WO2023088444A1 - 一种抗hiv-1 p24的抗体及其制备方法和用途 - Google Patents

一种抗hiv-1 p24的抗体及其制备方法和用途 Download PDF

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WO2023088444A1
WO2023088444A1 PCT/CN2022/132981 CN2022132981W WO2023088444A1 WO 2023088444 A1 WO2023088444 A1 WO 2023088444A1 CN 2022132981 W CN2022132981 W CN 2022132981W WO 2023088444 A1 WO2023088444 A1 WO 2023088444A1
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amino acid
acid sequence
seq
antibody
hiv
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French (fr)
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孟媛
姜瑢瑢
张嘉欣
熊俊文
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东莞市朋志生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the technical field of antibodies. More specifically, it relates to an anti-HIV-1 P24 antibody and its preparation method and use.
  • HIV Human Immunodeficiency Virus
  • AIDS Acquired Immunodeficiency Syndrome
  • Human immunodeficiency virus was first identified in the United States in 1981. It is a lentivirus that infects cells of the human immune system and is a type of retrovirus. At present, AIDS has not only become a public health problem that seriously threatens the health of our people, but also affects economic development and social stability.
  • HIV is the pathogen of AIDS, mainly through sexual contact, blood and mother-to-child transmission.
  • the number of HIV-infected patients has been on the rise.
  • the situation of the AIDS epidemic in my country is still severe, and sexual transmission is becoming the main route of transmission.
  • the determination of serum HIV antibody is a routine experimental method for diagnosing HIV infection, but the determination of HIV antibody has limitations: more than 70% of HIV-infected people can detect antibodies after 6 months of infection, and in the homosexual group, this number exceeds 80% %, the method of detecting antibodies increases the risk of HIV "window period" transmission; in addition, the newborn needs to produce antibodies one year after birth, and HIV antibodies from the mother will cause false positives; due to the persistence of HIV antibodies in the course of the disease, only It disappears in the late stage of AIDS and cannot be used as a stable indicator for treatment monitoring.
  • P24 is the main structural protein of HIV virus particles, and is the product of the structural gene GAG, which plays an important role in the packaging and maturation of the virus.
  • the amino acid sequence of P24 protein is highly conserved among HIV strains, and the lack of P24 will lead to the failure of normal virus assembly.
  • P24 protein is highly specific and has no cross-reaction with most other retroviruses. HIV infects the human body, and the virus marker that first appears in the blood of the infected person is the virus P24 protein. There is a long window period between the virus infection and the detection of HIV antibodies. Therefore, the detection of HIV-P24 antigen has been used in the early diagnosis of HIV infection, Patients' prognosis, screening and evaluation of anti-HIV drugs, and detection of mother-to-child transmission have played an important role.
  • HIV-1 P24 antigen adopts serological diagnostic methods, mainly including enzyme-linked immunoassay and chemiluminescence method based on double antibody sandwich, immune complex cleavage detection method, ultra-sensitive EIA method, enzyme-linked immunofluorescence method, etc.
  • the double-antibody sandwich method is commonly used to detect the HIV P24 antigen, which has good specificity. This method requires specific antibodies against the HIV P24 antigen, and the demand for monoclonal antibodies is mostly.
  • monoclonal antibody products targeting the HIV P24 antigen on the market there are defects in sensitivity, affinity and specificity.
  • the present invention provides an anti-HIV-1 P24 monoclonal antibody, at least one of which is improved in activity, affinity, sensitivity or specificity.
  • the present disclosure provides an anti-HIV-1 P24 antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include or are the same as SEQ ID NO: 15, SEQ ID NO The amino acid sequence consistent with HCDR1-HCDR3 of any one of heavy chain variable regions shown in: 26; said LCDR1-LCDR3 includes or is identical to the light chain shown in any one of SEQ ID NO: 16, SEQ ID NO: 27-30 The consistent amino acid sequence of LCDR1-LCDR3 in the variable region.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.
  • the present disclosure provides an anti-HIV-1 P24 antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising the following complementarity determining regions:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of it;
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of it;
  • HCDR3 which comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 21, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in any one of SEQ ID NO: 4, 22, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in any one of SEQ ID NO:5, 23, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • the antibody or its functional fragments further comprise the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region; wherein, HFR1 includes the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence with at least 80% homology; HFR2 includes the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence with at least 80% homology therewith Sequence; HFR3 comprises the amino acid sequence shown in SEQ ID NO:9 or has at least 80% homology therewith; HFR4 comprises the amino acid sequence shown in SEQ ID NO:10 or has at least 80% homology therewith The amino acid sequence; LFR1 comprises the amino acid sequence shown in any one of SEQ ID NO:11,24 or has the aminoacid sequence of at least 80% homology therewith; LFR2 comprises the aminoacid sequence shown in SEQ ID NO:12 or has the amino acid sequence thereof An amino acid sequence of at least 80% homology
  • the antibody or its functional fragments also have the following framework regions:
  • HFR1 The amino acid sequence of HFR1 is as shown in SEQ ID NO: 7 or has at least 80% homology therewith;
  • amino acid sequence of HFR2 is as shown in SEQ ID NO: 8 or has at least 80% homology therewith;
  • amino acid sequence of HFR3 is as shown in SEQ ID NO:9 or has at least 80% homology therewith;
  • the HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or has at least 80% homology therewith;
  • amino acid sequence of LFR1 is as shown in any of SEQ ID NO: 11, 24, or has at least 80% homology therewith;
  • amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology therewith;
  • amino acid sequence of LFR3 is as shown in any of SEQ ID NO: 13, 25, or has at least 80% homology therewith;
  • the LFR4 amino acid sequence is as set forth in SEQ ID NO: 14 or has at least 80% homology thereto.
