A humanized nanoantibody against TNF-α and its application

Abstract

The present invention belongs to the technical field of immunology, and in particular, relates to a humanized nanoantibody against TNF-α and its application. The present invention provides a humanized nanoantibody against TNF-α, and a recombinant humanized nanoantibody against TNF-α prepared by expressing the gene of the humanized nanoantibody against TNF-α. The humanized nanoantibody against TNF-α prepared by the present invention can specifically recognize the TNF-α antigen, thereby accurately targeting the inflammatory site, reducing nonspecific reactions, and improving the therapeutic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology, and in particular relates to a humanized anti-TNF-α nano antibody and an application thereof. Background Art

[0002] Tumor necrosis factor-α (TNF-α), also known as cachexia, is a multifunctional cytokine that promotes cell apoptosis and growth and plays a key role in inflammation and immunity. TNF-α is mainly produced by activated mononuclear macrophages in the body, and many malignant tumor cells can also produce a small amount of TNF-α. TNF-α promotes the progression of malignant tumors by creating an inflammatory environment and acting as an endogenous promoter of tumors. The TNF-α precursor (i.e., membrane-associated TNF-α) is composed of 233 amino acids, including a signal peptide composed of 76 amino acid residues, while mature TNF-α (also known as secretory TNF-α) is formed by the removal of the signal peptide by tumor necrosis factor converting enzyme to form a structure with 157 amino acid residues, and its relative molecular weight is 1.7×10 4 . TNF-α acts in the form of a trimer, which is composed of three identical monomer subunits and belongs to type II membrane protein. The subunits attached to the trimer conduct signal transduction and exert their effects by binding to different TNF-α receptors. Studies have found that about 20% of cancers are related to chronic infections. TNF-α is a cell signaling protein involved in systemic inflammatory response and a pleiotropic cytokine. It has been identified as a key regulator of inflammatory response.

[0003] Tumor necrosis factor (TNF-α) blocking drugs are considered to be one of the highly effective means of treating rheumatoid arthritis (RA), and they also show significant effects in the treatment of chronic inflammatory diseases such as cervical spondylosis, psoriasis and Crohn's disease. Currently, there are three TNF antagonists widely used in clinical practice, namely etanercept, infliximab and adalimumab. These TNF-α inhibitors effectively neutralize the function of TNF-α 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 many side effects of these drugs in treating RA are still the main disadvantages, which hinder their widespread application in clinical practice.

[0004] Therefore, there is an urgent need for a new effective drug targeting TNF-α for the treatment of rheumatoid arthritis, which is economical and practical, and has weak adverse reactions. Nanobodies, as tiny functional antigen-binding fragments, have advantages that ordinary antibodies do not have, such as small molecular weight, good stability, high affinity, low immunogenicity, and strong tissue penetration. They can make up for the defects of monoclonal antibody drugs and have great application value and development prospects in disease diagnosis and treatment as well as in the food field. Summary of the invention

[0005] The purpose of the present invention is to provide a humanized anti-TNF-α nanobody, encoding gene, recombinant nanobody, recombinant vector, recombinant strain and application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a humanized anti-TNF-α nanobody, wherein the humanized nanobody comprises a humanized framework region FR and an antigenic determinant complementary region CDR;

[0008] The complementary region CDR of the antigen determinant includes CDR1, CDR2 and CDR3;

[0009] 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.

[0010] Preferably, the frame region FR includes FR1, FR2, FR3 and FR4;

[0011] 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.

[0012] Preferably, the amino acid sequence of the humanized Nanobody is as shown in SEQ ID NO.1.

[0013] The second object of the present invention is to provide a gene encoding a humanized anti-TNF-α nanobody, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] The third object of the present invention is to provide a recombinant humanized anti-TNF-α nanobody, comprising the nanobody and a His tag expressed by fusion with the humanized nanobody.

[0015] Preferably, the amino acid sequence of the recombinant humanized antibody is as shown in SEQ ID NO.3.

