Humanized anti-TNF-α nanobody, recombinant nanobody, recombinant expression vector, recombinant engineered bacteria and their applications

Abstract

The present invention belongs to the field of immunology technology and specifically relates to a humanized anti-TNF-α nanobody, a recombinant nanobody, a recombinant expression vector, a recombinant engineered bacterium, and their applications. The humanized anti-TNF-α nanobody of the present invention is fused with a His tag to produce a humanized recombinant anti-TNF-α nanobody. Both the humanized anti-TNF-α nanobody and the recombinant nanobody can specifically recognize the TNF-α antigen and can be used in the molecular diagnosis of autoimmune diseases and tumors and in the preparation of autoimmune diseases and anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and specifically relates to a humanized anti-TNF-α nanobody, a recombinant nanobody, a recombinant expression vector, a recombinant engineered bacterium and applications thereof. Background Art

[0002] Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine with beneficial functions in immune regulation and host defense, but also deleterious proinflammatory and cytotoxic functions during inflammation. TNF-α plays a key role in several inflammatory diseases, including rheumatoid arthritis (RA), ulcerative colitis, and Crohn's disease. The TNF signaling pathway is mediated by binding to two cell surface receptors: TNF receptor type 1 (TNFR1 or p55), which is expressed in most tissues, and TNF receptor type 2 (TNFR2 or p75), which is inducible and typically present on cells of the immune system. TNFR1 induces proinflammatory cascades and apoptosis, while TNFR2 plays a role in cell survival, proliferation, and immune regulation. Binding of TNF-α to its two receptors mediates distinct signaling pathways, resulting in distinct biological functions. TNF-α specifically binds to TNFR1 and recruits the corresponding TRADD protein to mediate two signal transduction pathways: one is through the activation of receptor interacting protein (RIP), which is a crucial activator of NF-kB, thereby inducing NF-kB activation; the other is through the activation of FADD to cause cell apoptosis.

[0003] Nanobodies were first reported by Belgian scientists in Nature in 1993. They are a type of naturally light-chain-deficient antibody (VHH) present in the peripheral blood of alpacas. In 1995, they were found in cartilaginous fish such as nurse sharks, spotted sharks, and chimaeras. They are the smallest units known to date that can bind to target antigens. VHH has a molecular weight of only 15KD, so it is also called a nanobody (Nb). Nanobodies have unique advantages, including small molecular weight, good solubility, strong stability, high affinity, low immunogenicity, good tissue permeability in the body, and can easily pass through blood vessels or tissues to reach the target site. They have great room for development and are widely used. They can be used clinically for tumor treatment and as diagnostic tools.

[0004] Tumor necrosis factor (TNF-α) blockers are widely considered one of the most effective treatments for rheumatoid arthritis (RA). TNF inhibitors also have promising therapeutic effects on several other chronic inflammatory diseases, such as cervical spondylosis, psoriasis, and inflammatory bowel diseases like Crohn's disease. Currently, three TNF antagonists are used clinically: etanercept, infliximab, and adalimumab. TNF-α inhibitors block the interaction between TNF-α and its receptors TNFR1 and TNFR2, neutralizing TNF-α function and suppressing inflammatory gene expression. However, the high cost and numerous side effects of these drugs for RA remain major drawbacks, hindering their widespread use in clinical practice. The high cost is primarily due to the relatively labor-intensive and time-consuming production processes of existing drugs. These considerations have led to the search for more affordable and safer alternatives. Therefore, there is an urgent need for new, cost-effective, and effective drugs targeting TNF-α with minimal adverse reactions for the treatment of RA. Summary of the Invention

[0005] The purpose of the present invention is to provide a humanized anti-TNF-α nanobody, a recombinant nanobody, a recombinant expression vector, a recombinant engineered bacterium and applications thereof, wherein the humanized nanobody and the recombinant nanobody can specifically recognize the TNF-α antigen.

[0006] The present invention provides an anti-TNF-α humanized nanobody, which comprises a framework region FR and an antigenic determinant complementary region CDR, wherein the antigenic determinant complementary region CDR comprises CDR1, CDR2 and CDR3, and 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.

[0007] Preferably, the framework region FR includes FR1, FR2, FR3 and FR4, and the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0008] Preferably, the amino acid sequence of the humanized Nanobody is as shown in SEQ ID No.1.

