Nanobody specifically recognizing the fc fragment of igg1 antibodies and preparation and use thereof

Abstract

The application discloses a nano antibody capable of specifically recognizing an Fc segment of an IgG1 antibody and a preparation and application thereof. The nano antibody capable of specifically recognizing the Fc segment of the IgG1 antibody has an amino acid sequence shown in SEQ ID NO: 6. Four kinds of nano antibodies obtained by the application can be specifically combined with the Fc segment of an IgG1 immunoprotein.

Description

[0001] Cross-reference information This application is a divisional application of Chinese invention patent filed on December 9, 2022, with application number 2022115798678 and entitled "Nanobosomes that specifically recognize the Fc segment of IgG1 antibody and their preparation and application". Technical Field

[0002] This invention relates to the field of genetic engineering antibody technology, specifically to a nanobody that specifically recognizes the Fc segment of IgG1 antibody, and its preparation and application. Background Technology

[0003] Nanobodies, also known as single-domain heavy-chain antibodies (VHH antibodies, variable domain of heavy chain of heavy-chain antibody), are the smallest known antigen-binding antibody molecules, with a molecular weight of approximately 15 kDa. Compared to traditional antibodies, nanobodies, lacking a light chain structure, possess advantages such as small relative molecular mass, high affinity, high stability, low immunogenicity, and strong penetrability.

[0004] Immunoglobulin G (IgG) consists of two identical light chains and two identical heavy chains. Each chain contains a variable region (V region) and a constant region (C region). The variable region provides the antigen-binding site and specificity, while the constant region forms the framework of the immunoglobulin and has biological effector functions. Digestion of IgG with papain separates it into three functional regions: two identical antigen-binding fragments (Fab) and one crystallizable fragment (Fc). The Fc segment is located in the constant region and consists of half of the carboxyl terminus of the two heavy chains.

[0005] Currently, antibody purification mostly utilizes affinity chromatography, which leverages the affinity between specialized purification proteins and the Fc site of IgG. Due to the high production cost of these purified proteins, the purification cost of IgG is also high. IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4. While IgG1 is widely used clinically, there are still relatively few nanobody products targeting the IgG1 subtype. Summary of the Invention

[0006] To overcome the above-mentioned shortcomings, the present invention aims to provide a nanobody that specifically recognizes the Fc segment of IgG1 antibody, as well as its preparation and application.

[0007] The nanobody of the present invention specifically recognizes the Fc fragment of an IgG1 antibody, wherein its variable structural domain includes at least one group of the following complementarity-determining regions: ①CDR11, CDR12 and CDR13, The CDR11 contains the sequence VFNME (SEQ ID NO: 7). The CDR12 contains the sequence LISSGGSTNYADSVKG (SEQ ID NO: 8). The CDR13 contains the sequence RNGWRNI (SEQ ID NO: 9). ②CDR21, CDR22 and CDR23, The CDR21 contains the sequence AIGMG (SEQ ID NO: 10). The CDR22 contains the sequence LINSDGSTNYADFVKG (SEQ ID NO: 11). The CDR23 contains the sequence VARIGLGPYRDY (SEQ ID NO: 12). ③CDR31, CDR32, CDR33 The CDR31 contains the sequence TYTMG (SEQ ID NO: 13). The CDR32 contains the sequence TISSGGTTFYVASVKG (SEQ ID NO: 14). The CDR33 contains the sequence LPVGRWYGAEA (SEQ ID NO: 15). ④CDR41, CDR42, CDR43 The CDR41 contains the sequence IFNME (SEQ ID NO: 16). The CDR42 contains the sequence LISSGGSTNYADSVKG (SEQ ID NO: 17). The CDR43 contains the sequence RHIWRDI (SEQ ID NO: 18). The nanobody of the present invention that specifically recognizes the Fc segment of IgG1 antibody has an amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4 or SEQ ID NO: 6.

