A humanized nanobody against IL-6 and its recombinant vector and application

Abstract

The present invention belongs to the technical field of immunology, and specifically relates to a humanized nano antibody against IL-6, and a recombinant vector and application thereof. The present invention analyzes the screened anti-IL-6 nano antibody 4G10 sequence, and for screening the heavy chain of IL-6, the anti-IL-6 nano antibody 4G10 includes a framework region FR and an antigenic determinant complementary CDR, and the CDR1, CDR2, and CDR3 regions of the screened anti-IL-6 nano antibody 4G10 sequence are transplanted into the humanized hs2dAb skeleton to obtain the humanized nano antibody 4G10 against IL-6 of the present application; the recombinant nano antibody expressed by the gene encoding the humanized nano antibody 4G10 against IL-6 and the His tag gene fusion can 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 humanized nano-antibody and a recombinant vector and application thereof. Background Art

[0002] IL-6 (interleukin 6, IL-6) is released by many different types of cells, such as macrophages, T cells, and fibroblasts. 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 the intracellular signal transduction pathway on the target cells. Its signal transduction pathway is strictly regulated. If IL-6 signal transduction is dysregulated, it will cause chronic inflammation, autoimmune diseases, etc. In the classical pathway, IL-6 binds to its membrane-bound receptor mIL-6R and the membrane protein gp130 to form an IL-6-IL-6R-gp130 hexamer, triggering downstream signal transduction and gene expression. In the reverse pathway, the IL-6-sIL-6R complex binds to gp130 and initiates intracellular signal transduction. Next, JAK-STAT, RAS-RAF and other pathways are activated, promoting cell proliferation, differentiation, oxidative stress and immune regulation. However, when IL-6 levels increase, due to the ubiquitous presence of gp130, the signal is widely expressed. IL-6 activates signal transducer and activator of transcription 3 (STAT3) through JAK kinases (JAKs). The STAT3 signaling pathway plays an important role in autoimmune diseases, tumor cell proliferation, angiogenesis, and tumor immune escape. Small molecule inhibitors targeting IL-6 / STAT3 will become potential drugs for the treatment of inflammatory diseases and tumors.

[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 silver sharks. They are the smallest units known to bind to target antigens. VHH has a molecular weight of only 15KD, so it is also called nanobody (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] So far, more than 100 mouse monoclonal antibodies have been humanized through CDR transplantation. Basic principle: The variable region of the heavy and light chains of antibodies is mainly composed of CDR and framework region (FR). Among them, the 6 CDRs in the variable region are responsible for recognizing and binding to antigens. They are in direct contact with the antigen and determine the specificity of the antibody. The framework region is the other part outside the variable region, which mainly plays a supporting role in CDR, and their amino acid composition and arrangement are relatively difficult to change. Therefore, the CDR of mouse monoclonal antibody can be transplanted to the framework region of human monoclonal antibody, which may make human monoclonal antibody obtain the same antigen specificity as mouse monoclonal antibody, and can minimize the heterogeneity of mouse monoclonal antibody, thus obtaining modified antibodies. Compared with chimeric antibodies, modified antibodies further reduce the proportion of mouse-derived parts in antibodies and reduce human anti-mouse antibodies, but there are still antibodies that may lead to the production of anti-idiotypic antibodies and have some limitations, such as the relatively complex construction method, which is time-consuming and labor-intensive to operate; there are great problems in the crystal structure of antibodies and the microstructure of computer-simulated antibodies; there are still many problems in reducing immunogenicity and maintaining antigen binding activity.

[0005] Therefore, it is urgent to find a humanized anti-IL-6 antibody that can maintain high immunological activity. Summary of the invention

[0006] The object of the present invention is to provide a humanized anti-IL-6 nanobody. The humanized nanobody provided by the present invention can not only specifically bind to IL-6, but also block the binding of IL-6 to its receptor while maintaining affinity with IL-6.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides an anti-IL-6 humanized nanobody, wherein the nanobody comprises complementary determining regions CDR1-CDR3, and the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NOs.13-15.

[0009] Preferably, the Nanobody further comprises four framework regions FR1-FR4 alternately connected to the complementary determining regions CDR1-CDR3, and the amino acid sequences of FR1-FR4 are shown in SEQ ID NO.5-8.

[0010] Preferably, the amino acid sequence of the Nanobody is as shown in SEQ ID NO.1.