[0009] The present invention also provides a gene encoding a humanized anti-TNF-α nanobody, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0010] The present invention also provides a recombinant anti-TNF-α nanobody, comprising the nanobody described in the above technical solution and a His tag expressed by fusion with the humanized nanobody.

[0011] The present invention also provides a gene encoding the recombinant nanobody described in the above technical solution, wherein the gene of the recombinant nanobody comprises a gene encoding the humanized nanobody described in the above technical solution and a gene encoding the His tag.

[0012] The present invention also provides a biomaterial expressing a humanized anti-TNF-α nanobody, the biomaterial comprising a recombinant expression vector and / or a recombinant engineered bacterium; the recombinant expression vector comprising an initial vector and an expression gene inserted into the initial vector, the expression gene inserted into the initial vector comprising a gene encoding the humanized nanobody described in the above technical solution or a gene encoding the recombinant nanobody described in the above technical solution;

[0013] The recombinant engineering bacteria include the recombinant expression vector and the initial strain.

[0014] Preferably, the initial vector comprises a plasmid vector.

[0015] Preferably, the recombinant engineered bacteria include Escherichia coli.

[0016] The present invention also provides the use of the humanized nanoantibody described in the above technical solution, the gene encoding the anti-TNF-α humanized nanoantibody described in the above technical solution, the recombinant nanoantibody described in the above technical solution, the gene encoding the recombinant nanoantibody described in the above technical solution, and the biomaterial described in the above technical solution in the preparation of one or more of the anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents.

[0017] Beneficial effects:

[0018] The present invention provides an anti-TNF-α humanized nanobody, the humanized nanobody comprising a framework region FR and an antigenic determinant complementary region CDR, the antigenic determinant complementary region CDR comprising CDR1, CDR2 and CDR3, the amino acid sequences of CDR1, CDR2 and CDR3 being shown as SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, respectively. The humanized recombinant nanobody against TNF-α prepared by fusion expression of the humanized nanobody of the present invention with a His tag, the humanized nanobody and the recombinant nanobody both being able to specifically recognize the TNF-α antigen, and being able to 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

[0019] Figure 1 This is the first round DNA electrophoresis diagram of the Nanobody in Example 1;

[0020] Figure 2 This is the second round DNA electrophoresis diagram of the Nanobody in Example 1;

[0021] Figure 3 The insertion rate results of the colony PCR test library in Example 1;

[0022] Figure 4 This is a schematic diagram of screening specific single positive clones using phage-ELISA in Example 1;

[0023] Figure 5 This is an SDS-PAGE electrophoresis staining image during the purification process of the anti-TNF-α-1A3 humanized nanobody in Example 2;

[0024] Figure 6 This is the SDS-PAGE electrophoresis staining of the purified anti-TNF-α-1A3 humanized Nanobody in Example 2;

[0025] Figure 7 This is a western blot result of the anti-TNF-α-1A3 humanized nanobody in Example 3;

[0026] Figure 8 This is the result of ELISA test on the affinity between anti-TNF-α-1A3 humanized nanobody and TNF-α antigen in Example 4;

[0027] Figure 9 This is a graph showing the affinity determination between the anti-TNF-α-1A3 humanized nanobody and the TNF-α antigen in Example 5. DETAILED DESCRIPTION

[0028] The present invention provides an anti-TNF-α humanized nanobody, which comprises a framework region FR and an antigenic determinant complementary region CDR, wherein the antigenic determinant complementary region CDR comprises CDR1, CDR2 and CDR3, and 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.

[0029] The amino acid sequences of CDR1, CDR2 and CDR3 of the present invention are as follows: CDR1: GFTLNDED (SEQ ID No. 13); CDR2: ISINTWGRS (SEQ ID No. 14); CDR3: ATGWHISSLSRDY (SEQ ID No. 15).

[0030] 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:

[0031] CDR1:5'-GGTTTCACTCTGAATGATGAAGAC-3' (SEQ ID No. 16);

[0032] CDR2:5'-ATCTCCATTAACACCTGGGGCCGTAGC-3' (SEQ ID No. 17);

[0033] CDR3:5'-GCCACCGGTTGGCACATCTCTTCGCTGAGTCGTGACTAT-3'

[0034] (SEQ ID No. 18).