  • the present disclosure provides an anti-HIV-1 P24 antibody or a functional fragment thereof, the antibody or its functionality comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the above-mentioned HCDR1-3 and the above-mentioned HFR1-4, and the light chain variable region includes the above-mentioned LCDR1-3 and the above-mentioned LFR1-4.
  • amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 26; the amino acid sequence of the light chain variable region is as shown in any of SEQ ID NO: 16, 27, 28, 29, 30 one shown.
  • the antibody or functional fragment thereof further comprises a constant region.
  • the constant region comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the ⁇ -type or ⁇ -type light chain constant region .
  • the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , turkey, fighting cock or human; optionally, the species source of the constant region is human.
  • the heavy chain constant region is SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;
  • the light chain constant region is SEQ ID NO: 18 or an amino acid sequence with SEQ ID NO: 17 NO:18 is an amino acid sequence with more than 80% homology.
  • the functional fragment is selected from any one of F(ab') 2 , Fab', Fab, Fv and scFv of the antibody.
  • the present disclosure provides an anti-HIV-1 P24 antibody or a functional fragment thereof, including a heavy chain and/or a light chain, and the heavy chain includes the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the The light chain includes the above-mentioned light chain variable region and the above-mentioned light chain constant region.
  • amino acid sequence of the heavy chain is shown in any of SEQ ID NO: 19, 31; the amino acid sequence of the light chain is shown in any of SEQ ID NO: 20, 32, 33, 34, 35.
  • the present disclosure also provides an antibody conjugate, which includes the above-mentioned antibody or a functional fragment thereof.
  • the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioactive isotope , fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, One or more of glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label, drug.
  • a detectable label such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a
  • the coupling moiety is selected from solid phase supports, such as magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
  • the present disclosure also provides a nucleic acid encoding the antibody or a functional fragment thereof of any one of the above.
  • the present disclosure also provides a cell comprising the above-mentioned nucleic acid.
  • the present disclosure also provides a method for preparing any of the above-mentioned antibodies or functional fragments thereof, the method comprising culturing the above-mentioned cells.
  • the present disclosure also provides a reagent or a kit, the reagent or kit comprising any of the above-mentioned antibodies or functional fragments thereof or the above-mentioned antibody conjugates.
  • the present disclosure also provides a use of any one of the above-mentioned antibodies or functional fragments thereof, the above-mentioned antibody conjugates, the above-mentioned nucleic acids or the above-mentioned cells in the preparation of reagents or kits.
  • the reagent or kit is used for detecting HIV or HIV-1 P24 protein, diagnosing related diseases caused by HIV, and screening or evaluating anti-HIV drugs.
  • the present disclosure also provides an antibody or a functional fragment thereof, the antibody conjugate described above, the nucleic acid described above, the cell described above, or the reagent or kit described above, for use in It is used in detecting HIV or HIV-1 P24 protein, diagnosing related diseases caused by HIV, and screening or evaluating anti-HIV drugs.
  • the present disclosure also provides a method for detecting HIV or HIV-1 P24 protein, comprising: A) using any of the above-mentioned antibodies or functional fragments thereof, the above-mentioned The antibody conjugate, the above-mentioned nucleic acid, the above-mentioned cells, or the above-mentioned reagent or kit are contacted with the sample to be tested to form an immune complex; and B) detecting the presence of the immune complex, the immune The presence of a complex indicates the presence of said HIV or HIV-1 P24 protein in said test sample.
  • the present disclosure also provides a method for diagnosing related diseases caused by HIV, comprising: A) using any of the above-mentioned antibodies or functional fragments thereof, or the above-mentioned antibody conjugates under conditions sufficient for a binding reaction to occur; The conjugate, the above-mentioned nucleic acid, the above-mentioned cells or the above-mentioned reagent or kit are contacted with the sample from the subject to carry out the binding reaction; and B) detecting the immune complex generated by the binding reaction.
  • the present disclosure also provides a method for screening or evaluating anti-HIV drugs, comprising: using any of the above-mentioned antibodies or functional fragments thereof, the above-mentioned antibody conjugates, the above-mentioned nucleic acids, the above-mentioned
  • the above-mentioned cells or the above-mentioned reagents or kits detect HIV in the test sample before and after contacting the anti-HIV drug; determine the HIV content difference before and after the test sample contacts the anti-HIV drug, based on the The anti-HIV drug is differentially screened or evaluated.
