WO2022199106A1 - Chi3l1抗体及其应用、核酸、重组表达载体***和宿主细胞 - Google Patents

Chi3l1抗体及其应用、核酸、重组表达载体***和宿主细胞 Download PDF

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WO2022199106A1
WO2022199106A1 PCT/CN2021/134773 CN2021134773W WO2022199106A1 WO 2022199106 A1 WO2022199106 A1 WO 2022199106A1 CN 2021134773 W CN2021134773 W CN 2021134773W WO 2022199106 A1 WO2022199106 A1 WO 2022199106A1
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seq
chi3l1
nucleic acid
antibody
variable region
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French (fr)
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林晓涛
王刚
林标杨
程方明
吴力强
钱纯亘
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深圳市亚辉龙生物科技股份有限公司
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present application relates to the field of biotechnology, in particular to a CHI3L1 antibody and its application, nucleic acid, recombinant expression vector system and host cell.
  • Chitinase-3-like protein 1 (CHI3L1) is a secreted glycoprotein and one of the main indicators of liver fibrosis. CHI3L1 can also be used for the diagnosis, prognosis evaluation and treatment effect of liver cirrhosis As a marker for monitoring and disease course monitoring, the detection of CHI3L1 is of great significance for the prevention and treatment of liver disease.
  • the detection of CHI3L1 mainly uses CHI3L1 antibody, and the CHI3L1 antibody includes rabbit polyclonal antibody and mouse monoclonal antibody.
  • Rabbit polyclonal antibodies to CHI3L1 are generally prepared by immunizing rabbits with an immunogen and collecting serum. However, the specificity of rabbit polyclonal antibodies to CHI3L1 is poor.
  • the murine monoclonal antibody of CHI3L1 is generally prepared by immunizing mice with immunogens and then taking mouse spleen cells and mouse myeloma cells sp2/0 for cell fusion, and then subcloning detection to obtain positive cell lines, and collecting ascites fluid.
  • the monoclonal antibody of CHI3L1 can improve the problem of poor specificity of polyclonal antibody to a certain extent, but the specificity and affinity of the current monoclonal antibody of CHI3L1 need to be improved.
  • a CHI3L1 antibody comprising:
  • a light chain variable region includes a light chain complementarity determining region, wherein the amino acid sequence of the light chain complementarity determining region is shown in SEQ ID No.1 to SEQ ID No.3;
  • a heavy chain variable region includes a heavy chain complementarity determining region, wherein the amino acid sequence of the heavy chain complementarity determining region is shown in SEQ ID No.4 to SEQ ID No.6.
  • the above-mentioned CHI3L1 antibody has high affinity for CHI3L1, good specificity, and high sensitivity when applied to detect CHI3L1.
  • the above-mentioned CHI3L1 antibody can be produced by fermentation engineering technology, which improves the batch-to-batch difference of the current CHI3L1 monoclonal antibody due to individual differences in mice.
  • nucleic acid encoding the CHI3L1 antibody as described above.
  • a recombinant expression vector system comprising the nucleic acid described above.
  • a host cell transformed with the above-mentioned recombinant expression vector system.
  • a preparation method of CHI3L1 antibody comprising:
  • nucleic acid fragment encoding the light chain variable region of the CHI3L1 antibody with the amino acid sequence shown in SEQ ID No. 7 and the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody with the amino acid sequence shown in SEQ ID No. 8 were expressed by an expression vector Fragments are introduced into host cells;
  • the CHI3L1 antibody was harvested.
  • CHI3L1 antibody use of the above-mentioned CHI3L1 antibody, the above-mentioned nucleic acid, the above-mentioned recombinant expression vector system or the above-mentioned host cell in preparing a reagent for detecting CHI3L1 is provided.
  • a CHI3L1 detection kit comprising the above-mentioned CHI3L1 antibody.
  • Figure 1 is the map of plasmid pFUSE2-CLIg-mk
  • Figure 2 is the map of plasmid pFUSE-CHIg-mG1;
  • FIG. 3 is the reaction kinetics curve of the CHI3L1 recombinant antibody prepared in Example 1 in Example 2.
  • FIG. 3 is the reaction kinetics curve of the CHI3L1 recombinant antibody prepared in Example 1 in Example 2.
  • an “antibody” is a class of immunoglobulins that bind specifically to an antigen.
  • antibodies exist as one or more Y-shaped monomers, each Y-shaped monomer is composed of 4 polypeptide chains, including two identical heavy chains and two identical light chains, the light and heavy chains are based on named after their molecular weight.
  • the top of the Y-shaped structure is the variable region, which is the antigen-binding site.
  • Each heavy chain has two regions, the constant region and the variable region.
  • the constant region of all antibodies of the same type is the same, but there are differences between antibodies of different types.
  • Each light chain also has two consecutive domains, a constant region and a variable region.
  • Vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40.
  • “Host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Sf9 cells, etc. Insect cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • S2 Drosophila cells or Sf9 cells etc.
  • Insect cells or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • CHI3L1 antibody includes a light chain variable region and a heavy chain variable region
  • the light chain variable region includes a light chain complementarity determining region (VLCDR) and a light chain framework region (VLFR)
  • the heavy chain can be
  • the variable region includes the heavy chain complementarity determining region (VHCDR) and the light chain framework region (VHFR), wherein: the amino acid sequence of the light chain complementarity determining region is shown in SEQ ID No.1 to SEQ ID No.3; the heavy chain complementarity determining region The amino acid sequence of is shown in SEQ ID No.4 ⁇ SEQ ID No.6. It has been verified that the above CHI3L1 antibody has high affinity for CHI3L1 and good specificity for CHI3L1, and when it is applied to detect CHI3L1, it has high sensitivity.
  • amino acid sequence shown in SEQ ID No.1 is: RSSQTIVHSNGNTYLE; the amino acid sequence shown in SEQ ID No.2 is: KVSNRFS; the amino acid sequence shown in SEQ ID No.3 is: FQGSHVPPT; The amino acid sequence shown in ID No.4 is: SYWIE; the amino acid sequence shown in SEQ ID No.5 is: EILPGSGSTNCNEKFKG; the amino acid sequence shown in SEQ ID No.6 is: SDDYYDY.
  • the amino acid sequence of the light chain framework region is shown in SEQ ID No. 13 to SEQ ID No. 16; the amino acid sequence of the heavy chain framework region is shown in SEQ ID No. 17 to SEQ ID No. 20.
  • the four framework regions are separated by three complementarity determining regions, such that framework regions alternate with complementarity determining regions.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8
  • the light chain variable region and the heavy chain can be The variable regions are connected by disulfide bonds.