[0011] The present invention also provides a process for constructing the above-mentioned humanized nanobody, comprising transplanting the complementary determining regions CDR1-CDR3 described in claim 1 into the humanized hs2dAd framework.

[0012] The present invention also provides a nucleotide molecule, the nucleotide sequence of which is shown as SEQ ID NO.2.

[0013] The present invention also provides a recombinant vector, which comprises the above-mentioned nucleotide molecule and an initial vector.

[0014] Preferably, the initial vector is a pCZN1 plasmid vector.

[0015] The present invention also provides a recombinant bacterium, which comprises the above-mentioned recombinant vector.

[0016] The present invention also provides the use of the humanized nano-antibody in the preparation of autoimmune disease or anti-tumor drugs or tumor diagnostic reagents.

[0017] Beneficial effects of the present invention:

[0018] The recombinant humanized anti-IL-6 nanobody provided by the present invention is expressed only by camel-derived nanobody genes, can be stably expressed using the recombinant strain, can specifically recognize IL-6 antigen, and can be applied to molecular diagnosis of autoimmune diseases and tumors and preparation of autoimmune disease and anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] 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;

[0021] 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;

[0022] Figure 3 This is the result diagram of colony PCR analysis to identify the insertion rate of the target fragment;

[0023] Figure 4The schematic diagram of screening specific single positive clones by phage-ELISA method (phage-ELISA): 1 is IL-6 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;

[0024] Figure 5 The purified anti-IL-6-4G10 humanized nanobody, wherein the left figure is the SDS-PAGE electrophoresis staining diagram of the post-crushing treatment sample, the outflow sample and the elution sample; the right figure is the SDS-PAGE electrophoresis staining diagram of the purified anti-IL-6-4G10 humanized nanobody;

[0025] Figure 6 This is a western blot image of anti-IL-6-4G10 humanized nanobody.

[0026] Figure 7 This is the affinity test chart of anti-IL-6-4G10 humanized nanobody and IL-6 antigen (indirect ELISA).

[0027] Figure 8 This is the affinity determination diagram of anti-IL-6-4G10 humanized nanobody and IL-6 antigen (BLI). DETAILED DESCRIPTION

[0028] The present invention provides an anti-IL-6 humanized nanobody 4G10, the amino acid sequence of the nanobody (4G10-AA) is shown in SEQ ID NO.1; specifically as follows:

[0029] EVQLQASGGGFVQPGGSLRLSCAASGLDFDDYNMGWFRQAPGKEREFVSAISDWNGRTHYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAAYYTPFCYSGTDYDYWGQGTQVTVSS

[0030] In the present invention, the humanized anti-IL-6 nanobody includes a humanized framework region FR and an antigen determinant complementary region CDR; in the present invention, the humanized framework region FR includes FR1, FR2, FR3 and FR4; the specific amino acid sequence is as follows:

[0031] FR1-AA:EVQLQASGGGFVQPGGSLRLSCAAS(SEQ ID NO.5)

[0032] FR2-AA:MGWFRQAPGKEREFVSA(SEQ ID NO.6)

[0033] FR3-AA:YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC(SEQ ID NO.7)

[0034] FR4-AA:WGQGTQVTVSS(SEQ ID NO.8)

[0035] The corresponding nucleotide sequences are as follows:

[0036] FR1: GAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGTGCAGCCGG GTGGTAGCCTGCGTCTGAGCTGCGCAGCATCA (SEQ ID NO.9)

[0037] FR2: ATGGGCTGGTTTCGTCAGGCCCCGGGTAAAGAACGTGAATTTG TGAGTGCA (SEQ IDNO.10)

[0038] FR3: TATTATGCAGATAGTGTGAAAGGCCGTTTTACCATTAGTCGTGAT AATAGTAAGAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGA TACCGCAACCTATTATTGC (SEQ ID NO. 11)

[0039] FR4: TGGGGTCAGGGTACCCAGGTTACCGTTAGTAGC (SEQ ID NO. 12).

[0040] In the present invention, the complementary region CDR of the antigen determinant includes CDR1, CDR2 and CDR3; the specific amino acid sequence is as follows:

[0041] CDR1-AA:GLFFDDYN(SEQ ID NO.13)

[0042] CDR2-AA:ISDWNGRTH(SEQ ID NO.14)

[0043] CDR3-AA:AAAYYTPFCYSGTDYDY (SEQ ID NO. 15).