  • amino acid sequence involved in the disclosure of this application is as follows:
  • Figure 1 is the reduced SDS-PAGE results of 6F13RMb1 to 6F13RMb6 antibodies (the lanes are the reduced SDS-PAGE results of 6F13RMb1, 6F13RMb2, 6F13RMb3, 6F13RMb4, 6F13RMb5 and 6F13RMb6 from left to right).
  • Some embodiments of the present disclosure provide an anti-HIV-1 P24 antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include or are the same as SEQ ID NO: 15 1.
  • the amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region is shown.
  • the HCDR1-HCDR3 include or are amino acid sequences consistent with the HCDR1-HCDR3 of the heavy chain variable region shown in SEQ ID NO: 15; the LCDR1-LCDR3 include or are amino acid sequences consistent with SEQ ID NO: 16. The consistent amino acid sequence of LCDR1-LCDR3 of the light chain variable region shown in any one of SEQ ID NO:27-30.
  • the HCDR1-HCDR3 include or are amino acid sequences consistent with the HCDR1-HCDR3 of the heavy chain variable region shown in SEQ ID NO: 26; 16. The consistent amino acid sequence of LCDR1-LCDR3 of the light chain variable region shown in any one of SEQ ID NO:27-30.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • CDRs refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more or even all of the antibody, A region of major amino acid residues that contributes to the binding affinity of a functional or antigen-binding fragment thereof to the antigen or epitope it recognizes.
  • CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.
  • the heavy chain complementarity determining region is represented by "HCDR”, which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is represented by "LCDR", which includes LCDR1, LCDR2 and LCDR3.
  • CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. The accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues.
  • the system for defining the CDR is not particularly limited, and the CDR sequences defined by conventional systems in the art are within the protection scope of the present application.
  • the CDR definition method is referred to such as Kabat et al., U.S.Dept.of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) or Chothia et al., J Mol Biol 196:901-917 (1987).
  • Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can unambiguously map this Kabat numbering system to any variable region sequence, without reliance on any experimental data other than the sequence itself.
  • Kabat numbering refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983).
  • the polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, those of ordinary skill in the art are fully able to convert the sequence numbers of the sequence listing into Kabat numbers.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.
  • the HCDR1, HCDR2, and HCDR3 sequentially comprise amino acid sequences at positions 31-35, 50-65, and 95-100 of the heavy chain variable region, or sequentially such as 31 amino acid sequences of the heavy chain variable region. ⁇ 35, 50 ⁇ 65, 95 ⁇ 100 amino acid sequences;
  • the LCDR1, LCDR2 and LCDR3 sequentially comprise amino acid sequences at positions 24-34, 50-56, and 89-96 of the light chain variable region, or sequentially such as 24-34 and 50-56 of the light chain variable region , 89-96 amino acid sequence.
  • the numbering of the amino acid positions is based on the Kabat numbering system.
  • the antibody or functional fragment thereof comprises the following complementarity determining regions:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of it;
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of it;
  • HCDR3 which comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 21, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in any one of SEQ ID NO: 4, 22, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in any one of SEQ ID NO:5, 23, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • a "framework region” or "FR” region includes a heavy chain framework region and a light chain framework region, and refers to an antibody heavy chain variable region (which may be denoted as VH) and a light chain variable region (which may be denoted as VL)
  • VH antibody heavy chain variable region
  • VL light chain variable region
  • the antibody or its functional fragments further comprise the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and HFR3 of the light chain variable region. LFR4;
  • HFR1 includes the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology therewith;
  • HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto;
  • HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto;
  • HFR4 comprises an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto;
  • LFR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 24 or an amino acid sequence having at least 80% homology therewith;
  • LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto;
  • LFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 13, 25 or an amino acid sequence having at least 80% homology therewith;
  • LFR4 comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or a functional fragment thereof has the following framework regions:
  • HFR1 The amino acid sequence of HFR1 is as shown in SEQ ID NO: 7 or has at least 80% homology therewith;
  • amino acid sequence of HFR2 is as shown in SEQ ID NO: 8 or has at least 80% homology therewith;
  • amino acid sequence of HFR3 is as shown in SEQ ID NO: 9 or has at least 80% homology therewith;
  • amino acid sequence of HFR4 is as shown in SEQ ID NO: 10 or has at least 80% homology therewith;
  • amino acid sequence of LFR1 is as shown in any of SEQ ID NO: 11, 24, or has at least 80% homology therewith;
  • amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology therewith;
  • amino acid sequence of LFR3 is as shown in any of SEQ ID NO: 13, 25, or has at least 80% homology therewith;
  • the LFR4 amino acid sequence is as set forth in SEQ ID NO: 14 or has at least 80% homology thereto.
  • the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs according to The following combinations and permutations are obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • amino acid sequences of the framework regions of the antibodies or functional fragments thereof provided in the present disclosure may be identical to those of the above-mentioned corresponding framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12 , 13, or 14) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98% or 99% homology.
  • the antibody or its functional fragment has a KD ⁇ 10 -7 M, KD ⁇ 10 -8 M, KD ⁇ 10 -9 M, KD ⁇ 10 -10 M or KD ⁇ 10 -11 binding affinity to HIV-1 P24.
  • the antibody or functional fragment thereof binds HIV-1 P24 with an affinity of KD ⁇ 3.23 ⁇ 10 ⁇ 9 M or 9.42 ⁇ 10 ⁇ 10 M.
