Anti-IL-6 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 an anti-IL-6 nanobody, a recombinant nanobody, a recombinant expression vector, a recombinant engineered bacterium and its application. The anti-IL-6 nanobody of the present invention includes CDR1, CDR2 and CDR3, and the gene encoding the anti-IL-6 nanobody is fused with the His tag gene to express the recombinant nanobody. The anti-IL-6 nanobody and the recombinant nanobody can both specifically recognize the IL-6 antigen and can be applied to the molecular diagnosis of autoimmune diseases and tumors and 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 an anti-IL-6 nanobody, a recombinant nanobody, a recombinant expression vector, a recombinant engineered bacterium and applications thereof. Background Art

[0002] Interleukin 6 (IL-6) is released by many different types of cells. The hexameric complex composed of IL-6, its receptor (IL-6 receptor, IL-6R) and glycoprotein 130 (glycoprotein 130, gp130) mediates its biological effects and activates intracellular signal transduction pathways on target cells. Its signal transduction pathway is strictly regulated. If IL-6 signal transduction is dysregulated, it will cause chronic inflammation, autoimmune diseases, etc.

[0003] To date, two drugs targeting IL-6R, Tocilizumab and Sarilumab, are clinically available for the treatment of autoimmune diseases, including rheumatoid arthritis (RA). For example, Tocilizumab is a humanized monoclonal antibody (mAb) that was first approved in Japan in 2005 for the treatment of Castleman disease. Subsequently, Tocilizumab was approved by the FDA in 2010 for various indications, including moderate to severe active RA in adults who have had an inadequate response to one or more disease-modifying antirheumatic drugs (DMARDs). In contrast to the impressive results of IL-6R inhibitors, the development of RA therapeutics targeting IL-6 has remained stagnant.

[0004] Nanobodies are antibodies that naturally lack light chains (VHH) and are the smallest units known to 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. However, nanobody drugs against IL-6 antigens are still relatively rare in this field. Summary of the Invention

[0005] The object of the present invention is to provide an anti-IL-6 nanobody, a recombinant nanobody, a recombinant expression vector, a recombinant engineered bacterium and applications thereof, wherein the nanobody and the recombinant nanobody can specifically recognize the IL-6 antigen.

[0006] The present invention provides an anti-IL-6 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 Nanobody is as shown in SEQ ID No.1.

[0009] The present invention also provides a gene encoding an anti-IL-6 nanobody, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0010] The present invention also provides a recombinant anti-IL-6 nanobody, comprising the nanobody described in the above technical solution and a His tag expressed by fusion with the 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 nanobody described in the above technical solution and a gene encoding the His tag.

[0012] The present invention also provides a biomaterial expressing an anti-IL-6 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 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 nanoantibody described in the above technical solution, the gene encoding the anti-IL-6 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-IL-6 nanobody, comprising 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. The nanobody of the present invention comprises CDR1, CDR2, and CDR3, and a gene encoding the nanobody is fused with a His tag gene to express a recombinant nanobody. Both the nanobody and the recombinant nanobody are capable of specifically recognizing the IL-6 antigen, and are applicable 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 The absorbance value of a specific single positive clone screened by phage-ELISA in Example 1;

[0024] Figure 6 This is a Western Blot image of the purified anti-IL-6-4G10 nanobody in Example 2;

[0025] Figure 7 This is the result of ELISA detection of the affinity between anti-IL-6-4G10 nanobody and IL-6 antigen in Example 3;

[0026] Figure 8 This is a graph showing the affinity determination between the anti-IL-6-4G10 nanobody and the IL-6 antigen in Example 4. DETAILED DESCRIPTION

[0027] The present invention provides an anti-IL-6 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.

[0028] The amino acid sequences of CDR1, CDR2 and CDR3 of the present invention are as follows: CDR1: GLDFDDYN (SEQ ID No. 13); CDR2: IDWNGRT (SEQ ID No. 14); CDR3: AAAYYTPFCYSGTDYDY (SEQ ID No. 15).

[0029] 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:

[0030] CDR1:5'-GGATTGGATTTTGATGATTATAAC-3' (SEQ ID No. 16);

[0031] CDR2:5'-ATTGACTGGAATGGTAGAACA-3' (SEQ ID No. 17);

[0032] CDR3: 5'-GCGGCAGCTTACTACACTCCGTTCTGCTACTCTGGTACTGACT ACGACTAC-3' (SEQ ID No. 18).