[0044] In the present invention, the nucleotide sequences of CDR1, CDR2 and CDR3 are as follows:

[0045] CDR1:GGTTTAGATTTTGATGATTACAAC(SEQ ID NO.16)

[0046] CDR2: ATTAGCGATTGGAATGGCCGTACCCAT(SEQ ID NO.17)

[0047] CDR3: GCCGCCGCCTATTATACCCCGTTTTGCTATAGTGGTACCGATTATGATTAC (SEQ ID NO. 18).

[0048] In the present invention, the humanized anti-IL-6 nanobody is obtained by screening by the following method: a natural camel-derived nanobody phage display library is constructed using phage surface display technology, and then screening is performed based on biotinylated IL-6 antigen to obtain an IL-6-specific nanobody gene sequence, and the CDR1, CDR2, and CDR3 regions of the screened anti-IL-6 nanobody 4G10 sequence are transplanted into the humanized hs2dAb skeleton.

[0049] In the present invention, 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) using the cDNA as a template to perform nested PCR amplification 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.

[0050] In the present invention, total RNA from camel peripheral blood mononuclear cells is extracted, and the total RNA is reverse transcribed to obtain cDNA. The present invention has 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. In the present invention, the reverse transcription is preferably achieved by Thermo Scientific ReverAid First Strand cDNA Synthesis Kits.

[0051] After obtaining the cDNA, the present invention uses the cDNA as a template to perform nested PCR amplification to obtain the variable region fragment of the heavy chain antibody. In the present invention, 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 sequence of the first round of PCR is 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 sequence of the second round of PCR is 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 pCANTAB5e phage vector are respectively digested, and then connected to obtain a connection product. In the present invention, the digestion is preferably double digestion, and the digestion is preferably performed using restriction endonucleases SifI and Not I. In the present invention, the digestion procedure is preferably as follows: digestion at 37°C for 1h; digestion at 50°C for 1h. In the present invention, the connection temperature is preferably 16°C, and the connection time is preferably 4h.

[0053] After obtaining the ligation product, the present invention transfers the ligation product into competent cells to obtain a natural camel-derived nanoantibody phage display library. In the present invention, the competent cell is preferably an Escherichia coli competent cell TG1, and the Escherichia coli competent cell TG1 is preferably homemade by glycerol resuspension method; the transfer method is preferably electrotransformation. In the present invention, the transformation also includes a process of auxiliary phage rescue. The present invention has no special limitation on the process of electrotransformation and auxiliary phage assistance. For detailed steps, please refer to the embodiment.

[0054] The present invention has no particular limitation on the specific steps of screening anti-IL-6 nanobodies using the biotinylated IL-6 antigen, and conventional nanobody screening methods in the art may be used. For detailed steps, please refer to the examples.

[0055] The present invention also provides a gene encoding the humanized nanobody, the nucleotide sequence of the gene (4G10) is shown in SEQ ID NO.2, specifically as follows:

[0056] GAGGTTCAGCTGCAGGCAAGTGGCGGTGGTTTTGTGCAGCCGGGTGGTTTCACTGCGCCTGAGCTGCGCAGCATCAGGTTTAGATTTTGATGATTACAACATGGGCTGGTTTCGTCAGGCCCCGGGTAAAGAACGTGAATTTGTGAGTGCAATTAGCGATTGGAATGGCCGTACCCATTATTATGCAGA TAGTGTGAAAGGCCGTTTTACCATTAGTCGTGATAATAGTAAGAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCAACCTATTATTGCGCCGCCGCCTATTATACCCCGTTTTGCTATAGTGGTACCGATTATGATTACTGGGGTCAGGGTACCCAGGTTACCGTTAGTAGC.

[0057] The present invention provides a recombinant humanized nanobody against IL-6, comprising the humanized nanobody against IL-6 and His tag fusion expression. In the present invention, the amino acid sequence (4G10-His-AA) of the recombinant humanized nanobody is shown in SEQ ID NO.3, specifically as follows:

[0058] HHHHHHMEVQLQASGGGFVQPGGSLRLSCAASGLDFDDYNMGWFRQ APGKEREFVSAISDWNGRTHYYADSVKGRFTISRDNSKNTVYLQMNSLRAE DTATYYCAAAYYTPFCYSGTDYDYWGQGTQVTVSS.

[0059] The nucleotide full-length sequences or fragments thereof of various antibodies of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. The coding sequence of the heavy chain and the expression tag can also be fused together to form a fusion protein. The present invention provides a gene encoding the recombinant humanized nanobody, including the gene of the humanized nanobody and the His tag fusion expression gene.

