A nano-antibody against TNF-alpha and its preparation method and application

Abstract

The application belongs to the technical field of immunology, and particularly relates to an anti-TNF-alpha nanobody as well as a preparation method and application thereof. The application provides an anti-TNF-alpha nanobody, wherein the anti-TNF-alpha nanobody is prepared through gene expression of an anti-TNF-alpha nanobody. The anti-TNF-alpha nanobody prepared by the application can specifically recognize a TNF-alpha antigen, so as to accurately target an inflammation site, reduce non-specific reactions, and improve a treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, and particularly relates to an anti-TNF-α nanobody, its preparation method and application. Background Technology

[0002] Tumor necrosis factor-α (TNF-α), also known as cachexia, is a multifunctional cytokine that promotes cell apoptosis and growth, playing a crucial role in inflammation and immunity. TNF-α is primarily produced by activated monocytes and macrophages in vivo, although many malignant tumor cells can also produce small amounts. TNF-α promotes the progression of malignant tumors by acting as an endogenous promoter in the tumor and creating an inflammatory environment. Studies have found that approximately 20% of cancers are related to chronic infections. As a cellular signaling protein involved in systemic inflammatory responses and a pleiotropic cytokine, TNF-α has been identified as a key regulator of inflammatory responses.

[0003] Nanobodies, also known as heavy chain single-domain antibodies (VHHs), are a type of antibody discovered by scientists while studying the serum of camels. These antibodies differ significantly from traditional antibodies. They lack a light chain, containing only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Therefore, they have extremely small molecular weights, typically 2.5 nm in diameter and 4 nm in length, with a molecular weight of only 12–15 kDa, making them the smallest known active antigen-binding proteins. Nanobodies offer advantages such as simple structure, ease of genetic modification, small size, high antigen specificity, strong tissue penetration, and high stability. Furthermore, they can be mass-produced in bacterial expression systems, avoiding the high costs and long production cycles associated with cell production.

[0004] Tumor necrosis factor (TNF-α) blockers are considered one of the most effective treatments for rheumatoid arthritis (RA), and have also shown significant efficacy in treating chronic inflammatory diseases such as cervical spondylosis, psoriasis, and Crohn's disease. Currently, three TNF antagonists are widely used clinically: etanercept, infliximab, and adalimumab. These TNF-α inhibitors effectively neutralize TNF-α function by blocking the interaction between TNF-α and its receptors TNFR1 and TNFR2, thereby inhibiting the expression of inflammatory genes. However, the high cost and numerous side effects of these drugs in treating RA remain major drawbacks, hindering their widespread use in clinical practice.

[0005] Therefore, there is an urgent need for a new, effective drug targeting TNF-α for the treatment of rheumatoid arthritis that is economical, practical, and has few adverse reactions. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-TNF-α nanobody 1A5, its encoding gene, recombinant nanobody, recombinant vector, recombinant strain, and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first objective of this invention is to provide an anti-TNF-α nanobody, wherein the VHH chain of the TNF-α protein nanobody includes a framework region (FR) and a complementarity-determining region (CDR).

[0009] The framework region includes the following four amino acid sequences: FR1 as shown in SEQ ID NO.5, FR2 as shown in SEQ ID NO.6, FR3 as shown in SEQ ID NO.7, and FR4 as shown in SEQ ID NO.8;

[0010] The complementarity-determining region includes the following three amino acid sequences: CDR1 as shown in SEQ ID NO.13, CDR2 as shown in SEQ ID NO.14, and CDR3 as shown in SEQ ID NO.15.

[0011] Preferably, the amino acid sequence of the nanobody is shown in SEQ ID NO.1.

[0012] A second objective of the present invention is to provide a gene encoding an anti-TNF-α nanobody, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0013] A third objective of the present invention is to provide a recombinant nanobody against TNF-α, characterized in that it comprises the nanobody described in any one of claims 1-3.

[0014] Preferably, the amino acid sequence of the recombinant nanobody is shown in SEQ ID NO.3.

[0015] Preferably, the gene of the recombinant nanobody includes the gene encoding the nanobody according to any one of claims 1-3.

[0016] The fourth objective of this invention is to provide a method for preparing TNF-α nanobodies, characterized by comprising the following steps:

[0017] (1) The nucleotide sequence shown in SEQ ID NO.2 was cloned into the expression vector to obtain a recombinant plasmid. The recombinant plasmid was transformed into host cells to induce the expression of TNF-α protein nanobodies.