  • the detection of KD is carried out with reference to the method in the examples of the present disclosure.
  • amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 26;
  • amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 16, 27, 28, 29, or 30.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30.
  • the antibody or functional fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region” can be represented as CH; a "light chain constant region” can be represented as CL .
  • the heavy chain constant region is selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD heavy chain constant region, and the light chain constant region is selected from ⁇ type or ⁇ type Light chain constant region.
  • the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, gamecock or man.
  • the species origin of the constant region is human.
  • the heavy chain constant region sequence is such as SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;
  • the light chain constant region is SEQ ID NO: 17 ID NO: 18 or an amino acid sequence having more than 80% homology with SEQ ID NO: 18.
  • the constant region sequence provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
  • the above-mentioned functional fragment is selected from VHH, F(ab') 2 , Fab', Fab, Fv, Fd, scFv, single-chain antibody and diabody or domain antibody of the antibody any of the
  • Functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they were derived.
  • the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds.
  • enzymatic digestion including pepsin or papain
  • chemical reduction to split disulfide bonds On the basis of the structure of the intact antibody in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 31; the amino acid sequence of the light chain is as shown in any of SEQ ID NO: 20, 32, 33, 34, 35 one shown.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:32.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 33.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:34.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:35.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:31; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 32.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 33.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 34.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 35.
  • Some embodiments of the present disclosure also provide an anti-HIV-1 P24 antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising a heavy chain variable region and/or a light chain variable region, the heavy chain
  • the variable region includes the above-mentioned HCDR1-3 and the above-mentioned HFR1-4
  • the light chain variable region includes the above-mentioned LCDR1-3 and the above-mentioned LFR1-4.
  • amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 26;
  • amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 16, 27, 28, 29, or 30.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30.
  • the antibody or functional fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region” can be represented as CH; a "light chain constant region” can be represented as CL .
  • the heavy chain constant region is selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD heavy chain constant region, and the light chain constant region is selected from ⁇ type or ⁇ type Light chain constant region.
  • the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, gamecock or man.
  • the species origin of the constant region is human.
  • the heavy chain constant region sequence is such as SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;
  • the light chain constant region is SEQ ID NO: 17 ID NO: 18 or an amino acid sequence having more than 80% homology with SEQ ID NO: 18.
  • the constant region sequence provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
  • the above-mentioned functional fragment is selected from VHH, F(ab') 2 , Fab', Fab, Fv, Fd, scFv, single-chain antibody and diabody or domain antibody of the antibody any of the
  • Functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they were derived.
  • the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds.
  • enzymatic digestion including pepsin or papain
  • chemical reduction to split disulfide bonds On the basis of the structure of the intact antibody in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • Some embodiments of the present disclosure also provide an antibody to HIV-1 P24 or a functional fragment thereof, including a heavy chain and/or a light chain, and the heavy chain includes the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain includes the above-mentioned light chain variable region and the above-mentioned light chain constant region.
  • amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 31; the amino acid sequence of the light chain is as shown in any of SEQ ID NO: 20, 32, 33, 34, 35 one shown.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:32.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 33.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 34.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:35.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:31; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 32.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 33.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 34.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 31; the amino acid sequence of the light chain is shown in SEQ ID NO: 35.
  • Some embodiments of the present disclosure also provide an antibody conjugate comprising the above-mentioned antibody or a functional fragment thereof and a coupling moiety coupled thereto.
  • the coupling moiety is selected from a purification tag (such as a His tag); a detectable label, such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromogenic Group labels, electron-dense labels such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase
  • enzymes such as a fluorescent label, a chromogenic Group labels, electron-dense labels such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase
  • enzymes such as a fluorescent label, a chromogenic
  • the coupling moiety is selected from a solid phase carrier.
  • the solid support is selected from microspheres, plates or membranes.
  • the solid phase support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
  • the solid phase carrier is magnetic microspheres.
  • Some embodiments of the present disclosure also provide a reagent or a kit, the reagent or kit comprising the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.
  • Some embodiments of the present disclosure also provide a nucleic acid molecule encoding the above antibody or a functional fragment thereof.
  • Nucleic acids are usually RNA or DNA, and nucleic acid molecules can be single- or double-stranded.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA nucleic acid is used when it is ligated into a vector.
  • Some embodiments of the present disclosure also provide a vector containing the above nucleic acid molecule.
  • Some embodiments of the present disclosure also provide a recombinant cell containing the above vector.
  • Some embodiments of the present disclosure also provide a method for preparing an antibody or a functional fragment thereof, which includes: culturing the above-mentioned cells, and separating and purifying the antibody or a functional fragment thereof from the culture product.
  • Some embodiments of the present disclosure also provide a reagent or a kit, the reagent or kit comprising any of the above-mentioned antibodies or functional fragments thereof or the above-mentioned antibody conjugates.
  • Some embodiments of the present disclosure also provide any one of the above-mentioned antibodies or functional fragments thereof, the above-mentioned antibody conjugates, the above-mentioned nucleic acids or the above-mentioned cells in a preparation kit the use of.