[0060] In the present invention, the nucleotide sequence of the gene of the recombinant humanized Nanobody (4G10-His) is shown in SEQ ID NO.4, specifically as follows:

[0061] CACCACCACCACCACCACATGGAGGTTCAGCTGCAGGCAAGTGGCGGTGGTTTTGTGCAGCCGGGTGGTTCACTGCGCCTGAGCTGCGCAGCATCAGGTTTAGATTTTGATGATTACAACATGGGCTGGTTTCGTCAGGCCCCGGGTAAAGAACGTGAATTTGTGAGTGCAATTAGCGATTGGAATGGCCGTACCCAT TATTATGCAGATAGTGTGAAAGGCCGTTTTACCATTAGTCGTGATAATAGTAAGAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCAACCTATTATTGCGCCGCCGCCTATTATACCCCGTTTTGCTATAGTGGTACCGATTATGATTACTGGGGTCAGGGTACCCAGGTTACCGTTAGTAGC.

[0062] Among them, the nucleotide sequence of the His tag gene is shown in SEQ ID NO.23, specifically as follows: CACCACCACCACCACCAC.

[0063] The present invention also provides a recombinant vector for expressing humanized anti-IL-6 nanobody, comprising the gene of the recombinant humanized nanobody and an initial vector.

[0064] In the present invention, the initial vector is preferably a pCZN1 plasmid vector; the insertion site of the gene of the recombinant humanized nanobody is preferably located at the Nco I-Xho I site of the pCZN1 plasmid vector. The present invention has no special limitation on the preparation method of the recombinant vector, and the conventional recombinant vector preparation method in the art can be used.

[0065] The present invention also provides a recombinant strain expressing humanized nanobodies against IL-6, comprising the recombinant vector and Escherichia coli Arctic Express. In the present invention, the recombinant strain is preferably obtained by transferring the recombinant vector expressing humanized nanobodies against IL-6 into the Escherichia coli Arctic Express. The present invention does not specifically limit the specific preparation method of the recombinant strain, and the conventional preparation method of the recombinant strain in the art can be used.

[0066] All aspects of the various antibodies described herein can be used to prepare medicaments for preventing or treating various conditions and diseases described herein, especially diseases or conditions associated with cells expressing IL-6. In some embodiments, the conditions and diseases are autoimmune diseases or tumors.

[0067] 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.

[0068] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0069] RNA purification kit was purchased from TIANGEN; restriction endonucleases were purchased from NEB, and T4 DNA ligase was purchased from NEB.

[0070] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0071] Example 1 Construction of natural camel-derived nanoantibody gene library

[0072] 1. Extraction of total RNA from camel peripheral blood mononuclear cells

[0073] (1) Blood was collected from Bactrian camels, and 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.

[0074] (2) Add 0.2 mL of chloroform per 1 mL of Trizol, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0075] (3) Centrifuge the sample at 12000 rpm and 4°C for 15 min and transfer the upper layer to a new Eppendorf tube.

[0076] (4) Add an equal volume of isopropanol and place on ice for 20 min.

[0077] (5) Centrifuge the solution at 12,000 rpm and 4°C for 10 min and discard the supernatant.

[0078] (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).

[0079] (7) Centrifuge the solution in step (7) at 10,000 rpm for 10 min at 4°C, discard the supernatant, and repeat the previous step for one more wash.

[0080] (8) The solution obtained in step (7) was centrifuged at 10,000 rpm and 4°C for 10 min, the supernatant was discarded, and the solution was dried for 10-15 minutes.

[0081] (9) Add appropriate amount of ddH2O to dissolve the RNA precipitate, determine the RNA concentration, and store at -80°C.

[0082] The obtained RNA was purified using an RNA purification kit and reverse transcribed to obtain cDNA using the Thermo Scientific ReverAid FirstStrand cDNA Synthesis Kits.

[0083] 2. Using cDNA as a template, nested PCR was used to amplify the variable region fragment of the heavy chain antibody;

[0084] First round of PCR:

[0085] F-1: 5'-GTCCTGGCTGCTCTTCTACAAAG-3' (SEQ ID NO. 19)

[0086] R-1: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO. 20)

[0087] Table 1 First round PCR reaction system

[0088]

[0089] 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.

[0090] 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 As shown, the size of the fragment is about 700-900 bp, that is, there are about two electrophoresis bands of the nanobody gene.

