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CN-121991234-A - Nanobody capable of specifically recognizing Fc segment of IgG1 antibody, preparation and application thereof

CN121991234ACN 121991234 ACN121991234 ACN 121991234ACN-121991234-A

Abstract

The invention discloses a nano antibody specifically recognizing an Fc segment of an IgG1 antibody, and preparation and application thereof, and the nano antibody specifically recognizing the Fc segment of the IgG1 antibody has an amino acid sequence shown in SEQ ID NO. 5. The four nano antibodies obtained by the invention can be specifically combined with Fc segment of IgG1 immune protein.

Inventors

  • REN BAOYONG
  • LIU PENG

Assignees

  • 思格(苏州)生物科技有限公司

Dates

Publication Date
20260508
Application Date
20221209

Claims (10)

  1. 1. A nanobody specifically recognizing Fc segment of IgG1 antibody, characterized in that the variable domain of the nanobody comprises CDR1, CDR2 and CDR3, The CDR1 sequence is TYTMG (SEQ ID NO: 13); the CDR2 sequence is TISSGGTTFYVASVKG (SEQ ID NO: 14); The CDR3 sequence is LPVGRWYGAEA (SEQ ID NO: 15).
  2. 2. The nanometer antibody specifically recognizing the Fc segment of the IgG1 antibody is characterized in that the amino acid sequence of the variable structural domain of the nanometer antibody is shown as SEQ ID NO. 5.
  3. 3. The nanobody for specifically recognizing Fc segment of IgG1 antibody according to claim 1, wherein the nanobody is prepared by the following steps: S1, preparation of FC protein: Cloning the gene sequence of the FC protein to an expression vector by a molecular cloning method, and verifying that the molecular cloning construction is correct by sequencing; S2, animal immunization: Immunizing an alpaca by using the FC protein prepared in the step S1, and performing immunization by adopting a back intradermal and subcutaneous multipoint injection mode, wherein the first immunization adjuvant is a complete Freund adjuvant, and the subsequent immunization adjuvants are Freund incomplete adjuvants; s3, screening nanobodies and amplifying VHH genes: Extracting RNA of white blood cells of the animals immunized by S2, amplifying VHH genes, and constructing phage libraries; s4, screening of nanobody positive clones: s5, preparing the nano antibody.
  4. 4. The nanobody for specifically recognizing Fc segment of IgG1 antibody of claim 3, wherein in the 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 as SEQ ID NO. 2.
  5. 5. The nanobody for specifically recognizing Fc segment of IgG1 antibody according to claim 3, wherein the screening of nanobody positive clones in step S4 comprises the steps of: S41, acquisition of phage particles recognizing FC protein: labeling FC protein with a Biotin kit, adding the labeled protein into phage particles, incubating for 1h at a final concentration of 10 mug/ml at 37 ℃, adding 10 mu l of streptavidin magnetic beads into a phage library, incubating for 1h at 37 ℃, and capturing phage particles for identifying the FC protein; S42, elution of phage: transferring the eluate into a sterile centrifuge tube, performing gradient dilution on 10 mu L, measuring titer, calculating elutriation recovery rate, mixing the rest eluents, amplifying and purifying for next round of affinity elutriation; Mixing elutriation eluate with 5mL of E.coli TG1 culture in the early logarithmic growth stage, shaking culture at 37 ℃ for 45min at 220r/min, transferring to 20mL of 2 XYT-A liquid culture medium, shaking culture at 37 ℃ at 220r/min for 2h, adding M13K07 phage at 37 ℃ according to the ratio of cell: phage=1:20, standing for 15-30 min, shaking culture at 220r/min for 30-45 min, subpackaging the culture in a centrifuge tube at 4 ℃ at 3500r/min for 10min, re-suspending cell sediment in 25mL of 2 XYT-AK liquid culture medium at 30 ℃ for 250r/min, shaking culture at 4 ℃ at 12000r/min, transferring supernatant to new PEG-NaCl at 1/5 volume, adding PEG-NaCl at 4 ℃ for 1h after mixing uniformly, adding sediment at 12000r/min, removing supernatant at mLPBS, adding sediment at 1/5 volume for 200 mu.c, and performing amplification at 200 mu.m. for 20 min, or performing amplification, and performing amplification, namely, performing amplification, and performing measurement, namely, performing amplification, at 200 mu.m. for the following the steps; Single colonies were randomly picked from plates measuring the titer of the eluates from the final panning round with sterile toothpicks and inoculated in 1mL2 XYT-GA, shaking culture at 37℃at 220r/min for 12h. The culture was inoculated at 1% inoculum size into 2 XYT-GA, 37℃and 220r/min, and cultured until the logarithmic growth phase. Adding M13K07 phage according to the proportion of cell: phase=1:1, standing for 15min at 37 ℃, shaking culture for 30-45 min at 220r/min, 4 ℃ at 3500r/min, centrifuging for 10min, re-suspending the sediment with an equal volume of 2 XYT-AK at 30 ℃ and carrying out intense shaking culture for 12h, centrifuging the culture in a centrifuge tube at 4 ℃ and 10000rpm for 10min, collecting the supernatant, and detecting the supernatant by an ELISA (enzyme-linked immunosorbent assay) method, wherein positive clone judgment standard is the ratio of the OD value (S) of a test sample to the OD value (N) of a negative control; s43, carrying out sequence determination on the positive clone, carrying out multi-sequence comparison analysis on a sequencing result by adopting ClustalW software, and deducing the amino acid sequence of the nano antibody according to the nucleic acid sequence.
  6. 6. A nucleotide molecule encoding the nanobody of claim 1.
  7. 7. An expression vector comprising the nucleotide molecule of claim 6.
  8. 8. The use of the nanobody of claim 1 specifically recognizing the Fc-segment of IgG1 antibody for the isolation and purification of IgG1 antibody.
  9. 9. Use of a nanobody specifically recognizing the Fc-segment of an IgG1 antibody according to claim 1 for the isolation and purification of a fusion protein comprising the Fc-segment of an IgG1 antibody.
  10. 10. Use of a nanobody specifically recognizing the Fc-segment of an IgG1 antibody according to claim 1 for isolating the Fc-segment of an IgG1 antibody.