[0018] (2) Purify TNF-α protein nanobodies.

[0019] Preferably, the expression vector in step (1) is a pET-28a(+) plasmid vector.

[0020] The fifth objective of this invention is the application of the above-described nanobody, the above-described recombinant nanobody, or the product prepared by the above-described method of recombinant nanobody in one or more of the following: drugs for treating autoimmune diseases, antitumor drugs, diagnostic reagents for autoimmune diseases, and diagnostic reagents for tumors.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) The present invention expresses the gene of the anti-TNF-α nanobody 1A5 into a recombinant nanobody, which can specifically recognize the TNF-α antigen and can be applied to the molecular diagnosis of autoimmune diseases and tumors and the preparation of drugs for autoimmune diseases and anti-tumor diseases;

[0023] 2) Nanobodies are not easily decomposed naturally in vivo, have high stability, and have good cell penetration ability, enabling them to rapidly diffuse into tissues and specifically accumulate in target tissues. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is the first-round DNA electrophoresis image of the natural camel-derived nanobody gene library. From left to right, the DNA bands in the gel wells are: the first lane is a 2000bp marker (band sizes are 2000, 1000, 750, 500, 250, and 100bp respectively), and the second, third, and fourth lanes are PCR products with bands of approximately 700–900bp.

[0026] Figure 2 This is the second round of DNA electrophoresis images of the natural camel-derived nanobody gene library. From left to right, the DNA bands in the gel wells are: the first lane is a 2000bp marker (band size is the same as above), and the second, third, fourth, fifth, and sixth lanes are PCR products with bands of approximately 450bp.

[0027] Figure 3 A schematic diagram illustrating the colony PCR identification of phage VHH insertion rate;

[0028] Figure 4A schematic diagram of screening specific single positive clones using phage-ELISA: 1 is TNF-α antigen coated on an ELISA plate, 2 is phage supernatant, 3 is mouse anti-M13K07 antibody, 4 is goat anti-mouse IgG (AP) antibody, and 5 is TMB chromogenic solution.

[0029] Figure 5 The image shows the results of Phage-ELISA identification of single phage clones; PBS was used as a negative control and M13K07 as a positive control.

[0030] Figure 6 This is a schematic diagram of the purified anti-TNF-α-1A5 nanobody. The left image shows the SDS-PAGE electrophoresis staining of the fragmented sample, the eluent sample, and the eluted sample; the right image shows the SDS-PAGE electrophoresis staining of the purified anti-TNF-α-1A5 nanobody; lane 1 is 0.5 mg / mL BSA as the concentration standard, and lane 2 is the purified anti-TNF-α nanobody 1A5.

[0031] Figure 7 This is a schematic diagram of a Western blot of the anti-TNF-α-1A5 nanobody; in the diagram, lane M is the protein molecular standard and lane 1 is the TNF-α antigen.

[0032] Figure 8 This is a graph showing the affinity of the anti-TNF-α-1A5 nanobody for the TNF-α antigen.

[0033] Figure 9 Image showing the affinity assay (BLI) between the anti-TNF-α-1A5 nanobody and the TNF-α antigen; Detailed Implementation

[0034] The first objective of this invention is to provide an anti-TNF-α nanobody 1A5, wherein the VHH chain of the TNF-α protein nanobody includes a framework region FR and a complementarity-determining region CDR;

[0035] The frame region FR described in this invention preferably includes FR1, FR2, FR3 and FR4;

[0036] The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, as follows:

[0037] FR1:QVQLVESGGGLVEPGGSLRLSCAAS(SEQ ID NO.5)

[0038] FR2:MSWVRQAPGKGLEWVSE(SEQ ID NO.6)

[0039] FR3:NYAASVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYC(SEQ ID NO.7)

[0040] FR4:WGQGTQVTVSS (SEQ ID NO.8)

[0041] The nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the present invention are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively, as follows:

[0042] FR1:CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGGAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT (SEQ ID NO.9)

[0043] FR2:ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAG AA(SEQ IDNO.10)

[0044] FR3:AACTATGCAGCCTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGC CAAAAACACACTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTGT ATTACTGT(SEQ ID NO.11)

[0045] FR4: TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA (SEQ ID NO. 12).