[0016] Preferably, the gene of the recombinant Nanobody includes a gene encoding the humanized Nanobody.

[0017] The fourth object of the present invention is to provide a recombinant expression vector expressing a recombinant humanized nanobody against TNF-α, wherein the recombinant expression vector comprises an initial vector and a gene expressed by the initial vector;

[0018] The genes expressed by the inserted initial vector include genes encoding the humanized nanobody.

[0019] The fifth object of the present invention is to provide a recombinant strain expressing a recombinant humanized nanobody against TNF-α, wherein the recombinant strain comprises an initial strain and the recombinant expression vector;

[0020] The initial strain is Escherichia coli.

[0021] The sixth object of the present invention is to provide an application of the above-mentioned humanized nanoantibody, the gene encoding the humanized nanoantibody against TNF-α, the recombinant nanoantibody, the gene encoding the recombinant nanoantibody, the recombinant expression vector of the recombinant humanized nanoantibody or the recombinant strain of the recombinant humanized nanoantibody in the preparation of one or more of the anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The recombinant nanobody expressed by fusion of the gene of the anti-TNF-α humanized nanobody 1A5 and the His tag gene can specifically recognize the TNF-α antigen and can be applied to the molecular diagnosis of autoimmune diseases and tumors and the preparation of autoimmune disease and anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative labor.

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

[0026] Figure 2 This is the second round of DNA electrophoresis of the nanobody. The DNA bands in the gel wells from left to right are: the first lane is a 2000 bp marker (band size is the same as above), the second, third, fourth, fifth and sixth lanes are PCR products, and the band is about 450 bp;

[0027] Figure 3 Schematic diagram of the insertion rate of phage VHH identified by colony PCR;

[0028] Figure 4 The schematic diagram of screening specific single positive clones by phage-ELISA method (phage-ELISA): 1 is TNF-α antigen coated on the ELISA plate, 2 is phage supernatant, 3 is mouse anti-M13K07 antibody, 4 is goat anti-mouse IgG (AP) antibody, and 5 is TMB colorimetric solution;

[0029] Figure 5 Schematic diagram of purified anti-TNF-α-1A5 humanized nanobody; the left figure is the SDS-PAGE electrophoresis staining of the post-crushing treatment sample, the outflow sample and the elution sample; the right figure is the SDS-PAGE electrophoresis staining of the purified anti-TNF-α-1A5 humanized nanobody, lane 1 is 0.5 mg / mL BSA as a concentration standard, and lane 2 is the purified anti-TNF-α humanized nanobody 1A5;

[0030] Figure 6 It is a schematic diagram of western blot of anti-TNF-α-1A5 humanized nanobody; wherein, lane M is the protein molecule standard and lane 1 is the TNF-α antigen.

[0031] Figure 7 This is a graph showing the affinity test between the anti-TNF-α-1A5 humanized nanobody and the TNF-α antigen;

[0032] Figure 8 This is the affinity determination diagram of anti-TNF-α-1A5 humanized nanobody and TNF-α antigen (BLI); DETAILED DESCRIPTION

[0033] The first object of the present invention is to provide a humanized anti-TNF-α nanobody, wherein the humanized nanobody comprises a humanized framework region FR and an antigenic determinant complementary region CDR;

[0034] The complementary region CDR of the antigen determinant preferably includes CDR1, CDR2 and CDR3;

[0035] The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and are as follows:

[0036] CDR1:GFTFSSYW (SEQ ID NO.13)

[0037] CDR2:ISNQSGDTS (SEQ ID NO. 14)

[0038] CDR3:AKLGLSTWPYDY(SEQ ID NO.15);

[0039] The nucleotide sequences encoding the CDR1, CDR2 and CDR3 of the present invention are shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively, and are as follows:

[0040] CDR1:GGTTTCACGTTTTCTCTTACTGG(SEQ ID NO.16)

[0041] CDR2:ATTTCCAATCAAAGCGGCGACACCAGC(SEQ ID NO.17)

[0042] CDR3: GCGAAGCTGGGCCTGTCGACCTGGCCGTACGATTAT (SEQ ID NO. 18).