  • the amino acid sequence shown in SEQ ID No. 7 is: MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIK.
  • the light chain variable region of the CHI3L1 antibody is not limited to the polypeptide whose amino acid sequence is shown in SEQ ID No.7, but can also be other polypeptides (amino acid sequence) comprising the above-mentioned light chain complementarity determining region.
  • the heavy chain variable region of CHI3L1 antibody is not limited to the polypeptide whose amino acid sequence is shown in SEQ ID No.
  • the polypeptide of the region (the amino acid sequence is shown in SEQ ID No. 4 to SEQ ID No. 6).
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 9. ⁇ SEQ ID No.9 ⁇ :5’-ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3’ ⁇
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 10. ⁇ SEQ ID No.10 ⁇ :5’-ATGGATTGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAGGACTACTGGCTACACATTCAGTAGCTACTGGATAGAGT GGGTGAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAACTGCAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGTAAGATCGGATGATTACTACGACTACTGGGGCCAAGGCACCACTCTCACAGTCCTCA-3’ ⁇
  • the nucleic acid encoding the light chain variable region is not limited to the above, and can also be other light chains that can encode amino acid sequences such as those shown in SEQ ID No.7.
  • the nucleic acid of the variable region; the nucleic acid encoding the variable region of the heavy chain is not limited to the above, and can also be other nucleic acids that can encode the variable region of the heavy chain whose amino acid sequence is shown in SEQ ID No. 8.
  • the above-mentioned CHI3L1 antibody is composed of a light chain variable region whose amino acid sequence is shown in SEQ ID No. 7 and a heavy chain variable region whose amino acid sequence is shown in SEQ ID No. 8, wherein The light chain variable region and the heavy chain variable region are linked by disulfide bonds. At this time, the light chain variable region and the heavy chain variable region form an FV region having all antigen-binding sites through non-covalent bonding, and have antigen-binding ability.
  • the CHI3L1 antibody further includes a light chain constant region and a heavy chain constant region.
  • the light chain constant region is connected with the light chain variable region
  • the heavy chain constant region is connected with the heavy chain variable region
  • the heavy chain constant region is also connected with the light chain constant region to form a Y-shaped monomer structure.
  • amino acid sequence of the light chain constant region is shown in SEQ ID No. 11; and/or the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 12; it can be understood that, In other embodiments, the amino acid sequences of the heavy chain constant region and the light chain constant region are not limited to the above, and may also be other polypeptide fragments that can be used as constant regions.
  • an embodiment of the present application provides a nucleic acid encoding the above-mentioned CHI3L1 antibody.
  • the nucleic acid includes a nucleic acid encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 7 and a nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8 nucleic acid.
  • the nucleic acid encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 7 further comprises a transcription element.
  • a transcription element for example, promoter, terminator, etc.
  • the nucleic acid encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 7 may also not include transcription elements.
  • the nucleic acid encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 7 is inserted into a vector having a corresponding transcription element for expression.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No.7 is shown in SEQ ID No.9. It can be understood that, according to the degeneracy of codons, in other embodiments, the nucleotide sequence of the nucleic acid encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No.7 is not limited to the above, There are also other polynucleotides that can encode the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 7.
  • the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8 further comprises a transcription element.
  • a transcription element for example, promoter, terminator, etc.
  • the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8 may also not include transcription elements.
  • the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8 can be inserted into a vector having a corresponding transcription element for expression.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No.8 is shown in SEQ ID No.10. It can be understood that, according to the degeneracy of codons, in other embodiments, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8 is not limited to the above, It can also be other polynucleotides that can encode the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8.
  • the nucleic acid encoding the above-mentioned CHI3L1 antibody comprises the nucleotide sequence shown in SEQ ID No. 9. In some embodiments, the nucleic acid encoding the above-mentioned CHI3L1 antibody comprises the nucleotide sequence shown in SEQ ID No. 10.
  • nucleic acid fragment encoding the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No.7 and the heavy chain encoding the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No.8 The nucleic acid fragments of the variable region exist in different reagent systems (for example, the above two nucleic acid fragments are located on different expression vectors), or they can exist in the same reagent system at the same time (for example, the above two nucleic acid fragments are located on the same expression vector). ).
  • an embodiment of the present application also provides a recombinant expression vector system comprising the above nucleic acid.
  • the above-mentioned recombinant expression vector system comprises a recombinant expression vector comprising the above-mentioned nucleic acid fragment encoding the amino acid sequence of the CHI3L1 antibody light chain variable region shown in SEQ ID No. 7 and the above-mentioned encoding amino acid sequence such as SEQ ID No. 7.
  • the recombinant expression vector comprising the nucleic acid fragment of the light chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No.7 and the CHI3L1 encoding the amino acid sequence shown in SEQ ID No.8
  • Co-transfection of the recombinant expression vector of the nucleic acid fragment of the heavy chain variable region of the antibody into the host cell enables the host cell to produce the CHI3L1 antibody comprising the heavy chain variable region and the light chain variable region.
  • the above-mentioned recombinant expression vector system comprises a nucleic acid fragment comprising the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence shown in SEQ ID No. 7 and the above-mentioned encoding amino acid sequence as shown in SEQ ID No. 8 A recombinant expression vector for the nucleic acid fragment of the heavy chain variable region of the CHI3L1 antibody shown.
  • the above-mentioned recombinant expression vector system also includes a nucleic acid encoding a signal peptide. The signal peptide generated by the nucleic acid encoding the signal peptide after translation can enable the antibody produced by the host cell to be secreted outside the cell, so as to facilitate the collection of the antibody.
  • the recombinant expression vector in the above-mentioned recombinant expression vector system may be a prokaryotic expression vector or a eukaryotic expression vector. Further, the recombinant expression vector in the above-mentioned recombinant expression vector system is a eukaryotic expression vector. Further, the recombinant expression vector in the above-mentioned recombinant expression vector system is an expression vector for mammalian eukaryotic expression.
  • an embodiment of the present application also provides a host cell transformed with the above-mentioned recombinant expression vector system, capable of producing a CHI3L1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region
  • a host cell transformed with the above-mentioned recombinant expression vector system capable of producing a CHI3L1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region
  • the amino acid sequence of the region is shown in SEQ ID No. 7
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8.