[0008] Furthermore, the nanobody that specifically recognizes the Fc fragment of the IgG1 antibody is prepared by the following steps: S1: Preparation of FC protein: The gene sequence of the FC protein was cloned into an expression vector using molecular cloning methods, and the correctness of the molecular clone construction was verified by sequencing. S2: Animal Immunization: The FC protein prepared in step S1 was used to immunize alpacas by multiple intradermal and subcutaneous injections on the back. The adjuvant for the first immunization was complete Freund's adjuvant, and the adjuvant for subsequent immunizations was incomplete Freund's adjuvant. S3: Screening of nanobodies and amplification of the VHH gene: RNA was extracted from leukocytes of animals immunized with S2 and the VHH gene was amplified for phage library construction. S4: Screening of nanobody-positive clones: S5: Preparation of nanobodies.

[0009] Furthermore, in step S1, the gene sequence of the FC protein is SEQ ID NO: 1, and the nucleotide sequence for synthesizing the FC protein is shown in SEQ ID NO: 2.

[0010] Furthermore, in step S4, the screening of positive nanobody clones includes the following steps: S41: Obtaining phage particles that recognize FC proteins: The FC protein was labeled using a Biotin kit, and then the labeled protein was added to phage particles at a final concentration of 10 μg / ml. The mixture was incubated at 37°C for 1 h. Then, 10 μl of streptavidin magnetic beads were added to the phage library and incubated at 37°C for 1 h to capture phage particles that recognize the FC protein. S42: Eluting of bacteriophages: Enzymatic hydrolysis was used to elute specifically bound phages; the eluent was transferred to a sterile centrifuge tube; 10 µL was serially diluted, the titer was measured, and the recovery rate was calculated; the remaining eluent was mixed and amplified and purified for the next round of affinity panning. Mix the eluent with 5 mL of E. coli TG1 culture in the early logarithmic growth phase, incubate at 37°C with shaking at 220 rpm for 45 min, then transfer to 20 mL of 2×YT-A liquid medium and incubate at 37°C with shaking at 220 rpm for 2 h. Add M13K07 phage at a cell:phage ratio of 1:20, incubate at 37°C for 15-30 min, then incubate with shaking at 220 rpm for 30-45 min. Aliquot the culture into centrifuge tubes and incubate at 4°C, 3500 rpm for 10 min (2200 g for 15 min). Resuspend the cell pellet in 25 mL of 2×YT-AK liquid medium and incubate at 30°C, 250 rpm. Incubate overnight with shaking; centrifuge the overnight culture at 4°C, 12000 rpm for 10 min (7000 g, 15 min), transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG-NaCl, mix well, and incubate at 4°C for at least 1 h; centrifuge at 4°C, 12000 rpm for 10 min, remove the supernatant, resuspend the precipitate in 2 mL PBS, add 1 / 5 volume of PEG / NaCl, mix well, and incubate at 4°C for at least 1 h; centrifuge at 12000 rpm for 10 min, remove the supernatant, and resuspend the precipitate in 200 µL PBS, which is the amplification product. Measure the titer for the next round of panning or analysis. From the plate used to determine the titer of the eluent from the final round of panning, a single colony was randomly picked with a sterile toothpick and inoculated into 1 mL of 2×YT-GA. The culture was incubated at 37°C with shaking at 220 rpm for 12 h. A 1% inoculum was then added to 2×YT-GA and incubated at 37°C with shaking at 220 rpm until the early logarithmic growth phase. M13K07 phage was added at a cell:phage ratio of 1:1. The culture was incubated at 37°C for 15 min, then incubated with shaking at 220 rpm for 30–45 min. The culture was then centrifuged at 4°C with shaking at 3500 rpm for 10 min. The precipitate was resuspended in an equal volume of 2×YT-AK and incubated at 30°C with vigorous shaking for 12 h. The culture was then centrifuged in a centrifuge tube at 4°C with shaking at 10000 rpm for 10 min. The supernatant was collected for ELISA identification. The ELISA method for identifying positive clones uses the ratio of the OD value (S) of the test sample to the OD value (N) of the negative control. S43: Positive clones were sequenced, and ClustalW software was used to perform multiple sequence alignment analysis on the sequencing results. The amino acid sequence of the nanobody was deduced based on the nucleic acid sequence.