Description

Nanobody capable of specifically recognizing Fc segment of IgG1 antibody, preparation and application thereof Cross-reference information The application is based on the application of the Chinese patent application with the application number 2022115798678, 2022-12-09 and the application number 2022115798678, namely the nano antibody for specifically recognizing Fc segment of IgG1 antibody and the preparation and application thereof. Technical Field The invention relates to the technical field of genetic engineering antibodies, in particular to a nano antibody specifically recognizing an Fc segment of an IgG1 antibody, and preparation and application thereof. Background Nanobodies, also known as single domain heavy chain antibodies (VHH antibodies), are the smallest antigen-binding antibody molecules known to date, with a molecular weight of about 15KD. Compared with the traditional antibody, the nano antibody has the advantages of small relative molecular weight, high affinity, high stability, low immunogenicity, strong penetrability and the like because the nano antibody does not have a light chain structure. Immunoglobulin G (immunoglobulin G, igG) consists of two identical light chains and two identical heavy chains. Each chain contains a variable region (V region) that provides an antigen binding site and specificity, and a constant region (C region) that forms the framework of an immunoglobulin that has a biological effector function. IgG is digested with papain and divided into 3 functional regions, namely 2 identical antigen binding fragments (Fab) and one crystallizable fragment (FRAGMENT CRYSTALLINE, fc). The Fc segment is located in the constant region and consists of half of the carboxy-terminal ends of the two heavy chains. At present, affinity chromatography is often performed by utilizing affinity between a specific purified protein and the Fc portion of IgG when antibody purification is performed. The purification cost of IgG is also high due to the high production cost of purified proteins. IgG has four subtypes, igG1, igG2, igG3, and IgG4, of which IgG1 is widely used in clinical applications, but currently nanobody-related products directed against the IgG1 subtype remain few. Disclosure of Invention To overcome the above disadvantages, the present invention aims to provide a nanobody specifically recognizing the Fc-segment of IgG1 antibody, and its preparation and application. The nanobody of the invention specifically recognizes the Fc-segment of an IgG1 antibody, the variable domain of which comprises at least one set of complementarity determining regions: ① CDR11, CDR12 and CDR13, The CDR11 includes a sequence VFNME (SEQ ID NO: 7) The CDR12 includes a sequence LISSGGSTNYADSVKG (SEQ ID NO: 8) The CDR13 includes a sequence RNGWRNI (SEQ ID NO: 9) ② CDR21, CDR22 and CDR23, The CDR21 includes a sequence AIGMG (SEQ ID NO: 10) The CDR22 includes a sequence LINSDGSTNYADFVKG (SEQ ID NO: 11) The CDR23 includes a sequence VARIGLGPYRDY (SEQ ID NO: 12) ③CDR31,CDR32,CDR33, The CDR31 includes a sequence TYTMG (SEQ ID NO: 13) The CDR32 includes a sequence TISSGGTTFYVASVKG (SEQ ID NO: 14) The CDR33 includes a sequence LPVGRWYGAEA (SEQ ID NO: 15) ④CDR41,CDR42,CDR43, The CDR41 comprises a sequence IFNME (SEQ ID NO: 16) The CDR42 includes a sequence LISSGGSTNYADSVKG (SEQ ID NO: 17) The CDR43 includes a sequence RHIWRDI (SEQ ID NO: 18) The nano antibody specifically recognizing the Fc segment of the IgG1 antibody has the amino acid sequence shown in any one of SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 6. Furthermore, the nanobody specifically recognizing the Fc segment of the IgG1 antibody is prepared by the following steps: S1, preparation of FC protein: Cloning the gene sequence of the FC protein to an expression vector by a molecular cloning method, and verifying that the molecular cloning construction is correct by sequencing; S2, animal immunization: Immunizing an alpaca by using the FC protein prepared in the step S1, and performing immunization by adopting a back intradermal and subcutaneous multipoint injection mode, wherein the first immunization adjuvant is a complete Freund adjuvant, and the subsequent immunization adjuvants are Freund incomplete adjuvants; s3, screening nanobodies and amplifying VHH genes: Extracting RNA of white blood cells of the animals immunized by S2, amplifying VHH genes, and constructing phage libraries; s4, screening of nanobody positive clones: s5, preparing the nano antibody. Furthermore, in the step S1, the gene sequence of the FC protein is SEQ ID NO. 1, and the nucleotide sequence of the synthesized FC protein is shown as SEQ ID NO. 2. Further, in the step S4, the screening of nanobody positive clones includes the following steps: S41, acquisition of phage particles recognizing FC protein: labeling FC protein with a Biotin kit, adding the labeled protein into phage particles, incubating for 1h at a final concentration of 10 mug/ml at 37 ℃, adding 10 mu l of strep