  • the above-mentioned host cell is transformed with a nucleic acid fragment comprising the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence shown in SEQ ID No. 7 and the above-mentioned encoding amino acid sequence as shown in SEQ ID No. 8
  • a recombinant expression vector for the nucleic acid fragment of the heavy chain variable region of the CHI3L1 antibody is transformed with a nucleic acid fragment comprising the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence shown in SEQ ID No. 7 and the above-mentioned encoding amino acid sequence as shown in SEQ ID No. 8
  • a recombinant expression vector for the nucleic acid fragment of the heavy chain variable region of the CHI3L1 antibody is transformed with a nucleic acid fragment comprising the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence shown in SEQ ID No. 7 and the above-mentioned encoding amino acid sequence as shown in
  • the above-mentioned host cell is transformed with the above-mentioned recombinant expression vector comprising the nucleic acid fragment encoding the light chain variable region of the CHI3L1 antibody and the recombinant expression vector comprising the above-mentioned nucleic acid fragment encoding the heavy chain variable region of the CHI3L1 antibody.
  • the nucleic acid fragment encoding the light chain variable region of the CHI3L1 antibody and the nucleic acid fragment encoding the heavy chain variable region of the CHI3L1 antibody are placed on different expression vectors, and co-transfected into host cells, so as to control the lightness of the produced CHI3L1 antibody. Number of chain variable regions and heavy chain variable regions.
  • the host cell is a 293T cell.
  • the host cell is not limited to 293T cells, but can also be other cells.
  • one embodiment also provides a preparation method of the above-mentioned CHI3L1 antibody, the preparation method comprising:
  • the nucleic acid fragment encoding the light chain variable region of the CHI3L1 antibody with the amino acid sequence shown in SEQ ID No. 7 and the nucleic acid encoding the heavy chain variable region of the CHI3L1 antibody with the amino acid sequence shown in SEQ ID No. 8 were expressed by an expression vector The fragment is introduced into a host cell; the host cell is cultured; and the CHI3L1 antibody is harvested.
  • the recombinant expression vector comprising the nucleic acid fragment of the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence as described above as SEQ ID No.
  • the recombinant expression vector of the nucleic acid fragment of the heavy chain variable region of the CHI3L1 antibody is co-transfected into host cells; the transfected host cells are cultured; and the CHI3L1 antibody is harvested.
  • nucleic acid fragment containing the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence as above such as SEQ ID No. 7 and the heavy chain containing the CHI3L1 antibody encoding the amino acid sequence above such as SEQ ID No.
  • the recombinant expression vector of the nucleic acid fragment of the variable region is transfected into the host cell; the transfected host cell is cultured; and the CHI3L1 antibody is harvested.
  • the host cells or the culture medium for culturing the host cells are collected, and the primary product containing the CHI3L1 antibody is harvested; then the primary product is purified to prepare the purified CHI3L1 antibody.
  • the antibodies currently used for CHI3L1 detection are mainly rabbit polyclonal antibodies or mouse monoclonal antibodies.
  • Rabbit polyclonal antibodies are generally prepared by immunizing rabbits with an immunogen and collecting serum.
  • rabbit polyclonal antibodies have poor specificity.
  • Mouse-derived monoclonal antibodies are generally obtained by immunizing mice with immunogens and then taking mouse spleen cells and mouse myeloma cells sp2/0 for cell fusion, and then subcloning detection to obtain positive cell lines, and collecting ascites to prepare monoclonal antibodies.
  • monoclonal antibodies can improve the problem of poor antibody specificity to a certain extent, due to individual differences in mice, monoclonal antibodies are prone to batch-to-batch differences.
  • the preparation method of the above-mentioned CHI3L1 antibody by genetic engineering technology, the nucleic acid encoding the light chain variable region and the heavy chain variable region of the CHI3L1 antibody is introduced into the host cell for expression, so that the specificity of the obtained CHI3L1 antibody is good, and does not Batch-to-batch differences are likely to occur.
  • the above CHI3L1 antibody has good specificity and strong affinity for CHI3L1. Therefore, the above-mentioned CHI3L1 antibody, the above-mentioned nucleic acid, the above-mentioned recombinant expression vector system or the above-mentioned host cell can be used in the preparation of reagents for detecting CHI3L1. Therefore, an embodiment of the present application also provides an application of the above-mentioned CHI3L1 antibody, the above-mentioned nucleic acid, the above-mentioned recombinant expression vector system or the above-mentioned host cell in preparing a reagent for detecting CHI3L1.
  • an embodiment of the present application also provides a CHI3L1 detection kit
  • the CHI3L1 detection kit includes the above-mentioned CHI3L1 antibody for detecting CHI3L1.
  • the above-mentioned CHI3L1 antibody can be used as a capture antibody or as a labeled antibody.
  • the above-mentioned CHI3L1 detection kit further comprises at least one of a solid phase carrier and a buffer.
  • the solid phase carrier is magnetic beads or resin.
  • Buffers include phosphate buffer and glycine solution as sample dilution buffer and elution buffer, respectively.
  • the above-mentioned CHI3L1 detection kit includes the above-mentioned CHI3L1 antibody, which has good specificity to CHI3L1 and high detection sensitivity.
  • mice were intraperitoneally injected with 25 ⁇ g of immune protein, the spleen cells of the immunized mice were taken out, and 2.0 ⁇ 10 7 SP2/0 myeloma cells were mixed with 2.0 ⁇ 10 8
  • the spleen cells of the immunized Balb/c mice were mixed, centrifuged, the supernatant was discarded, and the spleen cells were shaken gently. Then, 20 mL of 1640 medium was added dropwise, centrifuged, the supernatant was discarded, washed again, centrifuged, and the supernatant was discarded to obtain hybridoma cells.
  • Hybridoma cells were selected on 96-well cell culture plates using HAT selection medium, and the total fusion rate was >95% detected under microscope. Coat the recombinant CHI3L1 protein to detect the supernatant of the monoclonal cell well, pick the cells in the well with OD450>0.8 for subcloning, and finally obtain the cell clone with a positive rate of CHI3L1 protein reaction >99%, which is used as the secretory anti-human CHI3L1 monoclonal cell clone. Hybridoma cell lines for cloning antibodies.
  • the antibody in the ascites fluid of each group of mice in step (4) was purified by protein G affinity chromatography.
  • OD value the eluate in the peak area is retained, and the eluate is collected after dialysis. It was identified as a purified monoclonal antibody by SDS-PAGE electrophoresis, and the purity was over 98%.