[0043] The framework region FR of the present invention preferably includes FR1, FR2, FR3 and FR4;

[0044] 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, and are as follows:

[0045] FR1:EVQLQASGGGFVQPGGSLRLSCAAS(SEQ ID NO.5)

[0046] FR2:MGWFRQAPGKEREFVSA(SEQ ID NO.6)

[0047] FR3:YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC(SEQ ID NO.7)

[0048] FR4:WGQGTQVTVSS (SEQ ID NO.8)

[0049] The nucleotide sequences encoding the 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, and are specifically as follows:

[0050] FR1:GAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCA (SEQ ID NO.9)

[0051] FR2:ATGGGTTGTTTCGTCAAGCACCGGGCAAAGAGCGCGAGTTCGTGTCCGCG (SEQ IDNO.10)

[0052] FR3:TACTACGCCGACAGCGTTAAAGGCCGTTTTACGATCAGCCGTGATAACTCG AAGAACACCGTGTATTTGCAAATGAACTCTTTGCGCGCTGAAGACACTGCGACCTA TTACTGC (SEQ ID NO. 11)

[0053] FR4:TGGGGTCAGGGCACCCAGGTTACCGTTAGCAGC (SEQ ID NO. 12).

[0054] In the present invention, the humanized Nanobody is preferably the humanized Nanobody 1A5, and the amino acid sequence of the humanized Nanobody 1A5 is preferably as shown in SEQ ID NO.1, specifically as follows:

[0055] EVQLQASGGGFVQPGGSLRLSCAASGFTFSSYWMGWFRQAPGKEREFVSAISNQSGDTSYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKLGLSTWPYDYWGQGTQVTVSS

[0056] The second object of the present invention is to provide a gene encoding a humanized nanobody against TNF-α, the nucleotide sequence of which is shown in SEQ ID NO.2, as follows:

[0057] GAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCAGGTTTCACGTTTCCTCTTACTGGATGGTTGGTTTCGTCAAGCACCGGGCAAAGAGCGCGAGTTCGTGTCCGCGATTTCCAATCAAAGCGGCGACACCAGCTAC TACGCCGACAGCGTTAAAGGCCGTTTTACGATCAGCCGTGATAACTCGAAGAACACCGTGTATTTGCAAATGAACTCTTTGCGCGCTGAAGACACTGCGACCTATTACTGCGCGAAGCTGGGCCTGTCGACCTGGCCGTACGATTATTGGGGTCAGGGCACCCAGGTTACCGTTAGCAGC

[0058] The method for obtaining the anti-TNF-α humanized nanobody 1A5 of the present invention comprises the following steps:

[0059] A natural camel-derived nanoantibody phage display library is constructed using phage surface display technology; then, the gene sequence of the anti-TNF-α nanoantibody 1A5 is screened based on biotinylated TNF-α antigen; the CDR1, CDR2, and CDR3 regions of the anti-TNF-α nanoantibody 1A5 sequence are transplanted into a humanized hs2dAb framework to obtain the anti-TNF-α humanized nanoantibody 1A5.

[0060] The method for constructing the natural camel-derived heavy chain antibody phage display gene library preferably includes the following steps: 1) extracting total RNA from camel peripheral blood mononuclear cells and reverse transcribing the total RNA to obtain cDNA; 2) performing nested PCR amplification using the cDNA as a template to obtain the variable region fragment of the heavy chain antibody; 3) respectively enzymatically cutting the variable region fragment of the heavy chain antibody and the pCANTAB5E phage vector, and then connecting to obtain a connection product; 4) transferring the connection product into the competent cell TG1 to obtain a natural camel-derived nanoantibody phage display library.