[0011] The present invention also provides a nucleotide sequence encoding the aforementioned nanobody.

[0012] The present invention also provides an expression vector comprising the aforementioned nucleotide sequence.

[0013] The present invention also provides a host cell comprising the aforementioned nucleotide sequence.

[0014] The present invention also provides the application of the aforementioned nanobodies that specifically recognize the Fc fragment of IgG1 antibodies in the detection, separation and purification of IgG1 antibodies.

[0015] The present invention also provides the application of the aforementioned nanobodies that specifically recognize the Fc fragment of IgG1 antibodies in the detection and purification of fusion proteins containing Fc fragments of IgG1 antibodies.

[0016] The present invention also provides the application of the aforementioned nanobodies that specifically recognize the Fc fragment of IgG1 antibodies in the detection and separation of Fc fragments of IgG1 antibodies.

[0017] The present invention has the following advantages: ①The present invention immunizes alpacas with the Fc segment protein of IgG1, which has the amino acid sequence SEQ ID NO: 1 and the gene sequence SEQ ID NO: 2. This enables the animal's immune system to produce immune cells that are specific to the Fc segment protein of IgG1. In turn, antibodies that specifically bind to the Fc segment protein of SEQ ID NO: 1 of the present application can be isolated from the immune cells of the immunized animal. ② This invention obtains immune cells that specifically bind to the Fc segment protein, and after VHH gene amplification and phage library construction, positive clones are obtained by screening with streptavidin magnetic beads. Positive clones of the Fc segment protein of IgG1 with the amino acid sequence SEQ ID NO: 1 and the gene sequence SEQ ID NO: 2 that specifically bind to the Fc segment protein of IgG1 are then extracted. After sequencing, the gene sequence and amino acid sequence are determined. The gene sequences of these four nanobodies that specifically recognize the Fc segment of IgG1 antibody can be successfully expressed in 293 cells, and four nanobodies are thus prepared.

[0018] ③ The four nanobodies obtained in this invention have good affinity with the Fc segment of IgG1 antibody, indicating that the nanobodies obtained in this invention can specifically bind to the Fc segment of IgG1 antibody. Attached Figure Description

[0019] Figure 1 Electrophoresis images of the four nanobodies in Example 5 of this invention; Figure 2(a) shows the affinity detection results of nanobody D2 in Example 5 of the present invention; Figure 2(b) shows the affinity detection results of nanobody D9 in Example 5 of the present invention; Figure 2(c) shows the affinity detection results of nanobody H5 in Example 5 of the present invention; Figure 2(d) shows the affinity detection results of nanobody D4 in Example 5 of the present invention. Detailed Implementation

[0020] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0021] Example 1: Preparation of FC protein (Fc fragment of IgG1) The nucleotide sequence of the FC protein (SEQ ID NO: 1) was cloned into the cDNA3.1 expression vector using molecular cloning methods, and the correct molecular clone construction was verified by sequencing.

[0022] The successfully constructed plasmid was used to obtain a plasmid for protein expression through a large extraction. The plasmid was then transiently transfected into 293F cells. After 6 days of transient transfection, the supernatant was collected and purified using protein A to obtain FC protein.

[0023] The gene sequence of the synthesized FC protein is shown in SEQ ID NO: 2.