  • Extract the total RNA of the cell line with the highest titer, and amplify the variable region of the antibody extract the total RNA of 1 ⁇ 10 6 hybridoma cells 1# with Trizol Reagent respectively, and then reverse-transcribe the total RNA into cDNA. Then use PCR amplification technology to amplify the nucleic acid fragment of the heavy chain variable region of hybridoma cell 1# expressing the heavy chain variable region of CHI3L1 antibody using primer 1 and primer 2 in Table 2, and use primer 3 and primer 4 in Table 2 to amplify the hybridoma Cell 1# expresses the nucleic acid fragment of the light chain variable region of the CHI3L1 antibody.
  • PCR amplification adopts hot start, and the reaction conditions are: 94°C 5min; 94°C 45s, 54°C 45s, 72°C 1min, 30 cycles; 72°C 7min.
  • Primer 1 5'-TGGRATGSAGCTGKGTMATSCTCTT-3' (SEQ ID No. 21)
  • Primer 2 5'-TGTYGTGCTAGCTGNRGAGACDGTGA-3'
  • Primer 3 5'-ATGGAGACACACTCCTGCTAT-3'
  • Primer 4 5'-GTCGACTTACGTTTKATTTCCARCTT-3' (SEQ ID No. 24)
  • Hybridoma cell 1# expressed the nucleic acid fragment of the variable region of the CHI3L1 antibody and the sequencing results of the variable region of the CHI3L1 antibody as shown in Table 3.
  • pFUSE2-CLIg-mk and pFUSE-CHIg-mG1 (the vector map shown in Figure 1 and Figure 2, respectively) expression vectors to obtain the ability to express CHI3L1 Recombinant plasmids of the variable region of the light chain of the antibody and the variable region of the heavy chain of the CHI3L1 antibody.
  • step (8) Transfecting the recombinant plasmid obtained in step (8) into mammalian cells (293T): HEK293 cells in logarithmic growth phase and with a viability rate higher than 90% are used as host cells. On the day of transfection, sample and count the cell density and viability. The cell density should be between 3 ⁇ 10 6 cells/mL and 5 ⁇ 10 6 cells/mL, and the viability rate should be higher than 90%. Adjust the cell density to 3 ⁇ 10 6 cells/mL, and the volume of each flask of cell fluid is 20 mL.
  • Antibody purification The culture supernatant of the transfected HEK293 cells was purified by protein G affinity chromatography. First prepare a protein G affinity chromatography column, equilibrate the column with PBS, take the cell supernatant and pass it through the column, then wash the column with PBS, elute with 50 nmol/L glycine hydrochloride solution, collect the eluate, and measure each collected The OD value of the tube, the eluate in the peak area is retained, and the eluate is collected after dialysis. The purified CHI3L1 recombinant antibody was identified by SDS-PAGE electrophoresis, and the purity was over 98%.
  • the kinetic curve obtained by the reaction was fitted by the software, and the affinity detection results are shown in Figure 3 below. According to the kinetic curve, the affinity KD of the obtained CHI3L1 recombinant antibody was 2.656 ⁇ 10 -10 , of which Kd was 2.893 ⁇ 10 -4 , and Ka was 1.089 ⁇ 10 6 .
  • the CHI3L1 rabbit polyclonal antibody (Shenzhen Yahuilong Biotechnology Co., Ltd.) was coated on the ELISA plate at a concentration of 2 ⁇ g/mL. After blocking with BSA, it was reacted with 96 negative blood for 1 h, and HRP-labeled with an appropriate concentration was added.
  • the CHI3L1 recombinant antibody prepared in Example 1 was developed using TMB, and the results are shown in Table 5.