[0046] The complementary regions (CDRs) of the antigenic determinants preferably include CDR1, CDR2, and CDR3;

[0047] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively, as follows:

[0048] CDR1:GFTFSSYW (SEQ ID NO.13)

[0049] CDR2:INQSGDTS(SEQ ID NO.14)

[0050] CDR3:AKLGLSTWPYDY(SEQ ID NO.15);

[0051] The nucleotide sequences encoding CDR1, CDR2, and CDR3 of this invention are shown in SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, respectively, as follows:

[0052] CDR1:GGATTCACCTTCAGCAGCTACTGG(SEQ ID NO.16)

[0053] CDR2:ATTAATCAAAGCGGTGATACTTCA(SEQ ID NO.17)

[0054] CDR3: GCAAAATTAGGTCTAAGTACCTGGCCGTATGACTAC (SEQ ID NO. 18).

[0055] The nanobody is preferably nanobody 1A5, and the amino acid sequence of nanobody 1A5 is preferably as shown in SEQ ID NO.1, specifically as follows:

[0056] QVQLVESGGGLVEPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSEINQSGDTSNYAASVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAKLGLSTWPYDYWGQGTQVTVSS

[0057] A second objective of this invention is to provide a gene encoding an anti-TNF-α nanobody, the nucleotide sequence of which is shown in SEQ ID NO.2, and is as follows:

[0058] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGGAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCAGCTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGAAATTAATCAAAGCGGTGATACTTCAAACT ATGCAGCCTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAAAACACACTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCAAAATTAGGTCTAAGTACCTGGCCGTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0059] The preferred method for obtaining the anti-TNF-α nanobody 1A5 includes the following steps:

[0060] A natural camel-derived nanobody phage display library was constructed using phage surface display technology. Then, based on biotinylated TNF-α antigen, the gene sequence of anti-TNF-α specific nanobody 1A5 was obtained through screening. The CDR1, CDR2, and CDR3 regions of the anti-TNF-α nanobody 1A5 sequence were transplanted into the hs2dAb backbone to obtain the anti-TNF-α nanobody 1A5.

[0061] The method for constructing the natural camel-derived heavy chain antibody phage display gene library includes the following steps: 1) extracting total RNA from camel peripheral blood mononuclear cells and reverse transcribing the total RNA to obtain cDNA; 2) using the cDNA as a template to perform nested PCR amplification to obtain the variable region fragment of the heavy chain antibody; 3) digesting the variable region fragment of the heavy chain antibody and the pCANTAB5E phage vector with enzymes respectively, and then ligating them to obtain the ligation product; 4) transforming the ligation product into competent TG1 cells to obtain the natural camel-derived nanobody phage display library.

[0062] The present invention does not specifically limit the method for extracting total RNA from camel peripheral blood mononuclear cells; conventional animal peripheral blood total RNA extraction methods in the art are acceptable. The reverse transcription is preferably performed using the Thermo Scientific ReverAid First Strand cDNA Synthesis Kit.

[0063] After obtaining the cDNA, nested PCR is performed using the cDNA as a template to obtain the variable region fragment of the heavy chain antibody. The nested PCR preferably includes two rounds of PCR: a first round of PCR is used to amplify the fragment between the heavy chain antibody guide peptide and antibody CH2, and the primer sequences for the first round of PCR are preferably shown in SEQ ID NO.19 and SEQ ID NO.20; a second round of PCR is used to amplify the fragment between the heavy chain antibody FR1 region and the long and short hinge regions, and the primer sequences for the second round of PCR are preferably shown in SEQ ID NO.21 and SEQ ID NO.22.

[0064] After obtaining the variable region fragment of the heavy chain antibody, the fragment is digested with enzymes and then ligated with the pCANTAB5e phage vector to obtain the ligation product. The enzyme digestion is preferably a double digestion using restriction endonucleases SifI and NotI. The preferred digestion program is: digestion at 37℃ for 1 hour; digestion at 50℃ for 1 hour; ligation temperature at 16℃ for 4 hours.

[0065] After obtaining the ligation product, the ligation product is transformed into competent cells to obtain a natural camel-derived nanobody phage display library. The competent cells are preferably *E. coli* competent cells TG1 prepared by glycerol resuspension. The transformation method is preferably electroporation, followed by a phage rescue process. This invention does not specifically limit the electroporation and phage rescue processes; detailed steps are described in the embodiments. This invention does not specifically limit the specific steps for screening anti-TNF-α nanobodies using the biotinylated TNF-α antigen; conventional nanobody screening methods in the art can be used; detailed steps are described in the embodiments.