[0061] The present invention extracts total RNA from camel peripheral blood mononuclear cells and reverse transcribes the total RNA to obtain cDNA. There is no special limitation on the method for extracting total RNA from camel peripheral blood mononuclear cells, and the conventional method for extracting total RNA from animal peripheral blood in the art can be used. Reverse transcription is preferably achieved by Thermo Scientific ReverAid First Strand cDNA Synthesis Kits.

[0062] After obtaining the cDNA, nested PCR amplification 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: the first round of PCR is used to amplify the fragment between the heavy chain antibody guide peptide and the antibody CH2, and the primer sequences of the first round of PCR are preferably as shown in SEQ ID NO.19 and SEQ ID NO.20; the 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 of the second round of PCR are preferably as shown in SEQ ID NO.21 and SEQ ID NO.22.

[0063] After obtaining the variable region fragment of the heavy chain antibody, the variable region fragment of the heavy chain antibody and the pCANTAB5e phage vector are respectively digested and then connected to obtain a connection product. The digestion is preferably double digestion, using restriction endonucleases SifI and Not I. The digestion procedure is preferably: digestion at 37°C for 1h; digestion at 50°C for 1h; connection temperature is 16°C, and time is 4h.

[0064] After obtaining the ligation product, the ligation product is transferred into competent cells to obtain a natural camel-derived nano-antibody phage display library. The competent cells are preferably homemade Escherichia coli competent cells TG1 by glycerol resuspension method; the transfer method is preferably electro-transformation. After transformation, the process of auxiliary phage rescue is also included. The present invention does not specifically limit the process of electro-transformation and auxiliary phage assistance. For detailed steps, please refer to the examples. The present invention does not specifically limit the specific steps of screening anti-TNF-α nano-antibodies with the biotinylated TNF-α antigen. Conventional nano-antibody screening methods in the field can be used. For detailed steps, please refer to the examples.

[0065] The third object of the present invention is to provide a recombinant humanized anti-TNF-α nanobody, including the nanobody.

[0066] Preferably, the amino acid sequence of the recombinant humanized antibody is shown in SEQ ID NO.3, as follows:

[0067] MGHHHHHHEVQLQASGGGFVQPGGSLRLSCAASGFTFSSYWMGWFRQAPGKEREFVSAISNQSGDTSYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKLGLSTWPYDYWGQGTQVTVSS

[0068] Preferably, the gene of the recombinant Nanobody includes a gene encoding the humanized Nanobody.

[0069] The nucleotide sequence of the recombinant humanized nanobody gene is shown in SEQ ID NO.4, as follows:

[0070] ATGGGTCACCACCACCACCACATGGAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCAGGTTATACCTTTAGTAGCGTTTGTATGGGTTGGTTTCGCCAGGCACCGGGCAAAGAACGCGAATTTGTGAGTGCCATTAGCTATACCTATACCGGTGCAACCTATTATG CAGATAGCGTTAAAGGTCGTTTCACCATTAGTCGTGATAATAGCAAAAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCCACCTATTATTGTGCAACCGATAGTTTTGGTGGCTGCCGCGCAAATCCGCTGAGTGATAGCGAATAATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTGAGCAGCTAATCTAGA

[0071] The nucleotide sequence of the His tag gene is shown in SEQ ID NO.23, which is as follows: ATGGGTCACCACCACCACCACCAC

[0072] The fourth object of the present invention is to provide a recombinant expression vector expressing a recombinant humanized nanobody against TNF-α, wherein the recombinant expression vector comprises a gene expressed by an initial vector inserted therein;

[0073] The genes expressed by the inserted initial vector include genes of the humanized Nanobodies;

[0074] The initial vector is a plasmid vector, preferably a pET-28a(+) plasmid vector; the insertion site of the gene of the recombinant humanized nanobody is preferably located at the NcoI-XhoI site of the pET-28a(+) plasmid vector.