  • the CHI3L1 recombinant antibody prepared in Example 1 was coated on an ELISA plate at a concentration of 1 ⁇ g/mL. After blocking with BSA, it was reacted with the CHI3L1 recombinant antibody diluted to different concentrations for 1 h. After washing the plate, an appropriate concentration of mouse anti-human IgG was added. -HRP was reacted for 30min, and TMB was used for color development. The results are shown in Table 6.

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Abstract

本申请涉及一种CHI3L1抗体及其应用、核酸、重组表达载体***和宿主细胞，该CHI3L1抗体包括轻链可变区，所述轻链可变区包括轻链互补决定区，其中所述轻链互补决定区的氨基酸序列如SEQ ID No.1～SEQ ID No.3所示；和重链可变区，所述重链可变区包括重链互补决定区，其中所述重链互补决定区的氨基酸序列如SEQ ID No.4～SEQ ID No.6所示。

Description

CHI3L1抗体及其应用、核酸、重组表达载体***和宿主细胞
相关申请的交叉引用
本申请要求于2021年3月22日提交中国专利局、申请号为202110303556.8、发明名称为“CHI3L1抗体及其应用、核酸、重组表达载体***和宿主细胞”的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本申请涉及生物技术领域，特别是涉及一种CHI3L1抗体及其应用、核酸、重组表达载体***和宿主细胞。
背景技术
壳多糖酶3样蛋白1(Chitinase-3-like protein 1，CHI3L1)是一种分泌型糖蛋白，是肝纤维化的主要指标之一，CHI3L1也可作为肝硬化的诊断、预后评估、治疗效果监测及病程监测的标记物，检测CHI3L1对肝病防治具有重要意义。
目前，CHI3L1的检测主要利用CHI3L1抗体，其中CHI3L1的抗体包括兔多克隆抗体和鼠源单克隆抗体。CHI3L1的兔多克隆抗体一般是通过免疫原免疫兔并收集血清而制备的兔多克隆抗体，然而，CHI3L1的兔多克隆抗体的特异性较差。CHI3L1的鼠源单克隆抗体一般是通过免疫原免疫小鼠后取小鼠脾脏细胞与小鼠骨髓瘤细胞sp2/0进行细胞融合，然后亚克隆检测获得阳性细胞株，收集腹水制得。CHI3L1的单克隆抗体能在一定程度上改善多克隆抗体特异性较差的问题，然而目前的CHI3L1的单克隆抗体的特异性和亲和力还有待提高。
发明内容
根据一些实施方式，提供一种CHI3L1抗体，所述CHI3L1抗体包括：
轻链可变区，所述轻链可变区包括轻链互补决定区，其中所述轻链互补决定区的氨基酸序列如SEQ ID No.1～SEQ ID No.3所示；和
重链可变区，所述重链可变区包括重链互补决定区，其中所述重链互补决定区的氨基酸序列如SEQ ID No.4～SEQ ID No.6所示。
上述CHI3L1抗体对CHI3L1的亲和力高，特异性好，应用于检测CHI3L1时灵敏度高。 此外，上述CHI3L1抗体可以通过发酵工程技术生产，改善目前CHI3L1的单克隆抗体由于小鼠个体差异而导致的批间差问题。
根据一些实施方式，提供一种核酸，其编码如上述的CHI3L1抗体。
根据一些实施方式，提供一种重组表达载体***，包括上述的核酸。
根据一些实施方式，提供一种宿主细胞，转化有上述的重组表达载体***。
根据一些实施方式，提供一种CHI3L1抗体的制备方法，包括：
通过表达载体将编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段和编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段导入宿主细胞；
培养所述宿主细胞；及
收获所述CHI3L1抗体。
根据一些实施方式，提供上述的CHI3L1抗体、上述的核酸、上述的重组表达载体***或上述的宿主细胞在制备检测CHI3L1的试剂中的应用。
根据一些实施方式，提供一种CHI3L1检测试剂盒，包括上述的CHI3L1抗体。
上述说明仅是本申请技术方案的概述，为了能够更清楚了解本申请的技术手段，并可依照说明书的内容予以实施，以下以本申请的较佳实施方式并配合附图详细说明如后。
附图说明
为了更清楚地说明本申请实施例或传统技术中的技术方案，下面将对实施例或传统技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为质粒pFUSE2-CLIg-mk图谱；
图2为质粒pFUSE-CHIg-mG1图谱；
图3为实施例2中实施例1制得的CHI3L1重组抗体的反应动力学曲线。
具体实施方式
为了便于理解本申请，下面将对本申请进行更全面的描述。但是，本申请可以以许多不同的形式来实现，并不限于本文所描述的实施方式。相反地，提供这些实施方式的目的是使本申 请公开内容更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施方式的目的，不是旨在于限制本申请。
术语定义
“抗体”是一类能与抗原特异性结合的免疫球蛋白。一般地，抗体以一个或者多个Y字形单体存在，每个Y字形单体由4条多肽链组成，包含两条相同的重链和两条相同的轻链，轻链和重链是根据它们的分子量大小来命名的。Y字形结构的顶端是可变区，为抗原结合部位。每个重链有两个区即恒定区和可变区，所有同一型的抗体其恒定区都是相同的，不同型的抗体之间则存在差异。每条轻链也有两个前后相连的结构域，即一个恒定区和一个可变区。
“载体”是指可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。
“宿主细胞”是指可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK 293细胞或人细胞等的动物细胞。
一实施方式提供一种CHI3L1抗体，CHI3L1抗体包括轻链可变区和重链可变区，轻链可变区包括轻链互补决定区(VLCDR)和轻链骨架区(VLFR)，重链可变区包括重链互补决定区(VHCDR)和轻链骨架区(VHFR)，其中：轻链互补决定区的氨基酸序列如SEQ ID No.1～SEQ ID No.3所示；重链互补决定区的氨基酸序列如SEQ ID No.4～SEQ ID No.6所示。经验证，上述CHI3L1抗体对CHI3L1的亲和力高，对CHI3L1的特异性好，将其应用于检测CHI3L1时，灵敏度高。
具体地，如SEQ ID No.1所示的氨基酸序列为：RSSQTIVHSNGNTYLE；如SEQ ID No.2所示的氨基酸序列为：KVSNRFS；如SEQ ID No.3所示的氨基酸序列为：FQGSHVPPT；如 SEQ ID No.4所示的氨基酸序列为：SYWIE；如SEQ ID No.5所示的氨基酸序列为：EILPGSGSTNCNEKFKG；如SEQ ID No.6所示的氨基酸序列为：SDDYYDY。
可选地，轻链骨架区的氨基酸序列如SEQ ID No.13～SEQ ID No.16所示；重链骨架区的氨基酸序列如SEQ ID No.17～SEQ ID No.20所示。当然，不论是在轻链可变区还是重链可变区，四个骨架区均被三个互补决定区间隔，以使得骨架区与互补决定区交替出现。
在其中一个实施方式中，轻链可变区的氨基酸序列如SEQ ID No.7所示，重链可变区的氨基酸序列如SEQ ID No.8所示，轻链可变区和重链可变区通过二硫键连接。具体地，如SEQ ID No.7所示的氨基酸序列为：MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIK。如SEQ ID No.8所示的氨基酸序列为：MDWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCRTTGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGSTNCNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCVRSDDYYDYWGQGTTLTVSS。