[0024] The sequence of SEQ ID NO: 1 is as follows: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The sequence of SEQ ID NO: 2 is as follows: GATAAGACACATACCTGTCCCCCATGCCCCGCCCCAGAGCTGCTGGGAGGCCCCTCTGTGTTCCTGTTCCCCCCAAAGCCAAAGGATACCCTGATGATCAGTCGGACCCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAATTCAATTGG TACGTGGATGGGGTCGAGGTGCACAACGCCAAGACCAAACCACGGGAAGAGCAGTACAACTCCACATACAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTATAAATGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCAATCGAAAAA ACTATTTCCAAGGCCAAGGGACAGCCCAGAGAGCCCCAGGTCTACACACTGCCTCCAAGTAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCATCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTAC AAGACCACCCCTCCAGTGCTGGATTCTGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGCTGCAGCAGGGGAATGTGTTCTCTTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACTCAGAAGAGCCTGTCTCTGTCCCCCGGGAAATAA Example 2: Animal Immunization One healthy alpaca was selected for immunization via multiple intradermal and subcutaneous injections on its back. The first immunization used complete Freund's adjuvant, while subsequent immunizations used incomplete Freund's adjuvant. The immunization schedule is shown in Table 1. Table 1 Animal Immunization Procedures

[0025] Example 3: Screening of nanobodies and amplification of the VHH gene The screening process for the nanobodies of the present invention includes the following steps: 1. Isolation of PBMCs: Peripheral blood was collected from the animals after the third, fourth, and fifth immunizations in Example 2, and PBMCs were isolated. PBMC, the specific steps are as follows: ① Take 50 mL of fresh animal peripheral blood sample, dilute it with an equal volume of PBS, and slowly add it to an equal volume of lymphocyte separation medium. Centrifuge at 800 g for 20 min. ② Carefully aspirate the suspended white blood cells from the middle layer into a new centrifuge tube, add an equal volume of PBS, mix well, and centrifuge at 700 g for 20 min; ③ Discard the supernatant solution, resuspend the white blood cell pellet in 0.3 mL of PBS, count the cells, and then count them every 10... 7 Add 1 mL of Trizol solution to the cells, mix well, and store at -80°C for later use.

[0026] 2. RNA was extracted and the VHH (Variable domain of heavy chain antibody) gene was amplified, and phage library construction was performed.

[0027] The specific steps for RNA extraction are as follows: ①The leukocytes preserved in the Trizol solution obtained in step 1 above were centrifuged at 12,000g for 15 min at 4°C, and the supernatant was collected; ② Add chloroform at a ratio of 200 μl chloroform / ml Trizol, vortex to mix, and incubate at room temperature for 15 min (Note: Do not use a vortex mixer to avoid genomic DNA breakage). ③ Centrifuge at 12,000g for 15 minutes at 4℃; ④ Transfer the upper aqueous phase to another centrifuge tube (Note: Do not aspirate the middle interface; if extracting DNA and protein at the same time, retain the lower phenolic phase and store it in a 4°C refrigerator; if extracting only RNA, discard the lower phenolic phase). ⑤ Add isopropanol at a ratio of 0.5 ml isopropanol / ml Trizol, mix well, and let stand at room temperature for 5-10 minutes; ⑥ Centrifuge at 12,000g for 10 min at 4℃, discard the supernatant, and let the RNA settle at the bottom of the tube; ⑦ Add 75% ethanol at a ratio of 1 ml 75% ethanol / ml Trizol, gently shake the centrifuge tube to suspend the precipitate; centrifuge at 8,000g for 5 min at 4℃, and discard as much supernatant as possible; ⑧ Air dry at room temperature or vacuum dry for 5-10 minutes; ⑨ RNA samples can be dissolved in 50 μl of H2O.

[0028] The VHH gene was amplified, and phage libraries were constructed. cDNA was synthesized using a reverse transcription kit. The nanobody gene fragment was amplified using nanobody-specific primers. The fragment was then inserted into a phage plasmid via enzyme digestion and ligation, and electroporated into TG1 competent cells to construct a phage library.

[0029] Example 4: Screening of nanobody positive clones The FC protein was labeled using a Biotin kit, and then the labeled protein was added to phage particles at a final concentration of 10 μg / ml and incubated at 37°C for 1 h. Next, 10 μl of streptavidin magnetic beads were added to the phage library and incubated at 37°C for 1 h to capture phage particles that recognize the FC protein.