[0035] The framework region FR of the present invention preferably includes FR1, FR2, FR3 and FR4, and 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; the amino acid sequences of FR1, FR2, FR3 and FR4 are specifically as follows:

[0036] FR1:EVQLQASGGGFVQPGGSLRLSCAAS(SEQ ID No.5);

[0037] FR2:MGWFRQAPGKEREFVSA(SEQ ID No.6);

[0038] FR3:YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC(SEQ IDNo.7);

[0039] FR4: WGQGTQVTVSS (SEQ ID No. 8).

[0040] 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, and are as follows:

[0041] FR1:5'-GAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGTGCAGCCGG GTGGTAGCCTGCGTCTGAGCTGCGCAGCATCA-3' (SEQ ID No. 9);

[0042] FR2: 5'-ATGGGTTGTTTCGTCAAGCACCGGGTAAGGAGCGCGAATTTGTGAGGCCG-3' (SEQ ID No. 10);

[0043] FR3:5'-TATTACGCGGATTCCGTCAAAGGCCGTTTCACCATTAGCCGTGA TAATAGCAAGAACACGGTTTATCTGCAGATGAACAGCTTGCGCGCGGAGG ACACCGCTACCTACTACTGC-3' (SEQ ID No. 11);

[0044] FR4: 5'-TGGGGTCAGGGCACCCAGGTTACCGTTAGCAGC-3' (SEQ ID No. 12).

[0045] In the present invention, the humanized Nanobody is preferably the humanized Nanobody 1A3, and the amino acid sequence of the humanized Nanobody 1A3 is preferably as shown in SEQ ID No. 1, specifically: EVQLQASGGGFVQPGGSLRLSCAASGFTLNDEDMGWFRQAPGKEREFVSAIS INTWGRSYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCATGWHISSLSRDYWGQGTQVTVSS.

[0046] The present invention also provides a gene encoding an anti-TNF-α humanized nanobody, the nucleotide sequence of which is shown in SEQ ID No. 2, specifically 5'-GAGGTTCAGCTGCAGGCTAGC GGTGGTGGTTTTGTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCAGGTTTCACTCTGAATGATGAAGACATGGGTTGGTTTCGTCAAGCACCGGGTAAGGAGCGCGAATTTGTGAGCGCGATCTCCATTAACACCTGGGGCCGTAGCTATTACGCGGATTCCGTCAAAGGCCGTTTCACCATTAGCCGTGATAATAGCAAGAACACGGTTTATCTGCAGATGAACAGCTTGCGCGCGGAGGACACCGCTACCTACTACTGCGCCACCGGTTGGCACATCTCTTCGCTGAGTCGTGACTATTGGGGTCAAGGCACGCAGGTTACCGTTTCTTCC-3', SEQ ID The nucleotide sequence shown in No. 2 is the nucleotide sequence encoding the humanized Nanobody 1A3.

[0047] The method for obtaining the humanized anti-TNF-α Nanobody 1A3 of the present invention preferably comprises the following steps:

[0048] A natural camel-derived nanobody phage display library is constructed using phage surface display technology; the natural camel-derived nanobody phage display library is screened using a biotinylated TNF-α antigen to obtain the gene sequence of the anti-TNF-α nanobody 1A3; the gene sequence of the anti-TNF-α nanobody 1A3 is transplanted into a humanized hs2dAb backbone to obtain the anti-TNF-α humanized nanobody 1A3. The humanized hs2dAb backbone of the present invention preferably comprises the humanized framework regions FR1, FR2, FR3, and FR4 described in the above technical solution.

[0049] In the present invention, the method for constructing the natural camel-derived 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 digesting the variable region fragment of the heavy chain antibody and the phage vector, and then ligating them to obtain a ligation product; 4) transforming the ligation product into Escherichia coli competent cells to obtain a natural camel-derived nanoantibody phage display library.

[0050] The present invention preferably extracts total RNA from camel peripheral blood mononuclear cells and reverse transcribes the total RNA to obtain cDNA. The present invention does not specifically limit the method for extracting total RNA from camel peripheral blood mononuclear cells; conventional methods for extracting total RNA from animal peripheral blood in the art can be employed. In the present invention, reverse transcription is preferably performed using a Thermo Scientific ReverAid First Strand cDNA Synthesis Kit.