[0075] The fifth object of the present invention is to provide a recombinant strain expressing a recombinant humanized nanobody against TNF-α, wherein the recombinant strain comprises an initial strain and the recombinant expression vector;

[0076] The initial strain is Escherichia coli, preferably Escherichia coli Arctic Express; there is no special limitation on the preparation method of the recombinant vector and the recombinant strain, and the conventional recombinant vector preparation method in the art can be used.

[0077] The sixth object of the present invention is to provide an application of the above-mentioned humanized nanoantibody, the gene encoding the humanized nanoantibody against TNF-α, the recombinant nanoantibody, the gene encoding the recombinant nanoantibody, the recombinant expression vector of the recombinant humanized nanoantibody or the recombinant strain of the recombinant humanized nanoantibody in the preparation of one or more of the anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents.

[0078] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0079] Example 1

[0080] Construction of a gene library of natural camel-derived nanoantibodies

[0081] (1) Extracting total RNA from camel peripheral blood mononuclear cells, the specific steps are as follows:

[0082] ① Blood collection from Bactrian camels was performed by professionals. Peripheral blood mononuclear cells of non-immunized camels were separated using Percoll cell separation solution. The separated peripheral blood mononuclear cells were washed three times with PBS and 1 mL of Trizol solution was added.

[0083] ② Add 0.2 mL of chloroform to every 1 mL of Trizol, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0084] ③ Centrifuge the sample at 12000rpm, 4℃ for 15min and transfer the upper layer to a new Eppendorf tube.

[0085] ④ Add an equal volume of isopropanol and mix on ice for 20 min.

[0086] ⑤ Centrifuge the solution at 12000rpm, 4℃ for 10min and discard the supernatant.

[0087] ⑥ Add 1 mL of 75% ethanol prepared with DEPC water to wash the precipitate (add at least 1 mL of ethanol for every 1 mL of Trizol).

[0088] ⑦ Centrifuge the solution from the previous step at 10,000 rpm for 10 min at 4°C, discard the supernatant, and repeat the previous step for one more wash.

[0089] ⑧ Centrifuge at 10000rpm, 4℃ for 10min, discard the supernatant and dry for 10-15 minutes.

[0090] ⑨Add appropriate amount of ddH2O to dissolve RNA precipitate, determine RNA concentration, and store at -80℃.

[0091] The RNA was purified using the RNA purification kit provided by TIANGEN and reverse transcribed using the Thermo Scientific ReverAid First Strand cDNA Synthesis Kits to obtain cDNA.

[0092] (2) Using cDNA as a template, nested PCR was used to amplify the variable region fragment of the heavy chain antibody;

[0093] First round of PCR:

[0094] Upstream primer: 5'-GTCCTGGCTGCTCTTCTACAAAG-3' (SEQ ID NO.19)

[0095] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO.20)

[0096] First round PCR reaction system

[0097]

[0098] The reaction conditions for the first round of PCR amplification were: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, 32 cycles; 72°C for 10 min.

[0099] The fragment between the heavy chain antibody guide peptide and antibody CH2 was amplified. This pair of primers was used to amplify the vh-CH1-CH2 fragment of 900 bp and the vhh-CH2 fragment of about 700 bp. The results were as follows: Figure 1 The fragment size was shown to be approximately 700-900 bp, ie, there were approximately two electrophoresis bands of the nanobody gene.

[0100] Second round of PCR:

[0101] The first round PCR product was used as template

[0102] Upstream primer: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3' (SEQ ID NO.21)

[0103] Downstream primer: 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3' (SEQ IDNO.22)

[0104] Second round PCR reaction system

[0105]

[0106]

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

[0108] The pCANTAB5e phage vector and VHH fragment were digested with restriction endonucleases SifI and Not I (purchased from NEB), and the two fragments were connected with T4 DNA ligase (purchased from NEB). The specific enzyme digestion system and ligation system are as follows:

[0109]

[0110] Enzyme digestion conditions: 37℃ for 1h, then 50℃ for 1h

[0111] Connection system

[0112]