[0091] Second round of PCR:

[0092] Using the first round PCR product as template,

[0093] F-2: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGG G-3' (SEQ ID NO. 21)

[0094] R-2: 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3' (SEQ ID NO. 22)

[0095] Table 2 Second round PCR reaction system

[0096]

[0097] 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 fragment and short fragment) 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 As shown, the size of the fragment is about 450 bp, that is, the electrophoresis band of the nanobody gene is about 450 bp.

[0098] The pCANTAB5E phage vector and VHH fragment were digested with restriction endonucleases SifI and Not I, and the two fragments were connected with T4 DNA ligase. The specific enzyme digestion system and connection system are as follows:

[0099] Table 3 Enzyme digestion system

[0100]

[0101] Enzyme digestion conditions: digestion at 37°C for 1 h, then at 50°C for 1 h.

[0102] Table 4 Connection system

[0103]

[0104] Ligation conditions: 16°C for 4 h, followed by overnight ligation at 4°C.

[0105] 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 .

[0106] The insertion rate of the natural camel-derived nanobody phage display library was detected by colony PCR. The primers used were the second-round PCR primers, and the annealing temperature was 55°C. The results are as follows Figure 3 As shown, the insertion rate reached more than 95% (target fragment insertion rate = the number of colonies containing the target fragment / the number of all colonies).

[0107] 3. The preparation method of competent cells TG1 is as follows:

[0108] Take out the E. coli TG1 glycerol culture from the -80℃ refrigerator, streak it on a 2×YT solid plate and culture it at 37℃ for 10h, pick a single colony and inoculate it into 3mL 2×YT liquid culture medium, and culture it at 37℃ with shaking at 200rpm overnight. The next day, amplify the culture liquid at a ratio of 1:100 and culture it in a conical flask containing 200mL 2×YT culture medium, and continue to culture it at 37℃ until the OD 600 When the concentration is about 0.4, collect the bacterial solution 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 pellet with the same volume of cold pure water, centrifuge, and repeat once. Then resuspend the bacterial pellet with pre-cooled 10% glycerol and centrifuge. Use 1mL 10% glycerol (pre-cooled pure water) to suspend the bacterial pellet and divide it into pre-cooled 1mL Ep tubes, 100μl per tube, and then immediately transfer to -80℃ refrigerator for storage, which is the competent cell TG1.

[0109] 4. The steps for helper phage rescue are as follows:

[0110] (1) Take 100 μL of the library and inoculate it into 50 mL of 2×YT / Amp / Glu medium. Cultivate at 37°C, 200 rpm, and shake until the OD value reaches the logarithmic phase. 600 About 0.4 to 0.5.

[0111] (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.

[0112] (3) Centrifuge the culture at 9000 rpm for 10 min at room temperature, discard the supernatant to precipitate the cells, resuspend them in 200 mL of 2×YT / Amp / Kana culture medium, and culture them at 37°C, 200 rpm, overnight.

[0113] (4) The culture medium was centrifuged at 9000 rpm for 10 min at 4°C, and the supernatant was taken. 1 / 5 volume of PEG / NaCl was added and the culture medium was allowed to stand at 4°C for 6 h.

[0114] (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, dispense it into 1.5 mL Eppendorf tubes, and store it at 4°C.

[0115] Example 2 Screening for anti-IL-6 nanobodies:

[0116] The phage library (1×10 13 phage) and 50 μL streptavidin magnetic beads were incubated 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 biotinylated IL-6 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, and incubated on a rotating table at room temperature for 30 minutes, and the 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 / L Tris-HCl at 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 will be continuously enriched, thereby achieving the purpose of using phage display technology to screen IL-6 specific antibodies in the antibody library.

[0117] Phage-ELISA method to screen specific single positive clones:

[0118] Screening principle diagram Figure 4 As shown, the specific method is as follows:

[0119] 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.

[0120] Dilute IL-6 antigen to 10 μg / mL with coating solution, add 100 μL to each well, coat overnight at 4°C, and set up negative and positive controls. The next day, wash three times with PBST, block with 2% PBSM at 37°C for 2h, wash three times with PBST, add 200 μL of pretreated VHH phage monoclonal supernatant, and incubate at 37°C for 1h. Add 1:5000 mouse anti-M13KO7 / HRP secondary antibody diluted with 0.1% PBST, incubate at 37°C 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.

[0121] 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-IL-6-4G10 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.