[0051] After obtaining the cDNA, the present invention preferably performs nested PCR amplification using the cDNA as a template to obtain the variable region fragment of the heavy chain antibody. The nested PCR described in the present invention preferably includes two rounds of PCR; the first round of PCR is used to amplify the fragment between the heavy chain antibody leader peptide and the antibody CH2, and the primer sequences for 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 for the second round of PCR are preferably as shown in SEQ ID No. 21 and SEQ ID No. 22.

[0052] After obtaining the variable region fragment of the heavy chain antibody, the variable region fragment of the heavy chain antibody and the phage vector are respectively enzymatically digested in the present invention, and then ligated to obtain a ligation product. The phage vector of the present invention is preferably pCANTAB5e phage. The enzymatic digestion of the present invention is preferably double enzyme digestion, and the enzymatic digestion is preferably performed using restriction endonucleases Sif I and Not I. The enzymatic digestion procedure of the present invention is preferably as follows: enzyme digestion at 37°C for 1 hour; enzyme digestion at 50°C for 1 hour. In the present invention, the temperature of the ligation is preferably 16°C, and the time of the ligation is preferably 4 hours.

[0053] After obtaining the ligation product, the present invention preferably transfers the ligation product into competent cells to obtain a natural camel-derived nanobody phage display library. In the present invention, the E. coli competent cells are preferably E. coli competent cells TG1, which are preferably prepared in-house using the glycerol resuspension method; the transfer method is preferably electroporation. In the present invention, the transformation also includes a helper phage rescue process. The present invention does not specifically limit the electroporation and helper phage rescue processes; detailed steps are described in the Examples.

[0054] The present invention has no particular limitation on the specific steps of screening anti-TNF-α nanobodies using the biotinylated TNF-α antigen. Conventional nanobody screening methods in the art can be used. For detailed steps, please refer to the examples.

[0055] The present invention also provides a recombinant anti-TNF-α nanobody, comprising the humanized anti-TNF-α nanobody described in the above technical solution and a His tag fused to the nanobody, wherein the amino acid sequence of the recombinant nanobody is preferably as shown in SEQ ID No. 3. The amino acid sequence shown in SEQ ID No. 3 of the present invention is as follows: MGHHHHHHEVQLQASGGGFVQPGGSLRLSCAASGFTLN DEDMGWFRQAPGKEREFVSAISINTWGRSYYADSVKGRFTISRDNSKNTVYL QMNSLRAEDTATYYCATGWHISSLSRDYWGQGTQVTVSS. The "MG" at the starting position of the amino acid shown in SEQ ID No. 3 of the present invention is the starting amino acid of the translation sequence.

[0056] The present invention also provides a gene encoding the recombinant nanobody described in the above technical solution, wherein the gene includes a gene encoding the humanized nanobody described in the above technical solution and a gene encoding the His tag. The nucleotide sequence of the gene of the recombinant Nanobody of the present invention is preferably as shown in SEQ ID No. 4, specifically: 5'-ATGGGTCACCACCACCACCACCACGAGGTTCAGCTGCAGGCTA GCGGTGGTGGTTTTGTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCAGGTTTCACTCTGAATGATGAAGACATGGGTTGGTTTCGTCAAGCACCGGGTAAGGAGCGCGAATTTGTGAGCGCGATCTCCATTAACACCTGGGGCCGTAGCTATTACGCGGATTCCGTCAAAGGCCGTTTCACCATTAGCCGTGATAATAGCAAGAACACGGTTTATCTGCAGATGAACAGCTTGCGCGCGGAGGACACCGCTACCTACTACTGCGCCACCGGTTGGCACATCTCTTCGCTGAGTCGTGACTATTGGGGTCAAGGCACGCAGGTTACCGTTTCTTCC-3'; wherein the nucleotide sequence of the gene encoding the His tag is as shown in SEQ ID No. 23, specifically as follows: 5'-CACCACCACCACCACCAC-3'.