  • the kit is used for detecting HIV or HIV-1 P24 protein, diagnosing related diseases caused by HIV, and screening or evaluating anti-HIV drugs.
  • Some embodiments of the present disclosure also provide an antibody or a functional fragment thereof, the antibody conjugate described above, the nucleic acid described above, the cell described above or the reagent described above or
  • the kit is used for detecting HIV or HIV-1 P24 protein, diagnosing related diseases caused by HIV, and screening or evaluating anti-HIV drugs.
  • Some embodiments of the present disclosure also provide a method for detecting HIV or HIV-1 P24 protein, comprising: A) using any of the above-mentioned antibodies or functional fragments thereof under conditions sufficient for a binding reaction to occur, The above-mentioned antibody conjugate, the above-mentioned nucleic acid, the above-mentioned cells or the above-mentioned reagent or kit are contacted with the sample to be tested to form an immune complex; and B) detecting the presence of the immune complex , the presence of the immune complex indicates the presence of the HIV or HIV-1 P24 protein in the test sample.
  • Some embodiments of the present disclosure also provide a method for diagnosing related diseases caused by HIV, comprising: A) using any of the above-mentioned antibodies or functional fragments thereof, the above-mentioned The above-mentioned antibody conjugates, the above-mentioned nucleic acids, the above-mentioned cells or the above-mentioned reagents or kits are contacted with the sample from the subject to perform a binding reaction; and B) detecting the immune complex generated by the binding reaction.
  • Some embodiments of the present disclosure also provide a method for screening or evaluating anti-HIV drugs, including: using any of the above-mentioned antibodies or functional fragments thereof, the above-mentioned antibody conjugates, the above-mentioned Nucleic acid, the above-mentioned cells, or the above-mentioned reagents or kits detect HIV in the test sample before and after contacting the anti-HIV drug; determine the HIV content difference before and after the test sample is exposed to the anti-HIV drug , screening or evaluating the anti-HIV drug based on the difference.
  • restriction enzymes and rTaq DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the present disclosure provides anti-HIV-1 P24 antibodies, reagents and kits for detecting HIV-1 P24.
  • the antibodies can specifically bind to HIV-1 P24 and have a high affinity for it.
  • the antibodies can be used to detect HIV or HIV- 1 P24 has good sensitivity or specificity.
  • the present disclosure provides a richer selection of antibodies for the detection of HIV-1 P24.
  • the inventor obtained hybridoma cell lines secreting anti-human HIV-1 P24 monoclonal antibody (6F13RMb1 antibody) through hybridoma preparation technology, extracted mRNA from the hybridoma cell line secreting anti-HIV-1 P24 monoclonal antibody, and passed RT -PCR method to obtain the DNA product, which is inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transformed into DH5 ⁇ competent cells. (Light chain) gene clones each 4 clones were sent to a gene sequencing company for sequencing.
  • VL light chain variable region
  • VH reconnected variable region
  • pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed.
  • the expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, EcoRI, etc., and named as pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector subsequently; according to the above steps 1- (2)
  • the obtained antibody variable region gene sequencing results in the pMD-18T vector, designed VL and VH gene-specific primers of the 6F13RMb1 antibody, with HindIII and EcoRI restriction sites and protective bases at both ends, and amplified by PCR.
  • the 0.74KB Light Chain gene fragment and the 1.46kb Heavy Chain gene fragment were amplified by the amplification method.
  • the Heavy Chain and Light Chain gene fragments were respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain Recombinant expression plasmids for Heavy Chain and Light Chain.
  • reaction OD was still greater than 1.0 after adding CHO cell supernatant and diluting 1000 times, and the reaction OD of wells without CHO cell supernatant was less than 0.1, indicating that the 6F13RMb1 antibody produced after plasmid transient transformation was active against HIV-1 P24 antigen.
  • Step 2-(2) Dilute the prepared plasmid with ultrapure water to 40 ⁇ g/100 ⁇ L, adjust the cell concentration of CHO cells to 1.43 ⁇ 10 7 cells/mL in a centrifuge tube, mix 100 ⁇ L of the above plasmid with 700 ⁇ L HO cells, and transfect Put into the electroporation cup for electroporation, and count the next day; 25 ⁇ mol/L MSX 96-well pressurized culture for about 25 days.
  • step 2-(3) After recovery, the cells obtained in step 2-(3) were first cultured in a 125mL shake flask, the inoculation volume was 30mL, the medium was 100% Dynamis medium, placed at a speed of 120r/min, the temperature was 37°C, and the carbon dioxide was 8% in the shaker. After culturing for 72 hours, inoculate the expanded culture at an inoculation density of 500,000 cells/mL. The expanded culture volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.
  • Shake flask parameters rotation speed 120r/min, temperature 37°C, carbon dioxide concentration 8%.
  • Fed-batch feeding Feed feeding starts every day when the shake flask is cultured for 72 hours.
  • HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day.
  • the protein A affinity chromatography column was used for affinity purification, and the purification steps were carried out by conventional methods in the art. 6.6 ⁇ g of the purified antibody was subjected to reducing SDS-PAGE.
  • the electropherogram is shown in Figure 1. Two bands were shown after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain). The results showed that the purified antibody was 6F13RMb1.