[0122] Example 3 Expression and purification of humanized nanobody IL-6-4G10-Nb in host bacteria Escherichia coli:

[0123] (1) The humanized nanobody IL-6-4G10-Nb sequence obtained by sequencing analysis was subcloned into the pCZN1 plasmid vector and transformed into Escherichia coli Arctic Express. A 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;

[0124] (2) The next day, the cells were inoculated at a ratio of 1:100 into 30 mL LB culture medium containing 50 μg / mL Kan and shaken at 220 rpm at 37°C until the bacterial OD 600 The pH value 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;

[0125] (3) The bacterial cells were collected and ultrasonically disrupted to obtain a crude liquid of inclusion body protein, which was then affinity purified using a Ni column to obtain a fusion protein.

[0126] The results are as follows Figure 5As shown, the purified anti-IL-6 humanized nanoantibody 4G10, wherein the left figure is the SDS-PAGE electrophoresis staining diagram of the purification process of the anti-IL-6 humanized nanoantibody 4G10: wherein lane M is the protein molecule standard, lanes 1-2 are the post-crushing treatment samples and the outflow samples, respectively, and lanes 3-4 are the elution samples; the right figure is the SDS-PAGE electrophoresis staining diagram of the purified anti-IL-6 humanized nanoantibody 4G10, 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-IL-6 humanized nanoantibody 4G10.

[0127] Example 4 Specificity verification of anti-IL-6 humanized nanobody 4G10

[0128] Western blot was performed using IL-6 antigen (the primary antibody was purified anti-IL-6 humanized nanobody 4G10, and the secondary antibody was anti-mouse His / HRP). The results are as follows Figure 6 As shown, the western blot of IL-6 antigen: wherein lane M is the protein molecule standard, and lane 1 is the IL-6 antigen. This shows that the anti-IL-6 humanized nanobody 4G10 provided by the present invention can specifically bind to the IL-6 antigen.

[0129] Example 5 ELISA method to detect the specific binding of IL-6 humanized nanobody 4G10 and IL-6 antigen

[0130] IL-6 antigen was diluted to 2 μg / mL with ELISA coating solution, and 100 μL was added to each well of 96-well ELISA 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 for blocking at 37°C for 2 h.

[0131] After blocking, wash the plate 3 times with PBST and pat dry. Add 100 μL of the experimental group and control group samples to each well, incubate at 37°C for 1 hour, wash the plate 3 times with PBST and pat dry. Dilute the mouse His antibody with 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. Dilute the goat anti-mouse HRP antibody with 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. The test results are as follows: Figure 7 As shown, the anti-IL-6-4G10-Nb humanized nanobody specifically binds to the IL-6 antigen.

[0132] Example 6 Anti-IL-6 humanized nanobody 4G10 and IL-6 antigen specificity detection

[0133] 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 6 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 Octet Red96e. The detection data were fitted according to the 1:1 binding model, and the binding and dissociation curves of the IL-6 nanobody 4G10 at each concentration with the IL-6 antigen were drawn to calculate the KD value.

[0134] The results are as follows Figure 8 The results show that its binding constant is 5.125×10 -4 , the dissociation constant is 4.999×10 -2 The calculated equilibrium dissociation constant (KD) value is 9.755×10 -6 , suggesting that the anti-IL-6 humanized nanoantibody 4G10 has a strong ability to specifically bind to the IL-6 antigen.

[0135] 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 IL-6, characterized in that: The nanobody includes complementary determining regions CDR1-CDR3, and the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO.13-15.

2. The humanized Nanobody according to claim 1, characterized in that The nanobody also includes four framework regions FR1-FR4 alternately connected to the complementary determining regions CDR1-CDR3, and the amino acid sequences of FR1-FR4 are shown in SEQ ID NO.5-8.

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

1.

4. The process for constructing the humanized nanobody according to claim 1, characterized in that: The method comprises transplanting the complementary determining regions CDR1-CDR3 described in claim 1 into a humanized hs2dAd framework.

5. A nucleotide molecule, characterized in that The nucleotide sequence is shown in SEQ ID NO.

2.

6. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide molecule according to claim 5 and an initial vector.

7. The recombinant vector according to claim 6, characterized in that The initial vector is a pCZN1 plasmid vector.

8. A recombinant bacterium, characterized in that: The recombinant bacteria include the recombinant vector according to claim 6.

9. Use of the humanized nanobody according to any one of claims 1 to 4 in the preparation of autoimmune disease or anti-tumor drugs or tumor diagnostic reagents.

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