[0113] Ligation conditions: 16°C for 4 hours, then overnight at 4°C

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

[0115] The preparation method of competent cell TG1 is as follows:

[0116] Take out the E. coli TG1 glycerol bacteria from the -80℃ refrigerator, streak inoculate on a 2×YT solid plate and culture at 37℃ for 10h, pick a single colony and inoculate in 3mL 2×YT liquid culture medium, and culture at 37℃, 200rpm overnight. The next day, amplify the bacterial liquid at a ratio of 1:100 and culture it in a conical flask containing 200mL 2×YT culture medium, continue to culture at 37℃ until OD600 is about 0.4, collect the bacterial liquid in a 50mL centrifuge tube, let it stand on ice for 1h, centrifuge at 9000rpm, 4℃ for 10min, discard the supernatant and resuspend the bacterial precipitate with the same volume of cold pure water, centrifuge, and repeat once. Then resuspend the bacterial cells with pre-cooled 10% glycerol and centrifuge. Use 1 mL of 10% glycerol (prepared with pre-cooled pure water) to suspend the bacterial precipitate and dispense it into pre-cooled 1 mL Eppendorf tubes, 100 μl per tube, and then immediately transfer to a -80°C refrigerator for storage, which is the competent cell TG1.

[0117] The helper phage rescue steps are as follows:

[0118] ① Take 100 μL of the library and inoculate it into 50 mL of 2×YT / Amp / Glu medium. Incubate at 37°C, 200 rpm, and shake until the OD600 is about 0.4-0.5 in the logarithmic phase.

[0119] ② Add helper phage M13KO7 at a multiplicity of infection of 20:1 to the culture medium, mix well and incubate at 37°C for 30 min.

[0120] ③ Centrifuge the culture medium at room temperature, 9000rpm, for 10min, discard the supernatant to precipitate the bacteria, resuspend in 200mL of 2×YT / Amp / Kana culture medium, and culture at 37℃200rpm overnight.

[0121] ④ Centrifuge the culture medium at 4°C, 9000 rpm for 10 min, take the supernatant, add 1 / 5 volume of PEG / NaCl, and let it stand at 4°C for 6 h.

[0122] 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, divide it into 1.5 mL Eppendorf tubes, and store it at 4°C.

[0123] At the same time, the insertion rate of the library was detected by colony PCR. The primers used were the second round PCR primers, and the annealing temperature was 55°C. The results were as follows Figure 3 The insertion rate was shown to be above 95% (target fragment insertion rate = number of colonies containing the target fragment / total number of colonies).

[0124] Screening process for anti-TNF-α nanoantibodies

[0125] The phage library (1×10 13phage) and 50 μL streptavidin magnetic beads were incubated on a rotating table at room temperature for 1 hour, and then phage antibodies were collected; 500 μL of pre-cut phage antibodies were added to two 1mL centrifuge tubes that had been blocked with 2% PBSM, and 500 μL of 5 μg biotinylated TNF-α antigen diluted with PBS was added to one centrifuge tube, and 500 μL of PBS buffer was added to the other centrifuge tube as a negative control, and incubated on a rotating table at room temperature for 1 hour, and then 50 μL of pre-blocked streptavidin magnetic beads were added, incubated on a rotating table at room temperature for 30 minutes, and magnetic beads were collected. The magnetic beads were washed 7 times with PBST, 2 times with PBSM, and 1 time with PBS. Glycine at pH = 2.7 was added for elution, and 1 mol / LTris-HCl at pH = 9.1 was neutralized. The above neutralization solution was added to 5 mL of TG1 in the logarithmic growth phase (OD600 was 0.5) to produce and purify phages for the next round of screening. After three rounds of screening, positive clones were continuously enriched, thereby achieving the purpose of screening TNF-α specific antibodies in the antibody library using phage display technology.