[0030] Enzymatic hydrolysis was used to elute the specifically bound phages; the eluent was transferred to a sterile centrifuge tube; 10 µL was serially diluted, the titer was measured, and the recovery rate was calculated; the remaining eluent was mixed and amplified and purified for the next round of affinity panning. Mix the eluent with 5 mL of E. coli TG1 culture in the early logarithmic growth phase, incubate at 37°C with shaking at 220 rpm for 45 min, then transfer to 20 mL of 2×YT-A liquid medium and incubate at 37°C with shaking at 220 rpm for 2 h. Add M13K07 phage at a cell:phage ratio of 1:20, incubate at 37°C for 15-30 min, then incubate with shaking at 220 rpm for 30-45 min. Aliquot the culture into centrifuge tubes and incubate at 4°C with shaking at 3500 rpm for 10 min (2200 g for 15 min). Resuspend the cell pellet in 25 mL of 2×YT-AK (liquid AK agar medium) and incubate overnight at 30°C with shaking at 250 rpm. Incubate the overnight culture at 4°C... Centrifuge at 12000 rpm for 10 min (7000 g, 15 min), transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG-NaCl, mix well, and incubate at 4℃ for at least 1 h; at 4℃, centrifuge at 12000 rpm for 10 min, remove the supernatant, resuspend the precipitate in 2 mL PBS, add 1 / 5 volume of PEG / NaCl, mix well, and incubate at 4℃ for at least 1 h; centrifuge at 12000 rpm for 10 min, remove the supernatant, and resuspend the precipitate in 200 µL PBS, which is the amplification product. Measure the titer for the next round of screening or analysis.

[0031] From the plate used to determine the titer of the eluent from the final round of panning, a single colony was randomly picked with a sterile toothpick and inoculated into 1 mL of 2×YT-GA (liquid casein agar), and incubated at 37°C with shaking at 220 rpm for 12 h. Then, a 1% inoculum was added to 2×YT-GA and incubated at 37°C with shaking at 220 rpm until the early logarithmic growth phase. M13K07 phage was added at a cell:phage ratio of 1:1. The culture was incubated at 37°C for 15 min, followed by shaking at 220 rpm for 30–45 min. The culture was then centrifuged at 4°C and 3500 rpm for 10 min. The precipitate was resuspended in an equal volume of 2×YT-AK and incubated at 30°C with vigorous shaking for 12 h. The culture was then transferred to centrifuge tubes and centrifuged at 4°C and 10000 rpm for 10 min. The supernatant was collected for ELISA identification. The ELISA method for identifying positive clones was based on a positive clone determination criterion: the ratio of the OD value (S) of the test sample to the OD value (N) of the negative control (S / N) ≥ 2.1. The positive clones were sequenced, and multiple sequence alignment analysis was performed using ClustalW software. The amino acid sequence of the nanobody was deduced from the nucleic acid sequence.

[0032] Example 5: Preparation of Nanobodies The amino acid sequence of the nanobody screened in Example 4 was cloned into the cDNA3.4 vector by molecular cloning, and an HIS tag was added to the C segment of the nanobody sequence.

[0033] The plasmids successfully constructed above were transiently transfected into 293 cells. After 6 days, the supernatant was collected and the target antibody protein was purified using a nickel column.

[0034] The proteins were identified by SDS-PAGE, and the results are as follows: Four nanobodies capable of binding to the Fc segment of human IgG1 were successfully expressed and prepared.

[0035] The results of SDS-PAGE reduction gel analysis are shown in the attached figure. Figure 1 As shown in the figure, from left to right are H5, MARKER, D2, D9 and D4 nanobodies.

[0036] Accordingly, the amino acid sequences of the nanobodies prepared by the method of the present invention are as follows: the amino acid sequence of D2 is SEQ ID NO: 3, the amino acid sequence of D9 is SEQ ID NO: 4, the amino acid sequence of H5 is SEQ ID NO: 5, and the amino acid sequence of D4 is SEQ ID NO: 6.