可以理解的是，在其他实施方式中，CHI3L1抗体的轻链可变区不限于氨基酸序列如SEQ ID No.7所示的多肽，还可以是其他包含上述轻链互补决定区的多肽(氨基酸序列如SEQ ID No.1～SEQ ID No.3所示)；CHI3L1抗体的重链可变区不限于氨基酸序列如SEQ ID No.8所示的多肽，还可以是其他包含有上述重链互补决定区的多肽(氨基酸序列如SEQ ID No.4～SEQ ID No.6所示)。
可选地，编码轻链可变区的核酸的核苷酸序列如SEQ ID No.9所示。如SEQ ID No.9所示的核苷酸序列为：5’-ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3’。
可选地，编码重链可变区的核酸的核苷酸序列如SEQ ID No.10所示。如SEQ ID No.10所示的核苷酸序列为：5’-ATGGATTGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAGGACTACTGGCTACACATTCAGTAGCTACTGGATAGAGT GGGTGAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAACTGCAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGTAAGATCGGATGATTACTACGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3’。
可以理解的是，根据密码子的简并性，在其他实施方式中，编码轻链可变区的核酸不限于上述，还可以是其他能够编码氨基酸序列如SEQ ID No.7所示的轻链可变区的核酸；编码重链可变区的核酸不限于上述，还可以是其他能够编码氨基酸序列如SEQ ID No.8所示的重链可变区的核酸。
在其中一个可选地具体示例中，上述CHI3L1抗体由氨基酸序列如SEQ ID No.7所示的轻链可变区和氨基酸序列如SEQ ID No.8所示的重链可变区组成，其中轻链可变区和重链可变区通过二硫键连接。此时，轻链可变区和重链可变区通过非共价键作用形成具有全部抗原结合位点的FV区，具有抗原结合能力。
在一些实施方式中，CHI3L1抗体还包括轻链恒定区和重链恒定区。其中，轻链恒定区与轻链可变区连接，重链恒定区与重链可变区连接，且重链恒定区还与轻链恒定区连接，以形成Y字型单体结构。
在一个可选地具体示例中，轻链恒定区的氨基酸序列如SEQ ID No.11所示；及/或，重链恒定区的氨基酸序列如SEQ ID No.12所示；可以理解的是，在其他实施方式中，重链恒定区和轻链恒定区的氨基酸序列不限于上述，还可以是其他可以作为恒定区的多肽片段。
此外，本申请一实施方式提供了一种编码上述CHI3L1抗体的核酸。具体地，该核酸包括编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸和编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸。
在一些实施方式中，上述编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸还包括转录元件。例如启动子、终止子等。可以理解的是，在一些实施方式中，上述编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸也可以不包括转录元件。此时，在使用时，将编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸***到具有相应转录元件的载体中进行表达即可。
可选地，编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸的核苷酸序列如SEQ ID No.9所示。可以理解的是，根据密码子的简并性，在其他实施方式中，编码 氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸的核苷酸序列不限于上述，还可以其他可以编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的多核苷酸。
在一些实施方式中，上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸还包括转录元件。例如启动子、终止子等。可以理解的是，在一些实施方式中，上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸也可以不包括转录元件。此时，在使用时，将编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸***到具有相应转录元件的载体中进行表达即可。
可选地，上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸的核苷酸序列如SEQ ID No.10所示。可以理解的是，根据密码子的简并性，在其他实施方式中，编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸的核苷酸序列不限于上述，还可以是其他可以编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的多核苷酸。
在一些实施方式中，编码上述CHI3L1抗体的核酸包括SEQ ID No.9所示的核苷酸序列。在一些实施方式中，编码上述CHI3L1抗体的核酸包括SEQ ID No.10所示的核苷酸序列。
需要说明的是，上述核酸中，编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段和编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段存在于不同试剂体系中(例如上述两个核酸片段分别位于不同的表达载体上)，也可以同时存在于同一个试剂体系中(例如上述两个核酸片段位于同一表达载体上)。
另外，本申请一实施方式还提供了一种重组表达载体***，该重组表达载体***包括上述核酸。
在其中一个实施方式中，上述重组表达载体***包括包含上述编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段的重组表达载体和包含上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段的重组表达载体。在使用时，将包含上述编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段的重组表达载体和包含上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段的重组表达载体共转染入宿主细胞，使得宿主细胞能够产生包括重链可变区和轻链可变区的CHI3L1抗体。
在其中一个实施方式中，上述重组表达载体***包括包含上述编码氨基酸序列如SEQ ID  No.7所示的CHI3L1抗体的轻链可变区的核酸片段和上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段的重组表达载体。进一步地，上述重组表达载体***还包含编码信号肽的核酸。通过编码信号肽的核酸经翻译后产生的信号肽，可以使得宿主细胞产生的抗体能够分泌到细胞外，便于收集抗体。
可以理解，上述重组表达载体***中的重组表达载体可以是原核表达载体，也可以是真核表达载体。进一步地，上述重组表达载体***中的重组表达载体为真核表达载体。更进一步地，上述重组表达载体***中的重组表达载体为用于哺乳动物真核表达的表达载体。
此外，本申请一实施方式还提供了一种宿主细胞，该宿主细胞转化有上述重组表达载体***，能够产生包括重链可变区和轻链可变区的CHI3L1抗体，其中，轻链可变区的氨基酸序列如SEQ ID No.7所示，重链可变区的氨基酸序列如SEQ ID No.8所示。