[0033] 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:

[0034] FR1:HVQLVESGGTSVQTGSSLTLSCIVS(SEQ ID No.5);

[0035] FR2:MGWFRQAPGEEREGVAL(SEQ ID No.6);

[0036] FR3:HYTDSVKGRFSISRDNAKNTVLLQMNVLQPEDTAMYYC(SEQ IDNo.7);

[0037] FR4: WGQGTQVTVSS (SEQ ID No. 8).

[0038] 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:

[0039] FR1:5'-CATGTGCAGCTGGTGGAGTCTGGGGGAACATCGGTGCAGACA GGGAGCTCTCTGACACTCTCCTGTATAGTCTCG-3' (SEQ ID No. 9);

[0040] FR2: 5'-ATGGGCTGGTTCCGCCAGGCTCCAGGGGAGGAGCGCGAGGGA GTAGCGTTG-3' (SEQ ID No. 10);

[0041] FR3:5'-CATTACACAGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAG ACAACGCCAAGAACACGGTGCTTCTGCAAATGAATGTCCTTCAACCTGAA GACACTGCCATGTACTACTGT-3' (SEQ ID No. 11);

[0042] FR4: 5'-TGGGGACAGGGAACACAGGTGACAGTGTCTTCA-3' (SEQ ID No. 12).

[0043] In the present invention, the Nanobody is preferably Nanobody 4G10, and the amino acid sequence of the Nanobody 4G10 is preferably as shown in SEQ ID No. 1, specifically: HVQLVESGGTSVQTGSSLTLSCIVSGLDFDDYNMGWFRQAPGEEREGVALID WNGRTHYTDSVKGRFSISRDNAKNTVLLQMNVLQPEDTAMYYCAAAYYTPFCYSGTDYDYWGQGTQVTVSS.

[0044] The present invention also provides a gene encoding an anti-IL-6 nanobody, the nucleotide sequence of the gene is shown in SEQ ID No. 2, specifically 5'-CATGTGCAGCTGGTGGAGTCTGGGGGA ACATCGGTGCAGACAGGGAGCTCTCTGACACTCTCCTGTATAGTCTCGGGATTGGATTTTGATGATTATAACATGGGCTGGTTCCGCCAGGCTCCAGGGGAGGAGCGCGAGGGAGTAGCGTTGATTGACTGGAATGGTAGAACACATTACACAGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAGACAACGCCAAGAACACGGTGCTTCTGCAAATGAATGTCCTTCAACCTGAAGACACTGCCATGTACTACTGTGCGGCAGCTTACTACACTCCGTTCTGCTACTCTGGTACTGACTACGACTACTGGGGACAGGGAACACAGGTGACAGTGTCTTCA-3'.

[0045] The method for obtaining the anti-IL-6 Nanobody 4G10 of the present invention preferably comprises the following steps:

[0046] A natural camel-derived nanoantibody phage display library was constructed using phage surface display technology; screening was performed in the natural camel-derived nanoantibody phage display library based on biotinylated IL-6 antigen to obtain the anti-IL-6 nanoantibody 4G10 gene sequence, and then the anti-IL-6 nanoantibody 4G10 was obtained.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The present invention has no particular limitation on the specific steps of screening anti-IL-6 nanobodies using the biotinylated IL-6 antigen. Conventional nanobody screening methods in the art can be used. For detailed steps, please refer to the examples.

[0053] The present invention also provides a recombinant anti-IL-6 nanobody, comprising the anti-IL-6 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: MGHHHHHHHVQLVESGGTSVQTGSSLTLSCIVSGLDFDDYNMGWF RQAPGEEREGVALIDWNGRTHYTDSVKGRFSISRDNAKNTVLLQMNVLQPE DTAMYYCAAAYYTPFCYSGTDYDYWGQGTQVTVSS. In the amino acid sequence shown in SEQ ID No. 3 of the present invention, "MG" has the function of translation initiation.