[0037] Specifically as follows: The underlined regions represent the complementarity-determining regions of the variable structural domains of each nanobody. The sequence of SEQ ID NO: 3 is: QVQLVESGGGLVQAGGSLKLSCTASGIAFS VFNME WYRQAPGKQRDLVA LISSGGSTNYADSVKG RFTISRDNAKNLVYLQMNSLKPEDTAVYYCRA RNGWRNI WGQGTQVTVSSHHHHHHHH ; The sequence of SEQ ID NO:4 is: QLQLVESGGGLVQAGGSLLRLSCAASESTFS AIGMG WYRQVPGKQREMVA LINSDGSTNYADFVKG RFTISRDTVLNAVYLQMNSLKPEDTAVYYCKT VARIGLGPYRDY WGQGTQVTVSSHHHHHHHH

[0038] The sequence of SEQ ID NO: 5 is: QVQLVETGGGLVQAGGSLNLSCAASGITFS TYTMG WYRQAPGKQRELVA TISSGGTTFYVASVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCNT LPVGRWYGAEA WGQGTQVTVSSHHHHHHHH ; The sequence of SEQ ID NO: 6 is: QVQLVETGGGLVQAGGSLKLSCTASGIAFS IFNME WYRQAPGKQRDLVA LISSGGSTNYADSVKG RFTISRDNAENTVYLQMNSLKPEDTAVYYCRA RHIWRDI WGQGTQVTVSSHHHHHHHH .

[0039] Example 5: Affinity Detection Antigen affinity was determined using an Octet RED384 sensor with a Protein A sensor. Antigen FC samples were diluted with a pH 7.4 running buffer to concentrations of 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM. Load time, baseline time, binding time, and dissociation time were all set at 180 s and 180 s, respectively. The regeneration buffer was 50 μM pH 1.7 Gly-HCl. The experimental results are shown in Table 2 and Figures 2(a)-2(d). Table 2 Comparison of affinity test results for four types of nanobodies

[0040] The detection experiments proved that the four nanobodies screened and prepared above can effectively bind to human FC protein, and their affinities are as follows: D2 nanobody has an affinity of 2.580E-09, D9 nanobody has an affinity of 4.326E-09, H5 nanobody has an affinity of 1.885E-09, and D4 nanobody has an affinity of 8.756E-09.

[0041] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A nanobody that specifically recognizes the Fc fragment of an IgG1 antibody, characterized in that: The variable structural domains of the nanobody include CDR1, CDR2, and CDR3. The CDR1 sequence is IFNME (SEQ ID NO: 16); The CDR2 sequence is LISSGGSTNYADSVKG (SEQ ID NO: 17); The CDR3 sequence is RHIWRDI (SEQ ID NO: 18).

2. A nanobody that specifically recognizes the Fc fragment of an IgG1 antibody, characterized in that, The amino acid sequence of the variable domain of the nanobody is shown in SEQ ID NO:

6.

3. The nanobody that specifically recognizes the Fc fragment of IgG1 antibody according to claim 1, characterized in that: It is prepared by the following steps: S1: Preparation of FC protein: The gene sequence of the FC protein was cloned into an expression vector using molecular cloning methods, and the correctness of the molecular clone construction was verified by sequencing. S2: Animal Immunization: The FC protein prepared in step S1 was used to immunize alpacas by multiple intradermal and subcutaneous injections on the back. The adjuvant for the first immunization was complete Freund's adjuvant, and the adjuvant for subsequent immunizations was incomplete Freund's adjuvant. S3: Screening of nanobodies and amplification of the VHH gene: RNA was extracted from leukocytes of animals immunized with S2 and the VHH gene was amplified for phage library construction. S4: Screening of nanobody-positive clones: S5: Preparation of nanobodies.

4. The nanobody that specifically recognizes the Fc fragment of IgG1 antibody according to claim 3, characterized in that: In step S1, the amino acid sequence of the FC protein is SEQ ID NO: 1, and the nucleotide sequence of the synthesized FC protein is shown in SEQ ID NO:

2.