在其中一个实施方式中，上述宿主细胞转化有包含上述编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段和上述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段的重组表达载体。
在另一个实施方式中，上述宿主细胞转化有包含上述编码CHI3L1抗体的轻链可变区的核酸片段的重组表达载体和包含上述编码CHI3L1抗体的重链可变区的核酸片段的重组表达载体。将编码CHI3L1抗体的轻链可变区的核酸片段和编码CHI3L1抗体的重链可变区的核酸片段置于不同的表达载体上，共转染到宿主细胞中，便于控制产生的CHI3L1抗体的轻链可变区和重链可变区的数量。
在其中一个实施方式中，宿主细胞为293T细胞。当然，在其他实施方式中，宿主细胞不限于293T细胞，还可以是其他细胞。
此外，一实施方式还提供了一种上述CHI3L1抗体的制备方法，该制备方法包括：
通过表达载体将编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段和编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段导入宿主细胞；培养此宿主细胞；及收获CHI3L1抗体。
在其中一个实施方式中，将包含有上述编码氨基酸序列如SEQ ID No.7的CHI3L1抗体的轻链可变区的核酸片段的重组表达载体和包含有上述编码氨基酸序列如SEQ ID No.8的CHI3L1抗体的重链可变区的核酸片段的重组表达载体共转染入宿主细胞；培养转染后的宿主细胞；及收获CHI3L1抗体。
在另一个实施方式中，将含有上述编码氨基酸序列如SEQ ID No.7的CHI3L1抗体的轻链 可变区的核酸片段和含有上述编码氨基酸序列如SEQ ID No.8的CHI3L1抗体的重链可变区的核酸片段的重组表达载体转染入宿主细胞；培养转染后的宿主细胞；收获CHI3L1抗体。
当然，在将转染后的宿主细胞培养一段时间后，收集宿主细胞或培养宿主细胞的培养液，收获含有CHI3L1抗体的初级产物；然后将初级产物纯化，制备纯化后的CHI3L1抗体。
目前用于CHI3L1检测的抗体主要是兔多克隆抗体或鼠源单克隆抗体。兔多克隆抗体一般是通过免疫原免疫兔并收集血清而制备的兔多克隆抗体，然而，兔多克隆抗体的特异性较差。鼠源单克隆抗体一般是通过免疫原免疫小鼠后取小鼠脾脏细胞与小鼠骨髓瘤细胞sp2/0进行细胞融合，然后亚克隆检测获得阳性细胞株，收集腹水制得单克隆抗体。虽然单克隆抗体能在一定程度上改善抗体特异性较差的问题，然而由于小鼠个体差异，单克隆抗体容易存在批间差的问题。上述CHI3L1抗体的制备方法，通过基因工程技术，将编码CHI3L1抗体的轻链可变区和重链可变区的核酸导入宿主细胞中进行表达，使得制得的CHI3L1抗体的特异性好，且不容易出现批间差。
上述CHI3L1抗体对CHI3L1的特异性好、亲和力强。因此，上述CHI3L1抗体、上述核酸、上述重组表达载体***或上述宿主细胞可应用于检测CHI3L1的试剂的制备中。因此，本申请一实施方式还提供了一种上述CHI3L1抗体、上述核酸、上述重组表达载体***或上述宿主细胞在制备检测CHI3L1的试剂中的应用。
另外，本申请一实施方式还提供了一种CHI3L1检测试剂盒，该CHI3L1检测试剂盒包括上述CHI3L1抗体，用于检测CHI3L1。
具体地，上述CHI3L1抗体可以作为捕获抗体，也可以作为标记抗体。
在其中一个实施方式中，上述CHI3L1检测试剂盒还包括固相载体和缓冲液中的至少一种。可选地，固相载体为磁珠或树脂。缓冲液包括磷酸盐缓冲液和甘氨酸溶液，分别作为样品稀释缓冲液和洗脱缓冲液。
上述CHI3L1检测试剂盒包括上述CHI3L1抗体，对CHI3L1特异性好，检测的灵敏度高。
以下结合具体实施例进行详细说明。以下实施例如未特殊说明，则不包括除不可避免的杂质外的其他组分。实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，按照常规条件，例如文献、书本中所述的条件或者生产厂家推荐的方法实现。以下所用的重组CHI3L1蛋白来自深圳市亚辉龙生物科技股份有限公司。
实施例1
(1)免疫：重组CHI3L1蛋白与弗氏佐剂等量混合充分乳化后各注射3只Balb/c小鼠， 每只小鼠25μg，共免疫3次，间隔时间15天。
(2)细胞融合、选择性培养、筛选：融合前3天，小鼠腹腔注射免疫蛋白25μg，取出免疫小鼠的脾脏细胞，将2.0×10 7个SP2/0骨髓瘤细胞与2.0×10 8个经免疫的Balb/c小鼠的脾细胞混匀，离心，弃上清，轻微振荡混匀，于37℃水浴中，在90秒内滴加1mL体积浓度为50％的PEG-1500水溶液，然后滴加20mL 1640培养基，离心，弃上清，再洗一次，离心，弃上清，得到杂交瘤细胞。使用HAT选择培养基在96孔细胞培养板上选择杂交瘤细胞，镜下检测总融合率＞95％。包被重组CHI3L1蛋白对单克隆细胞孔的上清进行检测，挑取OD450＞0.8的孔内细胞进行亚克隆，最终获得对CHI3L1蛋白反应阳性率＞99％的细胞克隆，作为分泌抗人CHI3L1单克隆抗体的杂交瘤细胞株。
(3)细胞克隆：对获得的阳性细胞株进行克隆，使用有限稀释法，克隆3次，最后得到7株产生高效价的抗人CHI3L1的单克隆抗体的杂交瘤细胞株(表1中编号为1#～7#)，扩大培养，冻存。
(4)腹水制备：6-8周龄雌性Balb/c小鼠用石蜡处理10天后，取杂交瘤细胞以2×10 6个细胞/只小鼠进行腹腔注射。10天后从小鼠腹腔中获取富含抗体的腹水，测定腹水效价＞10 5。
(5)纯化：采用protein G亲和层析法纯化步骤(4)的各组小鼠的腹水中的抗体。首先制备protein G亲和层析柱，用PBS平衡柱子后，取腹水过柱，然后用PBS清洗柱子，以50nmol/L的甘氨酸盐酸盐溶液洗脱，收集洗脱液，测定各收集管的OD值，保留峰值区的洗脱液，洗脱液经透析后收集。经SDS-PAGE电泳鉴定为纯化的单克隆抗体，纯度为98％以上。
(6)抗体效价检测：
将步骤(5)得到的各杂交瘤细胞株对应的抗体稀释成浓度为3μg/mL、1μg/mL、0.33μg/mL、0.11μg/mL、0.036μg/mL、0.012μg/mL，使用间接法ELISA检测抗体效价，ELISA检测结果见表1。
表1
抗体浓度 1# 2# 3# 4# 5# 6# 7#
3μg/mL 3.221 3.221 2.521 2.778 2.512 2.461 2.377
1μg/mL 3.125 2.335 2.265 2.115 1.635 1.528 1.468
0.33μg/mL 1.452 1.126 0.852 0.687 0.722 0.553 0.72
0.11μg/mL 0.558 0.335 0.358 0.286 0.348 0.251 0.341
0.036μg/mL 0.126 0.136 0.052 0.076 0.046 0.015 0.023
0.012μg/mL 0.045 0.042 0.023 0.022 0.032 0.015 0.016
1×PBS 0.012 0.011 0.009 0.011 0.012 0.012 0.011
(7)提取效价最高的细胞株的总RNA，并扩增抗体的可变区：用Trizol Reagent试剂分别提取1×10 6个杂交瘤细胞1#的总RNA，然后将总RNA逆转录成cDNA。接着采用PCR扩增技术利用表2中的引物1和引物2扩增杂交瘤细胞1#表达CHI3L1抗体的重链可变区的核酸片段，利用表2中的引物3和引物4扩增杂交瘤细胞1#表达CHI3L1抗体的轻链可变区的核酸片段。其中，PCR扩增采用热启动，反应条件：94℃ 5min；94℃ 45s，54℃ 45s，72℃ 1min，30个循环；72℃ 7min。
表2
引物1 5’-TGGRATGSAGCTGKGTMATSCTCTT-3’(SEQ ID No.21)
引物2 5’-TGTYGTGCTAGCTGNRGAGACDGTGA-3’(SEQ ID No.22)
引物3 5’-ATGGAGACACACTCCTGCTAT-3’(SEQ ID No.23)
引物4 5’-GTCGACTTACGTTTKATTTCCARCTT-3’(SEQ ID No.24)