[0054] The present invention also provides a gene encoding the recombinant nanobody described in the above technical solution, wherein the gene comprises a gene encoding the nanobody described in the above technical solution and a gene encoding the His tag. The nucleotide sequence of the gene encoding the recombinant nanobody of the present invention is preferably as shown in SEQ ID No. 4, specifically: 5'-CCATGGGCCACCATCACCACCACCCATGTGCAGCTGGTGGAGTCTGGGG GAACATCGGTGCAGACAGGGAGCTCTCTGACACTCTCCTGTATAGTCTCGGGATTGGATTTTGATGATTATAACATGGGCTGGTTCCGCCAGGCTCCAGGGGAGGAGCGCGAGGGAGTAGCGTTGATTGACTGGAATGGTAGAACACATTACACAGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAGACAACGCCAAGAACACGGTGCTTCTGCAAATGAATGTCCTTCAACCTGAAGACACTGCCATGTACTACTGTGCGGCAGCTTACTACACTCCGTTCTGCTACTCTGGTACTGACTACGACTACTGGGGACAGGGAACACAGGTGACAGTGTCTTCA-3′; wherein the nucleotide sequence of the gene encoding the His tag is shown in SEQ ID No. 23, specifically as follows: 5′-GCCACCATCACCACCACC-3′.

[0055] The present invention also provides a biomaterial for expressing an anti-IL-6 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 nanobody described in the above technical solution or a gene encoding the recombinant nanobody described in the above technical solution; the recombinant engineered bacterium comprising 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, and the preparation steps of conventional recombinant expression vectors and recombinant engineered bacteria in the art can be used.

[0056] The anti-IL-6 nanobody and the recombinant anti-IL-6 nanobody of the present invention can specifically recognize IL-6 and can be used for the preparation of anti-autoimmune disease drugs, anti-tumor drugs, autoimmune disease diagnostic reagents and tumor diagnostic reagents.

[0057] Based on the above technical advantages, the present invention also provides the use of the nanoantibodies described in the above technical solution, the genes encoding anti-IL-6 nanoantibodies described in the above technical solution, the recombinant nanoantibodies described in the above technical solution, the genes encoding the recombinant nanoantibodies described in the above technical solution, and the biomaterials described in the above technical solution in the preparation of one or more of the drugs for anti-autoimmune diseases, anti-tumor drugs, diagnostic reagents for autoimmune diseases and tumor diagnostic reagents, and more preferably in the preparation of drugs for anti-autoimmune diseases, anti-tumor drugs, diagnostic reagents for autoimmune diseases and tumor diagnostic reagents.

[0058] 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.

[0059] Example 1

[0060] Regarding the construction of natural camel-derived nanoantibody gene library:

[0061] Extract total RNA from camel peripheral blood mononuclear cells. The specific steps are as follows:

[0062] 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.

[0063] 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.

[0064] ③ Centrifuge the sample at 12000rpm and 4℃ for 15min, and transfer the upper layer to a new Eppendorf tube.

[0065] ④ Add an equal volume of isopropanol and mix on ice for 20 minutes.

[0066] ⑤ Centrifuge the solution in step ④ at 12000 rpm and 4°C for 10 min and discard the supernatant.

[0067] ⑥ Add 1 mL of 75% ethanol solution prepared with DEPC water to wash the precipitate (add at least 1 mL of 75% ethanol to every 1 mL of Trizol).

[0068] ⑦ Centrifuge the solution from the previous step at 10,000 rpm for 10 minutes at 4°C, discard the supernatant, and repeat the previous step to wash once more.

[0069] ⑧ Centrifuge at 10000 rpm, 4°C for 10 min, discard the supernatant, and dry for 10-15 min.

[0070] ⑨ Add appropriate amount of ddH2O to dissolve the RNA precipitate, determine the RNA concentration, and store at -80℃.

[0071] 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.

[0072] (2) Using cDNA as a template, nested PCR was used to amplify the variable region of the heavy chain antibody;

[0073] First round of PCR:

[0074] Upstream primer: 5′-GTCCTGGCTGCTCTTCTACAAAG-3′ (SEQ ID No. 19);

[0075] Downstream primer: 5′-GGTACGTGCTGTTGAACTGTTCC-3′ (SEQ ID No. 20);

[0076] The first-round PCR reaction system is shown in Table 1.

[0077] Table 1 First round PCR reaction system

[0078] Reagent name volume cDNA 2μL Mix 10 μL Upstream primer 1 μL Downstream primer 1 μL <![CDATA[ddH2O]]> Add to 20 μL

[0079] 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.