5. The nanobody that specifically recognizes the Fc fragment of IgG1 antibody according to claim 3, characterized in that: In step S4, the screening of positive nanobody clones includes the following steps: S41: Obtaining phage particles that recognize FC proteins: The FC protein was labeled using a Biotin kit, and then the labeled protein was added to phage particles at a final concentration of 10 μg / ml. The mixture was incubated at 37°C for 1 h. Then, 10 μl of streptavidin magnetic beads were added to the phage library and incubated at 37°C for 1 h to capture phage particles that recognize the FC protein. S42: Eluting of bacteriophages: Enzymatic hydrolysis was used to elute the specifically bound phages; the eluent was transferred to a sterile centrifuge tube; 10 µL was serially diluted, the titer was measured, and the panning recovery rate was calculated; the remaining eluent was mixed and then amplified and purified for the next round of affinity panning. Mix the eluent with 5 mL of E. coli TG1 culture in the early logarithmic growth phase, incubate at 37°C with shaking at 220 rpm for 45 min, then transfer to 20 mL of 2×YT-A liquid medium and incubate at 37°C with shaking at 220 rpm for 2 h. Add M13K07 phage at a cell:phage ratio of 1:20, incubate at 37°C for 15-30 min, then incubate with shaking at 220 rpm for 30-45 min. Aliquot the culture into centrifuge tubes and incubate at 4°C with centrifuge at 3500 rpm for 10 min. Resuspend the cell pellet in 25 mL of 2×YT-AK liquid medium and incubate at 30°C with centrifuge at 250 rpm. Incubate overnight with shaking; centrifuge the overnight culture at 4°C and 12000 rpm for 10 min, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG-NaCl, mix well, and incubate at 4°C for at least 1 h; centrifuge at 4°C and 12000 rpm for 10 min, remove the supernatant, resuspend the precipitate in 2 mL PBS, add 1 / 5 volume of PEG / NaCl, mix well, and incubate at 4°C for at least 1 h; centrifuge at 12000 rpm for 10 min, remove the supernatant, and resuspend the precipitate in 200 µL PBS, which is the amplification product. Measure the titer for the next round of panning or analysis. From the plate used to determine the titer of the eluent from the final round of panning, a single colony was randomly picked with a sterile toothpick and inoculated into 1 mL of 2×YT-GA. The culture was incubated at 37°C with shaking at 220 rpm for 12 h. A 1% inoculum was then added to 2×YT-GA and incubated at 37°C with shaking at 220 rpm until the early logarithmic growth phase. M13K07 phage was added at a cell:phage ratio of 1:

1. The culture was incubated at 37°C for 15 min, then incubated with shaking at 220 rpm for 30–45 min. The culture was then centrifuged at 4°C with shaking at 3500 rpm for 10 min. The precipitate was resuspended in an equal volume of 2×YT-AK and incubated at 30°C with vigorous shaking for 12 h. The culture was then centrifuged in a centrifuge tube at 4°C with shaking at 10000 rpm for 10 min. The supernatant was collected for ELISA identification. The ELISA method for identifying positive clones uses the ratio of the OD value (S) of the test sample to the OD value (N) of the negative control. S43: Positive clones were sequenced, and ClustalW software was used to perform multiple sequence alignment analysis on the sequencing results. The amino acid sequence of the nanobody was deduced based on the nucleic acid sequence.

6. A nucleotide molecule encoding the nanobody of claim 1.

7. An expression carrier, characterized in that, It comprises the nucleotide molecule of claim 6.

8. The application of the nanobody that specifically recognizes the Fc segment of IgG1 antibody as described in claim 1 in the isolation and purification of IgG1 antibody.

9. The use of the nanobody that specifically recognizes the Fc fragment of IgG1 antibody as described in claim 1 in the isolation and purification of fusion proteins containing the Fc fragment of IgG1 antibody.

10. The application of the nanobody that specifically recognizes the Fc fragment of IgG1 antibody as described in claim 1 in the separation of the Fc fragment of IgG1 antibody.

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