  • 6F13RMb1 obtained through the above steps, after sequencing and Kabat analysis, the heavy chain CDR1, CDR2, and CDR3 of 6F13RMb1 are shown in the amino acid sequence of SEQ ID NO: 1-3, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 15
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 16
  • the heavy chain has the amino acid sequence shown in SEQ ID NO: 16.
  • the amino acid sequence shown in ID NO:19, the light chain has the amino acid sequence shown in SEQ ID NO:20.
  • 6F13RMb1 designed mutation primers, repeated steps 1-(3) to 3-(2), and obtained 5 mutant antibodies after activity identification, named 6F13MRb2, 6F13MRb3, 6F13MRb4 , 6F13MRb5 and 6F13MRb 6.
  • the amino acid sequences of the heavy and light chains of the six antibodies from 6F13MRb1 to 6 (abbreviated as 6F13MRb1-6) are shown in Table 2.
  • the 6F13MRb1-6 antibody purified in Example 1 and the control antibody were diluted to 40 ⁇ g/mL with PBST, and the HIV-1 P24 antigen (purchased from Fapon) was serially diluted with PBST.
  • KD means the equilibrium dissociation constant, that is, affinity
  • Kon means the association rate constant
  • Kd means the dissociation rate constant
  • Example 1 The 6F13MRb1-6 antibodies purified in Example 1 were used as coating antibodies and paired with another strain of HIV p24 antibody (purchased from Feipeng) respectively, and the performance difference was detected on the enzyme-linked immunosorbent immunoassay platform, and 500 valued samples were tested , see Table 5 for specific properties.
  • Table 5 The lowest detection line of different antibodies and the correlation results with the sample determination
  • control antibody 5pg/ml 0.9899 6F13RMb1 2.5pg/ml 0.9921 6F13RMb2 2.5pg/ml 0.9936 6F13RMb3 1pg/ml 0.9997 6F13MRb4 2.5pg/ml 0.9942 6F13MAb5 1pg/ml 0.9984 6F13MRb6 1pg/ml 0.9991
  • the 6F13MRb1-6 antibodies obtained in Example 1 were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and the antibody samples were taken at 7 days, 14 days, and 21 days for state observation.
  • the activity of the 21-day antibody sample was detected, and the OD result was detected by the enzyme immunoassay.
  • step 2 of Example 2 The results showed that there was no obvious change in the protein state of the antibody under the three assessment conditions for 21 days, and the activity It also did not show a downward trend with the increase of the assessment temperature (Table 6). Therefore, it is further demonstrated that the 6F13RMb1-6 antibodies obtained in Example 1 are stable.
  • Antibody concentration 125 15.625 0 4°C
  • 21 days sample 1.385 0.375 0.012 -80°C
  • 21 days sample 1.342 0.367 0.021 37°C
  • 21 days sample 1.327 0.333 0.016
  • the anti-HIV-1 P24 antibody provided by the present disclosure has better affinity to HIV-1 P24, and the detection of HIV by using the antibody has better sensitivity or specificity.
  • the present disclosure provides a more excellent antibody selection for the detection of HIV. Therefore, the anti-HIV-1 P24 antibody, HIV detection reagent and kit provided by the present disclosure all have excellent practical performance and broad market application prospects.

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Abstract

本公开涉及一种抗HIV-1 P24的抗体及其制备方法和用途。本公开制备的抗HIV-1 P24的单克隆抗体对HIV-1 P24具有高亲和性、高反应活性、高灵敏度和特异性，为HIV-1 P24的检测提供了重要的原料来源。

Description

一种抗HIV-1 P24的抗体及其制备方法和用途
优先权信息
本公开请求2021年11月20日向中国国家知识产权局提交的、专利申请号为202111411387.6的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开属于抗体技术领域。更具体地，涉及一种抗HIV-1 P24的抗体及其制备方法和用途。
背景技术
