 mM NaCl, 10 - 20 mM imidazole, pH 8.0) to the recombinant E. coli precipitate after centrifugation (5000×g, 15 min, 4°C), and sonicate on ice (200 W, 3 s on / 5 s off, 10 min) until the bacterial solution becomes clear. Centrifuge at 12000×g for 30 min and collect the precipitate. Wash the precipitate twice with Tris buffer containing 2 M urea and 1% Triton X-100, then add denaturing buffer containing 6 M guanidine hydrochloride (50 mM Tris-HCl, pH 8.0, 10 mM DTT), stir at room temperature for 3 h to dissolve the inclusion bodies. Centrifuge at 15000×g for 25 min to remove insoluble matter and collect the supernatant. Add 7.5 mM DTT. Load the denatured protein into a dialysis bag and dialyze stepwise against urea concentrations of 8 M, 4 M, 2 M, 1 M, and 0 M, each dialysis for 5 h. The buffer formula during dialysis is 50 mM Tris-HCl, 0.5 M L-arginine, 1 mM GSH / GSSG, pH 8.0. Load the dialyzed sample onto Ni-NTA affinity chromatography, wash with buffer containing 20 - 50 mM imidazole for 10 - 15 CV to remove impurities, elute stepwise (250 / 300 / 500 mM imidazole), and collect the main peak. Elute with PBS to remove imidazole, and concentrate to the target concentration (1 mg / mL) using a 10 kDa ultrafiltration tube. Add TEV protease (1 U / mg protein) and incubate in a buffer of 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 8.0 at room temperature for 4 h. Pass through the Ni column twice to remove the His tag and protease, and obtain the purified target protein.

[0044] When purifying IFNα-2b, use a 7 kDa ultrafiltration tube for concentration.

[0045] The biological activity of interferon was determined by the reporter gene method. For the specific operation, refer to the second method of General Rule 3523 in the third part of the Chinese Pharmacopoeia 2020 Edition (https: / / shuju.qgyyzs.net / yd2020 / details15932GvoILqidTY53492.html). The biological activity of the fusion interferon was determined, and the results of its biological activity detection are shown in Table 3.

[0046] The results showed that the NB1-IFNα-2b obtained in the present invention had relatively ideal biological activity.

[0047] Table 3

[0048] Eight adult male SD rats weighing about 300 g and eight adult female SD rats weighing about 300 g were selected and randomly divided into two groups, with an equal number of male and female rats in each group. Each group was injected with 8×10 6 IU of IFNα-2b and the above-mentioned Nb1-IFNα-2b fusion protein; rats in the fusion protein group were bled from the tail vein at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, 52 h to collect serum. Rats in the IFNα-2 group were bled from the tail vein at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 h to collect serum for detecting the remaining interferon biological activity in the serum. The "blood drug activity-time curve" was plotted and data fitting was performed using software (as shown in Figure 3 and Figure 4 ), and the pharmacokinetic parameters were analyzed (as shown in Table 4).

[0049] Table 4

[0050] The results showed that the half-life t 1 / 2β of IFNα-2b was 1.79 h, and the t 1 / 2β of the fusion protein Nb1-IFNα-2b was 13.48 h, and the half-life was significantly prolonged.

[0051] As mentioned above, it is only the embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanobody that binds to bovine serum albumin, characterized in that, The amino acid sequence of the nanobody against bovine serum albumin is shown as SEQ ID NO.

1.

2. The nanobody binding bovine serum albumin according to claim 1, characterized in that The nanobody against bovine serum albumin can also bind to human serum albumin (HSA) and murine serum albumin (MSA).

3. A nucleic acid, characterized in that, The nucleic acid encodes the nanobody against bovine serum albumin according to any one of claims 1 or 2.

4. The nucleic acid according to claim 3, wherein The nucleic acid sequence is shown as SEQ ID NO.2 or has 95% homology with the sequence shown as SEQ ID NO.

2.

5. A biological material, characterized in that, The biological material is selected from any one of the following: I. A vector containing the nucleic acid according to claim 3 or 4; II. A transformant containing the vector described in I; III. A recombinant cell capable of producing the nanobody against bovine serum albumin according to claim 1 or 2 and / or containing at least one of the nucleic acids according to claim 3 or 4.

6. Use of the nanobody against bovine serum albumin according to any one of claims 1 or 2, the nucleic acid according to any one of claims 3 or 4, or the biological material according to claim 5 in any of the following aspects: A1. Use in the preparation of products for prolonging the half-life of drugs; A2. Use in the preparation of immunoassay or diagnostic products.

7. The application according to claim 6, wherein The product for prolonging the half-life of drugs described in A1 includes at least one of a fusion protein composed of the nanobody against bovine serum albumin and a protein drug, a conjugate formed by connecting the nanobody against bovine serum albumin and a therapeutic agent, and a pharmaceutical composition of the nanobody against bovine serum albumin.

8. A method for preparing the nanobody binding bovine serum albumin according to claim 1, characterized in that, The method includes the step of introducing the nucleic acid according to claim 2 into a recombinant cell to express the nanobody against bovine serum albumin according to claim 1.

9. The method according to claim 8, wherein The method includes the step of introducing the nucleic acid according to claim 3 into a recombinant cell to express the nanobody against bovine serum albumin according to claim 1.

10. The method according to any one of claims 8-9, characterized in that, The recombinant cell is selected from at least one of Chinese hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

Images