[0080] Amplify the fragment between the heavy chain antibody guide peptide and antibody CH2. This pair of primers is used to amplify a VH-CH1-CH2 fragment of 900 bp and a VHH-CH2 fragment of about 700 bp. The results show that the size of the fragment is about 700-900 bp, that is, there are about two electrophoretic bands of the nanobody gene. Figure 1 As shown, in Figure 1 In the figure, the DNA bands in the gel wells from left to right 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.

[0081] Second round of PCR:

[0082] Using the first-round PCR product as a template,

[0083] Upstream primer:

[0084] 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3'

[0085] (SEQ ID No. 21);

[0086] Downstream primer:

[0087] 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3'

[0088] (SEQ ID No. 22);

[0089] The second-round PCR reaction system is shown in Table 2.

[0090] Table 2 Second round PCR reaction system

[0091]

[0092]

[0093] The second round of PCR amplification reaction conditions were: 94°C for 5 minutes; 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds, for 25 cycles; and 72°C for 10 minutes. The fragments between the heavy chain antibody FR1 region and the long and short hinge regions (long and short fragments) were amplified. A 400-bp camel heavy chain antibody heavy chain variable region VHH fragment was amplified from the 700-bp VHH-CH2 fragment. The results showed that the fragment size was approximately 450 bp, indicating that the electrophoresis band of the nanobody gene was approximately 450 bp. Figure 2 As shown, 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 bands are about 450 bp.

[0094] 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.

[0095] Table 3 Enzyme digestion system

[0096] Reagent name volume 450bp product / pCANTAB5e 12 μL NotI 1 μL SifI 1 μL 10×Buffer 2μL <![CDATA[ddH2O]]> Add to 20 μL

[0097] Enzyme digestion conditions: digestion at 37°C for 1 h, then at 50°C for 1 h.

[0098] Table 4 Connection system

[0099] Reagent name volume Enzyme digestion of pCANTAB5e 8μL Enzyme digestion target fragment 3μL Buffer 2.5 μL T4 DNA ligase 1.5 μL

[0100] Ligation conditions: 16°C for 4 hours, followed by overnight ligation at 4°C.

[0101] 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 .

[0102] The preparation method of competent cells TG1 is as follows:

[0103] Remove E. coli TG1 glycerol culture from the -80°C freezer, streak on 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 and incubate at 37°C with shaking at 200 rpm overnight. The next day, amplify the culture at a volume ratio of 1:100 in a conical flask containing 200 mL of 2×YT medium and continue incubating at 37°C until the OD reaches 0. 600When the p-value is approximately 0.4, collect the bacterial suspension in a 50mL centrifuge tube, let it stand on ice for 1 hour, centrifuge at 9000 rpm at 4°C for 10 minutes, discard the supernatant, resuspend the bacterial pellet in the same volume of cold pure water, and repeat the centrifugation. Then resuspend the bacterial pellet in pre-chilled 10% glycerol and centrifuge. Resuspend the bacterial pellet in 1mL of 10% glycerol (pre-chilled pure water) and divide the suspension into pre-chilled 1mL Eppendorf tubes, 100μL per tube, and immediately transfer to a -80°C freezer for storage. This is the competent cell TG1.

[0104] The helper phage rescue steps are as follows:

[0105] 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.

[0106] 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.

[0107] 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 (“ / ” indicates “and”) culture medium, and culture at 37°C at 200 rpm overnight.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Screening process for anti-IL-6 nanoantibodies:

[0112] The phage library (1×10 13phage) 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 IL-6 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 IL-6 specific antibodies in the antibody library using phage display technology.

[0113] Phage-ELISA method (phage-ELISA) screening of specific single positive clones:

[0114] Screening principle diagram Figure 4 As shown, 1 is IL-6 antigen coated on the enzyme labeling 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.

[0115] The specific method is as follows:

[0116] 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.

[0117] Dilute IL-6 antigen to 10 μg / mL in coating solution, add 100 μL to each well, and coat overnight at 4°C. Negative and positive controls should be established. The next day, wash three times with PBST, block with 2% PBSM at 37°C for 2 hours, and then 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 (catalog number 11973-MM05T-H) diluted in 0.1% PBST, 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 positive control well is defined when the OD value of the sample well is at least twice that of the control well. 96×4 monoclonal clones were selected from the plates after the third and fourth rounds of screening, and the monoclonal supernatants were prepared and identified by phage ELISA (Phage-ELISA). PBS was used as a negative control and M13K07 as a positive control. When the OD value ratio of the experimental group to the negative control group was ≥2.1, it was considered a positive clone. 40 monoclonal samples with higher absorbance values ​​were selected. The results are as follows Figure 5 The positive bacterial solution was taken for gene sequencing.