人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)，即艾滋病(AIDS，获得性免疫缺陷综合征)病毒，是造成人类免疫系统缺陷的一种病毒。1981年，人类免疫缺陷病毒在美国首次发现。它是一种感染人类免疫系统细胞的慢病毒(Lentivirus)，属逆转录病毒的一种。目前，艾滋病不仅已成为严重威胁我国人民健康的公共卫生问题，且已影响到经济发展和社会稳定。
HIV是艾滋病的病原体，主要通过性接触、血液和母婴传播。近年来，HIV感染患者数量一直呈上升趋势。根据卫生部统计，中国自1985年发现第一例艾滋病病人以来，截至2009年10月底，累计报告艾滋病病毒感染者和病人319877例。2009年当年新发艾滋病病毒感染者4.8万人，我国艾滋病疫情形势依然严峻，性传播正成为主要传播途径。而目前测定血清HIV抗体是诊断HIV感染的常规实验方法，但是测定HIV抗体有局限性：有超过70％的HIV感染者在感染6个月后才能检测出抗体，在同性恋群体，这个数字超过80％，检测抗体方法增加了HIV“窗口期”传播的危险；另外新生儿产生抗体需要出生1年后，来自母亲的HIV抗体会致使假阳产生；由于HIV抗体在疾病过程中的持续存在，只有到艾滋病晚期时消失，无法作为治疗监测的稳定指标。
P24是HIV病毒颗粒的主要结构蛋白，是结构基因GAG的产物，在病毒的包装和成熟过程中起重要作用。P24蛋白的氨基酸序列在HIV各毒株之间高度保守，缺失P24会导致病毒无法正常组装。P24蛋白特异性很强，与多数其他逆转录病毒无交叉反应。HIV感染人体，感染者血液中首先出现的病毒标志物为病毒P24蛋白，从病毒感染到检出HIV抗体之间存在较长的窗口期，因此检测HIV-P24抗原已在HIV感染的早期诊断、患者的预后判断、筛选和评价抗HIV的药物，以及发现母婴传播等方面发挥了重要作用。
HIV-1 P24抗原的检测采用血清学诊断方法，主要有基于双抗体夹心的酶联免疫检测法和化学发光法、免疫复合物裂解检测法、超敏感EIA法、酶联免疫荧光法等。目前普遍使用双抗体夹心法检测人类免疫缺陷病毒P24抗原，具有很好的特异性，该方法需要针对人类免疫缺陷病毒P24抗原的特异性抗体，且单克隆抗体需求居多。然而目前市场上针对的人类免疫缺陷病毒P24抗原的单克隆抗体产品虽然多，但是灵敏度、亲和力以及特异性都存在缺 陷。
发明内容
本发明提供了一种抗HIV-1 P24单克隆抗体，其活性、亲和力、灵敏度或特异性至少之一得到改善。
本公开提供了一种抗HIV-1 P24的抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1～HCDR3包括或为与SEQ ID NO:15、SEQ ID NO:26任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括或为与SEQ ID NO:16、SEQ ID NO:27～30任一项所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
可选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3(简称CDRs)由Kabat、Chothia、IMGT、AbM或Contact系统定义。
本公开提供了一种抗HIV-1 P24的抗体或其功能性片段，所述抗体或其功能性片段包含以下互补决定区：
HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由其组成；
HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3、21任一所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4、22任一所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5、23任一所示的氨基酸序列，或由其组成；
LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由其组成。
可选地，所述的抗体或其功能性片段还包含重链可变区的骨架区HFR1、HFR2、HFR3和HFR4，和轻链可变区的骨架区LFR1、LFR2、LFR3和LFR4；其中，HFR1包括如SEQ ID NO:7所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；HFR2包括如SEQ ID NO:8所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；HFR3包括如SEQ ID NO:9所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；HFR4包括如SEQ ID NO:10所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；LFR1包括如SEQ ID NO:11、24任一所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；LFR2包括如SEQ ID NO:12所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；LFR3包括如SEQ ID NO:13、25任一所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；LFR4包括如SEQ ID NO:14所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列。
可选地，所述抗体或其功能性片段还具有以下的骨架区：
HFR1氨基酸序列如SEQ ID NO:7所示或与其具有至少80％同源性；
HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80％同源性；
HFR3氨基酸序列如SEQ ID NO:9所示或与其具有至少80％同源性；
HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80％同源性；
LFR1氨基酸序列如SEQ ID NO:11、24任一所示，或与其具有至少80％同源性；
LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80％同源性；
LFR3氨基酸序列如SEQ ID NO:13、25任一所示，或与其具有至少80％同源性；
LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80％同源性。
本公开提供了一种抗HIV-1 P24的抗体或其功能性片段，所述抗体或其功能性包含重链可变区和/或轻链可变区，所述重链可变区包含上述的HCDR1～3和上述的HFR1～4，所述轻链可变区包含上述的LCDR1～3和上述的LFR1～4。
可选地，所述重链可变区氨基酸序列如SEQ ID NO:15、26任一所示；所述轻链可变区氨基酸序列如SEQ ID NO:16、27、28、29、30任一所示。
可选地，所述抗体或其功能性片段还包含恒定区。
可选地，所述恒定区包括重链恒定区和/或轻链恒定区。
可选地，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
可选地，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；可选地，所述恒定区的种属来源为人。
可选地，所述重链恒定区为SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；所述轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
可选地，所述功能性片段选自所述抗体的F(ab’) 2、Fab’、Fab、Fv和scFv中的任意一种。
本公开提供了一种抗HIV-1 P24的抗体或其功能性片段，包括重链和/或轻链，所述重链包括上述的重链可变区和上述的重链恒定区；所述轻链包括上述的轻链可变区和上述的轻链恒定区。
可选地，所述重链的氨基酸序列如SEQ ID NO:19、31任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、32、33、34、35任一所示。