[0066] A third objective of this invention is to provide a recombinant nanobody against TNF-α, comprising the nanobody.

[0067] Preferably, the amino acid sequence of the recombinant antibody is as shown in SEQ ID NO.3, specifically as follows:

[0068] MGHHHHHHQVQLVESGGGLVEPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSEINQSGDTSNYAASVKGRFTISRDNAKNTLYLQMNNLKPEDTA VYYCAKLGLSTWPYDYWGQGTQVTVSS

[0069] Preferably, the gene of the recombinant nanobody includes a gene encoding the nanobody.

[0070] The nucleotide sequence of the gene for the recombinant nanobody is shown in SEQ ID NO.4, and is as follows:

[0071] ATGGGACACCACCACCACCACCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGGAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCAGCTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGAAATTAATCAAAGCGGTG ATACTTCAAACTATGCAGCCTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAAAACACACTGTATCTGCAAATGAACAACCTGAAAACCTGAGGACACGGCCGTGTATTACTGTGCAAAATTAGGTCTAAGTACCTGGCCGTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0072] The nucleotide sequence of the His tag gene is shown in SEQ ID NO.23, and is as follows: ATGGGACACCACCACCACCACCAC

[0073] The fourth objective is to provide a method for preparing TNF-α nanobodies, comprising the following steps:

[0074] (1) The nucleotide sequence shown in SEQ ID NO.2 was cloned into the expression vector to obtain a recombinant plasmid. The recombinant plasmid was transformed into host cells to induce the expression of TNF-α protein nanobodies.

[0075] (2) Purify TNF-α protein nanobodies.

[0076] The recombinant expression vector includes an initial vector and a gene expressed by inserting into the initial vector; the initial vector is preferably a pET-28a(+) plasmid vector; the gene insertion site of the recombinant nanobody is preferably located at the Nco I-Xho I site of the pET-28a(+) plasmid vector. The initial strain is Escherichia coli, preferably Escherichia coli Arctic Express; the present invention does not have any particular limitation on the preparation method of the recombinant vector and the recombinant strain, and conventional recombinant vector preparation methods in the art can be used.

[0077] The gene expressed by the inserted initial vector includes the gene encoding the nanobody;

[0078] The fifth objective of this invention is the application of the above-described nanobody, the above-described recombinant nanobody, or the product prepared by the above-described method of recombinant nanobody in one or more of the following: drugs for treating autoimmune diseases, antitumor drugs, diagnostic reagents for autoimmune diseases, and diagnostic reagents for tumors.

[0079] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0080] Example 1

[0081] Construction of a natural camel-derived nanobody gene library

[0082] (1) Extract total RNA from camel peripheral blood mononuclear cells. The specific steps are as follows:

[0083] ① Blood collection from Bactrian camels was performed by professionals. Percoll cell separation solution was used to separate peripheral blood mononuclear cells from non-immunized camels. The separated peripheral blood mononuclear cells were washed three times with PBS and 1 mL of Trizol solution was added.

[0084] ② Add 0.2 mL of chloroform per 1 mL of Trizol, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0085] ③ Centrifuge the sample at 12000 rpm and 4℃ for 15 min, and transfer the upper layer to a new Ep tube.

[0086] ④ Add an equal volume of isopropanol and mix on ice for 20 minutes.

[0087] ⑤ Centrifuge the solution at 12000 rpm and 4℃ for 10 min, and discard the supernatant.

[0088] ⑥ Wash the precipitate with 1 mL of 75% ethanol prepared with DEPC water (add at least 1 mL of ethanol for every 1 mL of Trizol).

[0089] ⑦ Centrifuge the solution from the previous step at 10,000 rpm for 10 minutes at 4℃, discard the supernatant, and repeat the washing process once more.

[0090] ⑧ Centrifuge at 10000 rpm and 4℃ for 10 min, discard the supernatant, and dry for 10-15 minutes.

[0091] ⑨ Add an appropriate amount of ddH2O to dissolve the RNA precipitate, determine the RNA concentration, and store at -80℃.

[0092] RNA was purified using a purification kit provided by TIANGEN, and cDNA was obtained by reverse transcription using the Thermo Scientific ReverAid First Strand cDNA Synthesis Kit.