[0057] The present invention also provides a biomaterial for expressing a humanized anti-TNF-α nanobody, comprising a recombinant expression vector and / or a recombinant engineered bacterium; the recombinant expression vector comprises an initial vector and an expression gene inserted into the initial vector, wherein the expression gene inserted into the initial vector comprises a gene encoding the humanized nanobody described in the above technical solution or a gene encoding the recombinant nanobody described in the above technical solution; and the recombinant engineered bacterium comprises the recombinant expression vector and an initial strain. The initial vector of the present invention preferably comprises a plasmid vector, more preferably a pET-28a(+) plasmid vector; when the initial vector is a pET-28a(+) plasmid vector, the expression gene inserted into the initial vector is preferably inserted between the Nco I and Xho I restriction sites of the pET-28a(+) plasmid vector. The recombinant engineered bacterium of the present invention preferably comprises Escherichia coli, more preferably Escherichia coli Arctic Express. The present invention does not specifically limit the preparation methods of the recombinant expression vector and the recombinant engineered bacterium; conventional preparation steps for recombinant expression vectors and recombinant engineered bacteria in the art can be used. The anti-TNF-α recombinant humanized nanobody provided by the present invention can be stably expressed using the recombinant strain and can specifically recognize TNF-α antigen.

[0058] The humanized anti-TNF-α nanobody and the recombinant anti-TNF-α nanobody of the present invention can specifically recognize TNF-α and can be used for the preparation of anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents.

[0059] Based on the above technical advantages, the present invention also provides the use of the nanobody described in the above technical solution, the gene encoding the anti-TNF-α humanized nanobody described in the above technical solution, the recombinant nanobody described in the above technical solution, the gene encoding the recombinant nanobody described in the above technical solution, and the biomaterial described in the above technical solution in the preparation of one or more of the anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents, more preferably in the preparation of anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents. The autoimmune diseases described in the present invention preferably include but are not limited to rheumatoid arthritis.

[0060] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Regarding the construction of natural camel-derived nanoantibody gene library:

[0063] Extract total RNA from camel peripheral blood mononuclear cells. The specific steps are as follows:

[0064] ① Blood collection from Bactrian camels was performed by professionals. Peripheral blood mononuclear cells (PBMCs) from non-immunized camels were separated using Percoll cell separation medium. The separated PBMCs were washed three times with PBS and 1 mL of Trizol solution was added.

[0065] ② Add 0.2 mL of chloroform per 1 mL of Trizol, shake vigorously for 15 seconds, and let it stand at room temperature for 3 minutes.

[0066] ③ Centrifuge the sample at 12000rpm and 4℃ for 15min, and transfer the upper layer to a new Eppendorf tube.

[0067] ④ Add an equal volume of isopropanol and place on ice for 20 minutes.

[0068] ⑤ Centrifuge the solution at 12000 rpm and 4°C for 10 min and discard the supernatant.

[0069] ⑥ Add 1 mL of 75% ethanol solution prepared with DEPC water to wash the precipitate (add at least 1 mL of ethanol to every 1 mL of Trizol).

[0070] 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 to wash once more.

[0071] ⑧ Centrifuge at 10000 rpm, 4°C for 10 min, discard the supernatant, and dry for 10-15 min.

[0072] ⑨ Add appropriate amount of ddH2O to dissolve the RNA precipitate, determine the RNA concentration, and store at -80℃.

[0073] 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.

[0074] (2) Using cDNA as a template, nested PCR was used to amplify the variable region of the heavy chain antibody;

[0075] First round of PCR:

[0076] Upstream primer: 5'-GTCCTGGCTGCTCTTCTACAAAG-3' (SEQ ID No. 19)

[0077] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID No. 20)

[0078] The first-round PCR reaction system is shown in Table 1.

[0079] Table 1 First round PCR reaction system

[0080]

[0081]

[0082] 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, and 72°C for 45 s, for 32 cycles; and 72°C for 10 min.

[0083] The fragment between the heavy chain antibody guide peptide and the antibody CH2 was amplified. This pair of primers was used to amplify a VH-CH1-CH2 fragment of 900 bp and a VHH-CH2 fragment of approximately 700 bp. The results showed that the size of the fragment was approximately 700 to 900 bp, that is, there were approximately two electrophoretic bands of the nanobody gene.

[0084] Second round of PCR:

[0085] Using the first-round PCR product as a template,

[0086] Upstream primer:

[0087] 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3'

[0088] (SEQ ID No.21)

[0089] Downstream primer:

[0090] 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3'

[0091] (SEQ ID No.22)

[0092] The second round PCR reaction system is shown in Table 2.