[0126] Phage-ELISA to screen specific single positive clones

[0127] Screening principle diagram Figure 4 As shown, the specific method is as follows:

[0128] First, prepare the VHH phage monoclonal supernatant: randomly pick 180 single colonies from the solid plate after three rounds of screening and inoculate them into a 96-deep-well plate containing 2×YT medium containing 100 μg / mL ampicillin and 2% glucose at 220 rpm. Culture at 37°C overnight, take 50 μL of bacterial solution to a new 96-deep-well plate the next day, add 800 μL of 2×YT medium containing 100 μg / mL ampicillin and 2% glucose to each well, grow to the logarithmic phase, add helper phage M13K07 with a multiplicity of infection of 20:1, infect at 37°C for 30 min, centrifuge at 10000 rpm for 5 min, discard the supernatant, resuspend the bacteria with 800 μL of fresh 2×YT medium containing 100 ug / mL ampicillin and 50 ug / mL kanamycin, culture at 37°C, 220 rpm for 12 h, and the next day, centrifuge the bacterial solution at 10000 rpm for 5 min. The supernatant is the VHH phage monoclonal supernatant.

[0129] Dilute TNF-α antigen to 10μg / mL with coating solution, add 100μL to each well, coat overnight at 4℃, and set up negative and positive controls. The next day, wash three times with PBST, block with 2% PBSM at 37℃ for 2h, wash three times with PBST, add 200μL pretreated VHH phage monoclonal supernatant, and incubate at 37℃ for 1h. Add 1:5000 mouse anti-M13KO7 / HRP secondary antibody diluted with 0.1% PBST, incubate at 37℃ for 1h, wash off unbound antibodies, add TMB colorimetric solution, and read the absorbance value at 450nm wavelength on the microplate reader. When the OD value of the sample well is more than twice the OD of the control well, it is judged as a positive control well, and the positive bacterial solution is taken for gene sequencing.

[0130] Snapegene software was used for sequence analysis and Blast alignment, and strains with the same CDR1, CDR2, and CDR3 sequences were considered to be the same clone. The CDR1, CDR2, and CDR3 regions of the screened anti-TNF-α-1A5 nanobody were transplanted into the humanized hs2dAb backbone, and a His tag was added to facilitate protein purification and identification. Finally, the nanobody sequence shown in SEQ ID NO.1 was used for subsequent experiments.

[0131] Example 2

[0132] Expression and purification of humanized nanoantibody TNF-α-1A5-Nb in host bacteria Escherichia coli

[0133] (1) The humanized nanoantibody TNF-α-1A5-Nb sequence obtained by sequencing analysis was subcloned into the pET-28a(+) plasmid vector and transformed into Escherichia coli Arctic Express. The single clone on the transformation plate was picked and inoculated into a test tube containing 3 mL LB culture medium containing 50 μg / mL Kan, and shaken at 37°C 220 rpm overnight; (2) The next day, it was inoculated into the above culture medium at a ratio of 1:100, shaken at 37°C 220 rpm until the bacterial OD600 was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and shaken at 20°C 220 rpm overnight to induce the expression of fusion protein; (3) The bacteria were collected and ultrasonically disrupted to obtain the inclusion body protein crude liquid, and then the fusion protein was obtained by Ni column affinity purification. Figure 5 shown.

[0134] Example 3

[0135] Specificity verification of anti-TNF-α humanized nanobody 1A5:

[0136] Western blot was performed using TNF-α antigen (the primary antibody was purified anti-TNF-α humanized nanobody 1A5, and the secondary antibody was anti-mouse His / HRP), such as Figure 6As shown, the anti-TNF-α humanized nanobody 1A5 can specifically bind to the TNF-α antigen.