[0093] (2) Using cDNA as a template, nested PCR was used to amplify the variable region fragment of the heavy chain antibody;

[0094] First round of PCR:

[0095] Upstream primer: 5'-GTCCTGGCTGCTCTTCTACAAAG-3' (SEQ ID NO.19)

[0096] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO.20)

[0097] First round PCR reaction system

[0098]

[0099] The conditions for the first round of PCR amplification were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, for 32 cycles; 72℃ for 10 min.

[0100] The fragment between the heavy chain antibody guide peptide and antibody CH2 was amplified. This primer pair was used to amplify a 900bp vh-CH1-CH2 fragment and a 700bp vhh-CH2 fragment, respectively; the results showed that the fragment size was approximately 700-900bp, meaning there were approximately two nanobody gene electrophoresis bands. Figure 1 As shown.

[0101] Second round of PCR:

[0102] Using the first round of PCR products as templates

[0103] Upstream primer: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3' (SEQ ID NO. 21)

[0104] Downstream primer: 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3'(SEQ IDNO.22)

[0105] Second round PCR reaction system

[0106]

[0107] The second round of PCR amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, 25 cycles; 72℃ for 10 min. Fragments (long and short fragments) between the FR1 region and the long and short hinge regions of the heavy chain antibody were amplified to obtain approximately 400 bp of the camel heavy chain antibody heavy chain variable region vhh fragment from the 700 bp vhh-CH2 fragment. The results are as follows: Figure 2 The size of the fragment is approximately 450 bp, meaning the nanobody gene electrophoresis band is approximately 450 bp.

[0108] The pCANTAB5e phage vector and VHH fragment were digested with restriction endonucleases (SifI and NotI, purchased from NEB), and the two fragments were ligated using T4 DNA ligase (purchased from NEB). The specific digestion and ligation systems are as follows:

[0109]

[0110] Enzyme digestion conditions: Digest at 37℃ for 1 hour, then digest at 50℃ for 1 hour.

[0111] Connection system

[0112]

[0113] Connection conditions: Connect at 16℃ for 4 hours, then connect overnight at 4℃.

[0114] The ligation product was electroporated into electrocompetent TG1 cells to construct a natural camel-derived nanobody phage display library. After helper phage rescue, the library size reached 9.0 × 10⁻⁶. 13 .

[0115] The preparation method of competent TG1 cells is as follows: *E. coli* TG1 glycerol bacteria were taken from a -80℃ freezer and streaked onto a 2×YT solid plate. The culture was incubated at 37℃ for 10 h. A single colony was picked and inoculated into 3 mL of 2×YT liquid medium, and cultured overnight at 37℃ with shaking at 200 rpm. The next day, the bacterial culture was scaled up at a 1:100 ratio in an Erlenmeyer flask containing 200 mL of 2×YT medium. The culture was continued at 37℃ until the OD600 reached approximately 0.4. The bacterial culture was then collected in a 50 mL centrifuge tube, incubated on ice for 1 h, centrifuged at 9000 rpm at 4℃ for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended in an equal volume of cold pure water. This process was repeated once. Then, the bacterial pellet was resuspended in pre-chilled 10% glycerol and centrifuged. The bacterial precipitate was suspended in 1 mL of 10% glycerol (pre-cooled pure water) and dispensed into 1 mL Eppendorf tubes (100 μl per tube) that had been pre-cooled. The tubes were then immediately transferred to a -80°C freezer for storage. These were the competent cells TG1.

[0116] The steps for assisting phage rescue are as follows:

[0117] ① Take 100 μL of the library and inoculate it into 50 mL of 2×YT / Amp / Glu medium. Incubate at 37℃ and 200 rpm with shaking until the OD600 in the logarithmic phase is about 0.4 to 0.5.

[0118] ② Add helper phage M13KO7 with an infection multiplicity of 20:1 to the culture medium, mix well, and let stand at 37°C for 30 min.

[0119] ③ Centrifuge the culture medium at 9000 rpm for 10 min at room temperature, discard the supernatant precipitate of bacterial cells, resuspend in 200 mL of 2×YT / Amp / Kana culture medium, and incubate overnight at 37℃ and 200 rpm.

[0120] ④ Centrifuge the culture medium at 4℃ and 9000rpm for 10min, collect the supernatant, add 1 / 5 volume of PEG / NaCl, and let it stand at 4℃ for 6h.

[0121] ⑤ Centrifuge at 9000 rpm for 20 min, discard the supernatant, resuspend the precipitate with PBS (1 mL) to obtain the recombinant phage antibody library, aliquot into 1.5 mL Eppendorf tubes, and store at 4 °C.