[0093] Table 2 Second round PCR reaction system

[0094] Reagent name volume 700bp~900bp product 2μL Mix 25 μL Upstream primer 1 μL Downstream primer 1 μL <![CDATA[ddH2O]]> Add to 50 μL

[0095] The second round of PCR amplification reaction conditions were: 94°C for 5 minutes, followed by 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds, and finally 72°C for 10 minutes. The fragments between the heavy chain antibody FR1 region and the long and short hinge regions (the long and short fragments) were amplified. This fragment was used to amplify a 400-bp camelid heavy chain antibody heavy chain variable region VHH fragment from the 700-bp VHH-CH2 fragment. The results showed that this fragment was approximately 450 bp in size, indicating that the electrophoresis band of the nanobody gene was approximately 450 bp.

[0096] The pCANTAB5e phage vector and VHH fragment were digested with restriction enzymes SifI and NotI (purchased from NEB), and the two fragments were ligated using T4 DNA ligase (purchased from NEB). Specific enzyme digestion and ligation systems are shown in Tables 3-4, respectively.

[0097] Table 3 Enzyme digestion system

[0098] Reagent name volume 450bp product / pCANTAB5e 12 μL NotI 1 μL SifI 1 μL 10×Buffer 2μL <![CDATA[ddH2O]]> Up to 20 μL

[0099] Enzyme digestion conditions: digestion at 37°C for 1 h, then at 50°C for 1 h.

[0100] Table 4 Connection system

[0101] Reagent name volume Enzyme digestion of pCANTAB5e 8μL Enzyme digestion target fragment 3μL Buffer 2.5 μL T4 DNA ligase 1.5 μL

[0102] Ligation conditions: 16°C for 4 hours, followed by overnight ligation at 4°C.

[0103] The ligation products were electrotransformed into electrocompetent cells TG1 to construct a natural camel-derived nanobody phage display library. After helper phage rescue, the library capacity reached 9.0×10 13 .

[0104] The preparation method of competent cells TG1 is as follows:

[0105] Remove E. coli TG1 glycerol stock from a -80°C freezer, streak onto a 2×YT solid plate, and incubate at 37°C for 10 hours. Pick a single colony and inoculate it into 3 mL of 2×YT liquid medium. Incubate at 37°C with shaking at 200 rpm overnight. The next day, scale up the culture at a 1:100 ratio and transfer it to a conical flask containing 200 mL of 2×YT medium. Continue incubating at 37°C until the OD600 reaches approximately 0.4. Collect the culture into a 50 mL centrifuge tube, incubate on ice for 1 hour, and centrifuge at 9000 rpm for 10 minutes at 4°C. Discard the supernatant and resuspend the pellet in an equal volume of cold purified water. Repeat the centrifugation. Resuspend the pellet in pre-chilled 10% glycerol and centrifuge again. Use 1 mL of 10% glycerol (prepared with pre-cooled pure water) to suspend the bacterial pellet and dispense it into pre-cooled 1 mL Eppendorf tubes, 100 μl per tube, and immediately transfer to a -80°C refrigerator for storage. This is the competent cell TG1.

[0106] The helper phage rescue steps are as follows:

[0107] 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 OD value reaches the logarithmic phase. 600 About 0.4 to 0.5.

[0108] 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 minutes.

[0109] ③ Centrifuge the culture at room temperature at 9000 rpm for 10 min, discard the supernatant to precipitate the bacteria, resuspend in 200 mL of 2×YT / Amp / Kana culture medium, and culture at 37°C at 200 rpm overnight.

[0110] 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.

[0111] 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 at 4°C.

[0112] At the same time, the insertion rate of the library was detected by colony PCR. 24 single clones were randomly picked from the SOC plate after electroporation for 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 As shown, the results show that the target fragment insertion rate of the library reaches more than 95%, close to 100%, where the target fragment insertion rate = the number of colonies containing the target fragment / the total number of colonies.