(8)PCR产物委托上海生工进行测序，杂交瘤细胞1#表达CHI3L1抗体的可变区的核酸片段及CHI3L1抗体的可变区的测序结果如表3所示。
表3
Figure PCTCN2021134773-appb-000001
Figure PCTCN2021134773-appb-000002
(8)委托上海生工将杂交瘤细胞1#表达CHI3L1抗体的轻链可变区的核酸片段(核苷酸序列如SEQ ID No.9所示)和表达CHI3L1抗体的重链可变区的核酸片段(核苷酸序列如SEQ ID No.10所示)分别克隆到pFUSE2-CLIg-mk及pFUSE-CHIg-mG1(载体图谱分别如图1和图2所示)表达载体中获得能够表达CHI3L1抗体的轻链可变区和CHI3L1抗体的重链可变区的重组质粒。
(9)将步骤(8)得到的重组质粒转染到哺乳动物细胞(293T)中：采用处在对数生长期且活率高于90％的HEK293细胞作为宿主细胞。转染当天，取样计数细胞密度和活率，细胞密度应该在3×10 6cells/mL～5×10 6cells/mL，活率高于90％。调整细胞密度至3×10 6cells/mL，每瓶细胞液体积为20mL。用150mM的NaCl稀释20μg步骤(8)得到的重组质粒至总体积为0.5mL，温和混匀；用150mM的NaCl稀释100μL Sinofection转染试剂至总体积为0.5mL，温和混匀；将稀释好的重组质粒和转染试剂同时单独静置约5分钟后温和混匀，制得总体积1mL的转染液，之后室温静置10分钟，然后将转染液逐滴加入到细胞密度为3×10 6cells/mL的细胞培养液中，滴加的同时轻轻摇动培养瓶，摇匀后放回摇床继续培养。
(10)转染48小时后收集细胞上清，检测是否分泌CHI3L1抗体：采用间接法，检测细胞是否分泌重组抗体，包被重组CHI3L1蛋白(1μg/mL)，添加100μL细胞上清，二抗使用鼠抗 人IgG-HRP。如果ELISA检测OD值为阳性(OD值大于1.0且为空白对照OD值的3倍以上为阳性)，说明此转染细胞株分泌CHI3L1抗体，结果如表4所示。
表4
组别 检测孔1 检测孔2
细胞上清 2.5 2.6
空白对照 0.01 0.01
由表4可以看出，转染成功，转染后的HEK293细胞分泌CHI3L1抗体。
(11)抗体纯化：采用protein G亲和层析法纯化转染后的HEK293细胞的培养上清。首先制备protein G亲和层析柱，用PBS平衡柱子后，取细胞上清过柱，然后用PBS清洗柱子，以50nmol/L的甘氨酸盐酸盐溶液洗脱，收集洗脱液，测定各收集管的OD值，保留峰值区的洗脱液，洗脱液经透析后收集。经SDS-PAGE电泳鉴定为纯化的CHI3L1重组抗体，纯度为98％以上。
实施例2
亲和力检测
使用Biacore T200检测实施例1步骤(11)制备的CHI3L1重组抗体的亲和力：在CM5芯片上固定重组CHI3L1蛋白，使用不同浓度的待测CHI3L1重组抗体，按照合适的时间与芯片上CHI3L1蛋白进行反应，反应所得的动力学曲线通过软件进行拟合，亲和力检测结果如下图3所示。根据动力学曲线，获得CHI3L1重组抗体的亲和力KD为2.656×10 -10，其中Kd为2.893×10 -4，Ka为1.089×10 6。
实施例3
特异性检测
将CHI3L1兔多抗(深圳市亚辉龙生物科技股份有限公司)按照2μg/mL的浓度包被至酶标板上，BSA封闭后，与96例阴性血反应1h，加入合适浓度的HRP标记的实施例1制得的CHI3L1重组抗体，使用TMB进行显色，结果如表5所示。
表5 CHI3L1重组抗体特异性检测
Figure PCTCN2021134773-appb-000003
Figure PCTCN2021134773-appb-000004
由表5可以看出显示，96例阴性血的OD值都在0.2以下，特异性良好。
实施例4
灵敏度检测
将实施例1制得的CHI3L1重组抗体按照1μg/mL浓度包被至酶标板上，BSA封闭后，与稀释成不同浓度的CHI3L1重组抗体反应1h，洗板后加入合适浓的鼠抗人IgG-HRP反应30min，使用TMB进行显色，结果如表6所示。
表6重组抗体灵敏度检测结果
浓度(μg/mL) OD值
1 3.581
0.33 3.317
0.11 2.596
0.036 1.357
0.012 0.652
0.004 0.124
1×PBS 0.015
由表6可知，12ng/mL水平的抗原都能获得理想的信号，与阴性对照信噪比大于10，说明 实施例1制得的重组抗体的灵敏度高。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (17)

  1. 一种CHI3L1抗体，包括：
    轻链可变区，所述轻链可变区包括轻链互补决定区，其中所述轻链互补决定区的氨基酸序列如SEQ ID No.1～SEQ ID No.3所示；和
    重链可变区，所述重链可变区包括重链互补决定区，其中所述重链互补决定区的氨基酸序列如SEQ ID No.4～SEQ ID No.6所示。
  2. 根据权利要求1所述的CHI3L1抗体，其中，所述轻链可变区的氨基酸序列如SEQ ID No.7所示，所述重链可变区的氨基酸序列如SEQ ID No.8所示。
  3. 根据权利要求1或2所述的CHI3L1抗体，其中，所述CHI3L1抗体还包括轻链恒定区和重链恒定区，所述轻链恒定区的氨基酸序列如SEQ ID No.11所示，所述重链恒定区的氨基酸序列如SEQ ID No.12所示。
  4. 一种核酸，其编码如权利要求2～3任一项所述的CHI3L1抗体。
  5. 根据权利要求4所述的核酸，其中，所述核酸包括编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸和编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸。
  6. 根据权利要求5所述的核酸，其中，所述编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸还包括转录元件。
  7. 根据权利要求5所述的核酸，其中，所述编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸还包括转录元件。
  8. 根据权利要求4所述的核酸，其中，所述核酸包括SEQ ID No.9所示的核苷酸序列。
  9. 根据权利要求4或5所述的核酸，其中，所述核酸包括SEQ ID No.10所示的核苷酸序列。
  10. 一种重组表达载体***，包括权利要求4～9任一项所述的核酸。
  11. 根据权利要求10所述的重组表达载体***，其中，所述重组表达载体***还包含编码信号肽的核酸。
  12. 一种宿主细胞，转化有权利要求10或11所述的重组表达载体***。
  13. 一种制备CHI3L1抗体的方法，包括：
    通过表达载体将编码氨基酸序列如SEQ ID No.7所示的CHI3L1抗体的轻链可变区的核酸片段和编码氨基酸序列如SEQ ID No.8所示的CHI3L1抗体的重链可变区的核酸片段导入宿主 细胞；
    培养所述宿主细胞；及
    收获所述CHI3L1抗体。
  14. 根据权利要求13所述的方法，其中，所述方法包括：
    将包含有上述编码氨基酸序列如SEQ ID No.7的CHI3L1抗体的轻链可变区的核酸片段的重组表达载体和包含有上述编码氨基酸序列如SEQ ID No.8的CHI3L1抗体的重链可变区的核酸片段的重组表达载体共转染入宿主细胞；
    培养转染后的宿主细胞；及
    收获所述CHI3L1抗体。
  15. 权利要求1～3任一项所述的CHI3L1抗体、权利要求4～9任一项所述的核酸、权利要求10或11所述的重组表达载体***或权利要求12所述的宿主细胞在制备检测CHI3L1的试剂中的应用。
  16. 一种CHI3L1检测试剂盒，包括权利要求1～3任一项所述的CHI3L1抗体。
  17. 根据权利要求16所述的CHI3L1检测试剂盒，其中，所述试剂盒还包括固相载体和缓冲液中的至少一种。
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