[0122] Meanwhile, the insertion rate of the library was detected by colony PCR. Second-round PCR primers were used, and the annealing temperature was 55℃. The results were as follows: Figure 3 The insertion rate is shown to be over 95% (target fragment insertion rate = number of colonies containing the target fragment / total number of colonies).

[0123] Screening process for anti-TNF-α nanobodies

[0124] phage library (1×10) 13After incubating 50 μL of streptavidin magnetic beads with a bacteriophage on a rotating platform at room temperature for 1 h, the bacteriophage antibody was collected. In two 1 mL centrifuge tubes blocked with 2% PBSM, 500 μL of pre-thresholded bacteriophage antibody was added to each tube. 500 μL of 5 μg of biotinylated TNF-α antigen diluted in PBS was added to one tube, and 500 μL of PBS buffer was added to the other tube as a negative control. Both tubes were incubated on a rotating platform at room temperature for 1 h. Then, 50 μL of pre-blocked streptavidin magnetic beads were added, and the tubes were incubated on a rotating platform at room temperature for 30 min. The magnetic beads were collected. The beads were washed 7 times with PBST, 2 times with PBSM, and 1 time with PBS. Eluting was performed with Glycine at pH 2.7, and neutralization was performed with 1 mol / L Tris-HCl at pH 9.1. The above neutralization solution was added to 5 mL of TG1 (OD600 = 0.5) in the logarithmic growth phase to generate and purify phages for the next round of screening. After three rounds of screening, positive clones will be continuously enriched, thereby achieving the goal of screening TNF-α specific antibodies in the antibody library using phage display technology.

[0125] Phage-ELISA method for screening specific single positive clones

[0126] The principle diagram of the filtering process is as follows: Figure 4 As shown, the method is as follows: First, prepare the VHH phage monoclonal supernatant: randomly pick 180 single colonies from the solid plates after 3 rounds of screening and inoculate them into 96-well plates of 2×YT medium containing 100 μg / mL ampicillin and 2% glucose, incubate at 220 rpm. Incubate overnight at 37°C. The next day, transfer 50 μL of bacterial culture to a new 96-well plate. Add 800 μL of 2×YT medium containing 100 μg / mL ampicillin and 2% glucose to each well. After growth to the logarithmic phase, add helper phage M13K07 with an infection multiplicity of 20:1. Infect at 37°C for 30 min, centrifuge at 10,000 rpm for 5 min, discard the supernatant, and resuspend the bacteria in 800 μL of fresh 2×YT medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. Incubate at 37°C and 220 rpm for 12 h. The next day, centrifuge the bacterial culture at 10,000 rpm for 5 min. The supernatant is the supernatant of VHH phage monoclonal cells.

[0127] Dilute TNF-α antigen to 10 μg / mL with coating buffer, add 100 μL to each well, and coat overnight at 4°C. Set up negative and positive controls. The next day, wash three times with PBST, block with 2% PBSM at 37°C for 2 h, wash three times with PBST, add 200 μL of pretreated VHH phage monoclonal supernatant, and incubate at 37°C for 1 h. Add 1:5000 of murine anti-M13KO7 / HRP secondary antibody diluted with 0.1% PBST, incubate at 37°C for 1 h, wash away unbound antibody, add TMB chromogenic buffer, and read the absorbance at 450 nm using a microplate reader. A sample well with an OD value more than twice that of a control well is considered a positive control well. Figure 5 Gene sequencing was performed on positive bacterial cultures.

[0128] Sequence analysis and BLAST alignment were performed using Snapegene software. Strains with identical CDR1, CDR2, and CDR3 sequences were considered to be the same clone. The nanobody sequence shown in SEQ ID NO.1 was ultimately used for subsequent experiments.

[0129] Example 2

[0130] Expression and purification of nanobody TNF-α-1A5-Nb in host bacterium Escherichia coli

[0131] (1) The subclone pET-28a(+) plasmid vector of the nanobody sequence obtained by sequencing analysis was transformed into E. coli Arctic Express. Single colonies on the transformation plate were picked and inoculated into test tubes containing 3 mL LB medium of 50 μg / mL Kan and shaken at 220 rpm overnight at 37℃; (2) The next day, the cells were inoculated at 1:100 into 30 mL LB medium of 50 μg / mL Kan and shaken at 220 rpm at 37℃ until the bacterial OD600 was 0.6-0.8. IPTG was added to a final concentration of 0.5 mM and shaken at 220 rpm overnight at 20℃ to induce fusion protein expression; (3) The bacterial cells were collected and sonicated to obtain a crude solution of inclusion body protein, which was then purified by Ni column affinity to obtain the fusion protein, such as Figure 6 As shown.