[0113] Screening process for anti-TNF-α nanoantibodies:

[0114] The phage library (1×10 13 phage) were incubated with 50 μL of streptavidin magnetic beads on a rotating table at room temperature for 1 hour, and then the phage antibodies were collected; 500 μL of pre-cut phage antibodies were added to two 1 mL centrifuge tubes that had been blocked with 2% PBSM, and 500 μL of 5 μg of biotinylated TNF-α antigen diluted with PBS was added to one centrifuge tube. 500 μL of PBS buffer was added to the other centrifuge tube as a negative control, and incubated at room temperature for 1 hour on a rotating table. 50 μL of pre-blocked streptavidin magnetic beads were added, and the beads were incubated at room temperature for 30 minutes on a rotating table to collect the magnetic beads. The magnetic beads were washed 7 times with PBST, 2 times with PBSM, and 1 time with PBS. Glycine with pH = 2.7 was added for elution, and 1 mol / LTris-HCl with pH = 9.1 was neutralized. The above neutralization solution was added to 5 mL of TG1 (OD 600 is 0.5), phages are produced and purified for the next round of screening. After three rounds of screening, positive clones are continuously enriched, thereby achieving the purpose of screening TNF-α specific antibodies in the antibody library using phage display technology.

[0115] Phage-ELISA method (phage-ELISA) screening of specific single positive clones:

[0116] Screening principle diagram Figure 4 As shown, 1 is TNF-α antigen coated on the enzyme-labeled plate, 2 is phage supernatant, 3 is mouse anti-M13K07 antibody, 4 is goat anti-mouse IgG (AP) antibody, and 5 is TMB color development solution;

[0117] The specific method is as follows:

[0118] First, prepare the supernatant of VHH phage monoclonal clones: 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. The next day, take 50 μL of bacterial solution to a new 96-deep-well plate and 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 a multiplicity of infection of 20:1. Infect at 37°C for 30 minutes, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and 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 hours. The next day, centrifuge the bacterial solution at 10,000 rpm for 5 minutes. The supernatant is the VHH phage monoclonal supernatant.

[0119] Dilute TNF-α antigen to 10 μg / mL in coating buffer and add 100 μL to each well. 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 hours, and wash three times with PBST. Add 200 μL of pretreated VHH phage monoclonal supernatant and incubate at 37°C for 1 hour. Add a 1:5000 dilution of mouse anti-M13KO7 / HRP secondary antibody in 0.1% PBST and incubate at 37°C for 1 hour. Wash away any unbound antibody, add TMB colorimetric solution, and read the absorbance at 450 nm on a microplate reader. A sample well is considered a positive control well if its OD value is at least twice that of the control well. Positive bacterial cultures are then analyzed for gene sequencing.

[0120] Sequence analysis and Blast alignment were performed using Snapegene software, and strains with identical CDR1, CDR2, and CDR3 sequences were considered the same clone. The CDR1 (SEQ ID No. 13), CDR2 (SEQ ID No. 14), and CDR3 (SEQ ID No. 15) regions of the screened anti-TNF-α-1A3 nanobody were transplanted into the humanized hs2dAb backbone, and a His tag was added to facilitate protein purification and identification. The nanobody sequence shown in SEQ ID No. 1 was ultimately used for subsequent experiments, wherein the humanized framework region of the anti-TNF-α-1A3 humanized nanobody contained in SEQ ID No. 1 consists of FR1, FR2, FR3, and FR4, and 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.

[0121] Example 2

[0122] Expression and purification of humanized nanobody TNF-α-1A3-Nb in host bacteria Escherichia coli:

[0123] (1) The His-tagged humanized nanoantibody TNF-α-1A3-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 and 220 rpm overnight; (2) The next day, it was inoculated into 30 mL LB culture medium containing 50 μg / mL Kan at a ratio of 1:100, and shaken at 37°C and 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 and 220 rpm overnight to induce the expression of the fusion protein; (3) The bacterial cells were collected and ultrasonically disrupted to obtain a crude inclusion body protein solution, which was then affinity purified using a Ni column to obtain the fusion protein. Figure 5-6 It is a purified anti-TNF-α humanized nanobody 1A3, wherein Figure 5 The figure is an SDS-PAGE electrophoresis staining diagram of the purification process of the anti-TNF-α humanized nanobody 1A3: wherein lane M is the protein molecule standard, lanes 1-2 are the post-crushing treatment sample and the outflow sample, respectively, and lanes 3-4 are the elution samples; Figure 6 This is the SDS-PAGE electrophoresis staining of the purified anti-TNF-α humanized nanobody 1A3, wherein lane M is the protein molecule standard, lane 1 is 0.5 mg / mL BSA as the concentration measurement standard, and lane 2 is the purified anti-TNF-α humanized nanobody 1A3.