[0118] Sequence analysis and Blast alignment were performed using Snapegene software, and strains with identical CDR1 (SEQ ID No. 13), CDR2 (SEQ ID No. 14), and CDR3 (SEQ ID No. 15) sequences were considered the same clone. The Nanobody sequence shown in amino acid sequence SEQ ID No. 1 was ultimately used for subsequent experiments. The amino acid sequences of FR1, FR2, FR3, and FR4 in the framework region FR of the Nanobody shown in SEQ ID No. 1 are shown in SEQ ID No. 5 to SEQ ID No. 8, respectively. The nucleotide sequence encoding the amino acids shown in SEQ ID No. 1 is shown in SEQ ID No. 2.

[0119] Example 2

[0120] Expression and purification of nanobodies in host bacteria Escherichia coli:

[0121] (1) The His-tagged nanoantibody sequence obtained by sequencing analysis was subcloned into the pET-28a(+) plasmid vector and transformed into Escherichia coli Arctic Express. Single clones on the transformation plate were 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, the cells were 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 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 6 It is a purified anti-IL-6 nanoantibody. The figure is a Western Blot diagram of the purified anti-IL-6 nanoantibody, where lane M is the protein molecule standard, lane 1 is 0.5 mg / mL TNF-a as the concentration measurement standard, and lane 2 is the purified anti-IL-6-4G10 nanoantibody.

[0122] Example 3

[0123] ELISA method to detect the specific binding of IL-6 nanoantibody 4G10 to IL-6 antigen

[0124] Human IL-6 antigen (Human, product number IL6-H82Q9) was diluted to 2 μg / mL in ELISA coating buffer and 100 μL was added to each well of a 96-well microtiter plate for coating at 4°C. PBS 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 blocked with 300 μL of 5% PBSM per well at 37°C for 2 hours. After blocking, the plate was washed three times with PBST and patted dry. 100 μL of the experimental and control groups (using the HER-2-Nb antibody, disclosed in Chinese patent CN114437222A) was added to each well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, patted dry, and 100 μL of the mouse His antibody diluted 1:5000 in 0.05% PBST was added to each well. The plate was incubated at 37°C for 1 hour, washed, and patted dry. Dilute goat anti-mouse HRP antibody with 0.05% PBST at a volume ratio of 1:5000, add 100 μL to each well, incubate at 37°C for 1 hour, wash the plate and pat dry. Figure 7 As shown, the anti-IL-6-4G10-Nb nanobody specifically binds to the IL-6 antigen.

[0125] Example 4

[0126] Anti-IL-6 Nanobody 4G10 and IL-6 Antigen Specificity Detection:

[0127] The affinity of the nanobody was determined by biomembrane interferometry (BLI). The biotinylated humanized IL-6 antigen was bound to the Streptavidin (SA) probe, and six nanobody concentration gradients of 8000, 4000, 2000, 1000, 500, and 250 nmol / L were set. The binding and dissociation of the nanobody at each concentration with the biotinylated humanized IL-6 antigen were determined using OctetRed96e. The detection data were fitted according to a 1:1 binding model. The results are shown in Figure 2. Figure 8 The binding and dissociation curves of IL-6 nanoantibodies at various concentrations and IL-6 antigens were plotted and the KD values ​​were calculated. The results showed that the binding constant was 6.034×10 -5 , the dissociation constant is 2.161×10 -3 The calculated equilibrium dissociation constant (KD) value is 3.581×10 -8 , suggesting that the anti-IL-6-4G10 nanoantibody has a strong ability to specifically bind to IL-6 antigen.

[0128] 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. An anti-IL-6 nanobody, characterized in that The nanobody 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.

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

1.

4. A gene encoding an anti-IL-6 nanobody, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

2.

5. A recombinant nanobody against IL-6, characterized in that Comprising the Nanobody according to any one of claims 1 to 3 and a His tag expressed by fusion with the 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 Nanobody according to any one of claims 1 to 3 and a gene encoding the His tag.

7. A biomaterial expressing an anti-IL-6 nanobody, 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 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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