[0132] Example 3

[0133] Specificity validation of anti-TNF-α nanobody 1A5:

[0134] Western blot was performed using TNF-α antigen (with purified anti-TNF-α nanobody as the primary antibody and anti-mouse His / HRP as the secondary antibody). Figure 7 As shown, the anti-TNF-α nanobody can specifically bind to the TNF-α antigen.

[0135] Example 4

[0136] ELISA method for detecting the specific binding of TNF-α nanobody 1A5 to TNF-α antigen

[0137] TNF-α antigen was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL was added to each well of a 96-well ELISA plate for coating at 4℃. PBS coating served as a negative control. The next day, the plate was washed three times with 0.05% PBST, blotted dry with absorbent paper, and 300 μL of 5% PBSM was added to each well for blocking at 37℃ for 2 h. After blocking, the plate was washed three times with PBST and blotted dry. 100 μL of experimental and control samples were added to each well, and the plate was incubated at 37℃ for 1 h. The plate was washed three times with PBST and blotted dry. Mouse His antibody was diluted 1:5000 with 0.05% PBST, and 100 μL was added to each well. The plate was incubated at 37℃ for 1 h, then washed and blotted dry. Goat anti-mouse HRP antibody was diluted 1:5000 with 0.05% PBST, and 100 μL was added to each well. The plate was incubated at 37℃ for 1 h, then washed and blotted dry. The detection results are as follows: Figure 8 As shown, the anti-TNF-α-1A5-Nb nanobody specifically binds to the TNF-α antigen.

[0138] Example 5

[0139] Specificity detection of anti-TNF-α nanobody 1A5 and TNF-α antigen

[0140] The affinity of nanobodies was determined using biomembrane interference (BLI) technology. Biotinylated humanized TNF-α antigen was bound to a Streptavidin (SA) probe. Six nanobody concentration gradients (1000, 500, 250, 125, 62.5, and 31.25 nmol / L) were established. The binding and dissociation of each concentration of nanobodies with the biotinylated humanized TNF-α antigen were measured using Octet Red96e. The detection data were fitted using a 1:1 binding model, and binding and dissociation curves of TNF-α nanobodies at each concentration were plotted. The KD value was calculated. Results Figure 9 The display shows that its binding constant is 6.087 × 10⁻⁶. -4 The dissociation constant is 7.460 × 10⁻⁶. -3 The equilibrium dissociation constant (KD) was calculated to be 4.892 × 10⁻⁶. -7 The results suggest that the anti-TNF-α-1A5 nanobody has a strong ability to specifically bind to the TNF-α antigen.

[0141] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nanobody against TNF-α, characterized in that, The VHH chain of the anti-TNF-α nanobody includes a framework region (FR) and a complementarity-determining region (CDR). The frame region FR includes the following four amino acid sequences: FR1 as shown in SEQ ID NO.5, FR2 as shown in SEQ ID NO.6, FR3 as shown in SEQ ID NO.7, and FR4 as shown in SEQ ID NO.8; The complementarity-determining region (CDR) includes the following three amino acid sequences: CDR1 as shown in SEQ ID NO.13, CDR2 as shown in SEQ ID NO.14, and CDR3 as shown in SEQ ID NO.

15.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

3. A gene encoding an anti-TNF-α nanobody, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A recombinant nanobody against TNF-α, characterized in that, It consists of the nanobody and His tag as described in claim 1 or 2.

5. The recombinant nanobody according to claim 4, characterized in that, The amino acid sequence of the recombinant nanobody is shown in SEQ ID NO.

3.

6. A gene encoding the recombinant nanobody of claim 4, characterized in that, The gene includes a gene encoding the nanobody of claim 1 or 2.

7. The method for preparing the anti-TNF-α nanobody according to claim 1 or 2, characterized in that, Includes the following steps: (1) The nucleotide sequence shown in SEQ ID NO.2 was cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid was transformed into a host cell and expression was induced to obtain the anti-TNF-α nanobody. (2) Purify the anti-TNF-α nanobody obtained in step (1).

8. The preparation method according to claim 7, characterized in that, The expression vector in step (1) is the pET-28a(+) plasmid vector.

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