[0124] Example 3

[0125] Specificity verification of anti-TNF-α humanized nanobody 1A3:

[0126] Human TNF-α antigen (brand: arco; product number: TNA-H82E3) was used for western blot (the primary antibody was purified anti-TNF-α humanized nanobody 1A3, and the secondary antibody was anti-mouse His / HRP). Figure 7 This is a western blot of TNF-α antigen: Lane M is a protein molecule standard, and Lane 1 is a TNF-α antigen. This shows that the anti-TNF-α humanized Nanobody 1A3 provided by the present invention can specifically bind to the TNF-α antigen.

[0127] Example 4

[0128] ELISA to detect the specific binding of TNF-α humanized nanobody 1A3 to TNF-α antigen

[0129] TNF-α antigen (brand: arco; item number: TNA-H82E3) was diluted to 2 μg / mL with ELISA coating solution, and 100 μL was added to each well of a 96-well enzyme-labeled plate for coating at 4°C. 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 300 μL of 5% PBSM was added to each well and blocked at 37°C for 2 hours. After blocking, the plate was washed three times with PBST and patted dry. 100 μL of the experimental group and control group (using an unrelated nanoantibody HER-2, disclosed in Chinese patent CN114437222A) samples were added to each well and incubated at 37°C for 1 hour. The plate was washed three times with PBST and patted dry. The mouse His antibody was diluted with 0.05% PBST at a ratio of 1:5000, 100 μL was added to each well, incubated at 37°C for 1 hour, and the plate was washed and patted dry. Dilute goat anti-mouse HRP antibody in 0.05% PBST at a ratio of 1:5000, add 100 μL to each well, incubate at 37°C for 1 hour, wash the plate and pat dry. Figure 8 As shown, the anti-TNF-α-1A3-Nb humanized nanobody specifically binds to the TNF-α antigen.

[0130] Example 5

[0131] Anti-TNF-α humanized nanobody 1A3 and TNF-α antigen specificity detection:

[0132] The affinity of the nanobody was determined by biomembrane interferometry (BLI). The biotinylated humanized TNF-α antigen (brand: arco; item number: TNA-H82E3) was bound to the Streptavidin (SA) probe, and 6 nanobody concentration gradients of 2000, 1000, 500, 250, 125 and 62.5 nmol / L were set. The binding and dissociation of the nanobody at each concentration with the biotinylated humanized TNF-α antigen were determined using Octet Red96e. The detection data were fitted according to the 1:1 binding model, and the binding and dissociation curves of the TNF-α nanobody 1A3 at each concentration with the TNF-α antigen were drawn to calculate the KD value. The results are shown in Figure 2. Figure 9 Show: Its binding constant is 1.285×10 -4 , the dissociation constant is 4.233×10 -3 The calculated equilibrium dissociation constant (KD) value is 3.294×10 -7 , suggesting that the anti-TNF-α humanized nanobody 1A3 has a strong specific binding ability to TNF-α antigen.

[0133] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. A humanized anti-TNF-α nanobody, characterized in that The humanized nanobody includes a framework region FR and an antigenic determinant complementary region CDR, the antigenic determinant complementary region CDR includes CDR1, CDR2 and CDR3, and 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.

2. The humanized Nanobody according to claim 1, characterized in that The framework region FR includes FR1, FR2, FR3 and FR4, and 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 2, 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 nanobody against TNF-α, characterized in that Comprising the Nanobody according to any one of claims 1 to 3 and a His tag expressed in fusion with the humanized Nanobody.

6. A gene encoding the recombinant nanobody according to claim 5, characterized in that The gene of the recombinant Nanobody comprises a gene encoding the humanized Nanobody according to any one of claims 1 to 3 and a gene encoding the His tag.

7. A biomaterial expressing a humanized nanobody against TNF-α, characterized in that: The biological material includes a recombinant expression vector and / or a recombinant engineered bacterium; the recombinant expression vector includes an initial vector and an expression gene inserted into the initial vector, and the expression gene inserted into the initial vector includes a gene encoding the humanized Nanobody according to any one of claims 1 to 3 or a gene encoding the recombinant Nanobody according to claim 5; The recombinant engineering bacteria include the recombinant expression vector and the initial strain.

8. The biomaterial according to claim 7, characterized in that The initial vector includes a plasmid vector.

9. The biomaterial according to claim 7, characterized in that The recombinant engineering bacteria include Escherichia coli.

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