[0137] Example 4

[0138] ELISA method to detect the specific binding of TNF-α humanized nanobody 1A5 to TNF-α antigen

[0139] The TNF-α antigen was diluted to 2μg / mL with ELISA coating solution, and 100μL was added to each well of a 96-well ELISA plate for coating at 4℃. PBS coating was used as a negative control. The next day, the plate was washed three times with 0.05% PBST, patted dry with absorbent paper, and 300μL of 5% PBSM was added to each well and blocked at 37℃ for 2h. After blocking, the plate was washed three times with PBST and patted dry. 100μL of experimental and control group samples were added to each well, incubated at 37℃ for 1h, washed three times with PBST, patted dry, and mouse His antibody was diluted with 0.05% PBST at a ratio of 1:5000. 100μL was added to each well. After incubation at 37℃ for 1h, the plate was washed and patted dry. The sheep anti-mouse HRP antibody was diluted with 0.05% PBST at a ratio of 1:5000. 100μL was added to each well. After incubation at 37℃ for 1h, the plate was washed and patted dry. The test results are as follows: Figure 7 As shown, the anti-TNF-α-1A5-Nb humanized nanobody specifically binds to the TNF-α antigen.

[0140] Example 5

[0141] Anti-TNF-α humanized nanobody 1A5 and TNF-α antigen specificity detection

[0142] The affinity of the nanobody was determined by biomembrane interferometry (BLI). The biotinylated humanized TNF-α antigen was bound to the Streptavidin (SA) probe, and 6 nanobody concentration gradients of 1000, 500, 250, 125, 62.5 and 31.25 nmol / L were set. OctetRed96e was used to determine the binding and dissociation of the nanobody at each concentration with the biotinylated humanized TNF-α antigen. The detection data was fitted according to the 1:1 binding model, and the binding and dissociation curves of the TNF-α nanobody 1A5 at each concentration with the TNF-α antigen were plotted to calculate the KD value. The results are shown in Figure 8 Display: Its binding constant is 6.610×10 -5 , the dissociation constant is 1.0×10 -7 The calculated equilibrium dissociation constant (KD) value is 9.755×10 -12 , that is, the anti-TNF-α humanized nanoantibody 1A5 has a strong ability to specifically bind to the TNF-α antigen.

[0143] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A humanized nanobody against TNF-α, characterized in that: The humanized nanobody includes a humanized framework region FR and an antigen determinant complementary region CDR; The complementary region CDR of the antigen determinant includes CDR1, CDR2 and CDR3; The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 respectively.

2. The humanized Nanobody according to claim 1, characterized in that The frame region FR includes FR1, FR2, FR3 and FR4; 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.

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

1.

4. A gene encoding a humanized nanobody against TNF-α, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

5. A recombinant humanized nanobody against TNF-α, characterized in that: Comprising the Nanobody according to any one of claims 1 to 3 and a His tag expressed by fusion with the humanized Nanobody.

6. The recombinant humanized antibody according to claim 5, characterized in that: The amino acid sequence of the recombinant humanized antibody is shown in SEQ ID NO.

3.

7. A gene encoding the recombinant nanobody according to claim 5, characterized in that: The gene of the recombinant Nanobody includes a gene encoding the humanized Nanobody according to any one of claims 1 to 3.

8. A recombinant expression vector expressing a recombinant humanized nanobody against TNF-α, characterized in that: The recombinant expression vector includes an initial vector and a gene expressed by the initial vector; The gene expressed by the inserted initial vector includes the gene encoding the humanized Nanobody according to any one of claims 1 to 3.

9. A recombinant strain expressing a recombinant humanized nanobody against TNF-α, characterized in that: The recombinant strain comprises the initial strain and the recombinant expression vector according to claim 8; The initial strain is Escherichia coli.

10. Use of the humanized Nanobody according to any one of claims 1 to 3, the gene encoding the anti-TNF-α humanized Nanobody according to claim 4, the recombinant Nanobody according to claim 5, the gene encoding the recombinant Nanobody according to claim 7, the recombinant expression vector of the recombinant humanized Nanobody according to claim 8, or the recombinant strain of the recombinant humanized Nanobody according to claim 9 in the preparation of one or more of anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents.

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