本公开还提供了一种抗体偶联物，所述抗体偶联物包括上述的抗体或其功能性片段。
可选地，所述偶联部分选自纯化标签或可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记、药物中的一种或多种。
可选地，所述偶联部分选自固相载体，例如磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
本公开还提供了一种核酸，所述核酸编码上文任一项所述的抗体或其功能性片段。
本公开还提供了一种细胞，所述细胞包含上述的核酸。
本公开还提供了一种制备上文任一项所述的抗体或其功能性片段的方法，所述方法包括培养上述的细胞。
本公开还提供了一种试剂或试剂盒，所述试剂或试剂盒包含上述任一项所述的抗体或其功能性片段或上述的抗体偶联物。
本公开还提供了一种上述任一项所述的抗体或其功能性片段、上述的抗体偶联物、上述的核酸或上述的细胞在制备试剂或试剂盒中的用途。
可选地，所述试剂或试剂盒用于检测HIV或HIV-1 P24蛋白、诊断HIV引起的相关疾病、筛选或评估抗HIV药物。
本公开还提供了一种上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒，用于检测HIV或HIV-1 P24蛋白、诊断HIV引起的相关疾病、筛选或评估抗HIV药物中的用途。
本公开还提供了一种检测HIV或HIV-1 P24蛋白的方法，包括：A)在足以发生结合反应的条件下，采用上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒与待测样品接触以进行形成免疫复合物；以及B)检测所述免疫复合物的存在，所述免疫复合物的存在指示所述待测样品中所述HIV或HIV-1 P24蛋白的存在。
本公开还提供了一种诊断HIV引起的相关疾病的方法，包括：A)在足以发生结合反应的条件下，采用上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒与来自受试者的样品接触以进行结合反应；以及B)检测结合反应产生的免疫复合物。
本公开还提供了一种筛选或评估抗HIV药物的方法，包括：采用上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒对接触所述抗HIV药物前后的待测样品中的HIV进行检测；确定所述待测样品接触所述抗HIV药物前后的HIV含量差值，基于所述差值筛选或评估所述抗HIV药物。
本申请公开涉及的氨基酸序列如下：
Figure PCTCN2022132981-appb-000001
Figure PCTCN2022132981-appb-000002
Figure PCTCN2022132981-appb-000003
Figure PCTCN2022132981-appb-000004
附图说明
图1是6F13RMb1至6F13RMb6抗体的还原性SDS-PAGE结果(泳道从左至右依次为6F13RMb1、6F13RMb2、6F13RMb3、6F13RMb4、6F13RMb5和6F13RMb6的还原性SDS-PAGE结果)。
具体实施方式
本公开的一些实施方式提供了一种抗HIV-1 P24的抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1～HCDR3包括或为与SEQ ID NO:15、SEQ ID NO:26任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括或为与SEQ ID NO:16、SEQ ID NO:27～30任一项所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
在可选的实施方式中，所述HCDR1～HCDR3包括或为与SEQ ID NO:15所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括或为与SEQ ID NO:16、SEQ ID NO:27～30任一项所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
在可选的实施方式中，所述HCDR1～HCDR3包括或为与SEQ ID NO:26所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括或为与SEQ ID NO:16、SEQ ID NO:27～30任一项所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
在本文中，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体，双特异性或多特异性抗体，以及嵌合抗体，只要它们展示所需的生物学活性。
在本文中，术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区，指包含一种或多种或者甚至全部的对抗体、其功能性片段或抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在本公开具体实施方式中，CDRs是指所述抗体的重链和轻链的高度可变区。
在本文中，重链互补决定区用“HCDR”表示，其包括HCDR1、HCDR2和HCDR3；轻链互补决定区用“LCDR”表示，其包括LCDR1、LCDR2和LCDR3。本领域常用的CDR标示方法包括：Kabat编号方案、IMGT编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中，序列的积累导致了KABATMAN数据库的创建，Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。
对所述CDR进行定义的系统不受特别限制，本领域常规系统定义的CDR序列均在本申请的保护范围之内。例如CDR定义方法参见如Kabat等，U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)或Chothia等，J Mol Biol 196：901-917(1987)。示例性的定义的CDR列于下表1中。在给定抗体的可变区氨基酸序列的情况下，本领域技术人员可以常规地确定哪些残基包含特定CDR。
表1：CDR定义 1
CDR Kabat AbM 2 IMGT
HCDR1 31-35 26-35 26-35
HCDR2 50-65 50-58 51-56
HCDR3 95-102 95-102 93-102
LCDR1 24-34 24-34 27-32
LCDR2 50-56 50-56 50-51
LCDR3 89-97 89-97 89-97
1表1中所有CDR定义的编号是依据Kabat编号系统(参见下文)。
2如表1中使用的“AbM”具有小写“b”，是指通过Oxford Molecular的“AbM”抗体建模软件定义的CDR。
Kabat等还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以明确地将该Kabat编号系统对应到任何可变区序列，而不依赖于序列本身之外的任何实验数据。如本文所述，“Kabat编号”是指Kabat等，U.S.Dept.of Health and HumanServices，“Sequence  of Proteins of Immunological Interest”(1983)所述的编号系统。序列表中的多肽序列未根据Kabat编号系统编号。然而，本领域普通技术人员完全能够将序列表的序列编号转换为Kabat编号。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3(简称CDRs)由Kabat、Chothia、IMGT、AbM或Contact系统定义。
在可选的实施方式中，所述HCDR1、HCDR2和HCDR3依次包含重链可变区的31～35位、50～65位、95～100位氨基酸序列，或依次如重链可变区的31～35位、50～65位、95～100位氨基酸序列所示；
所述LCDR1、LCDR2和LCDR3依次包含轻链可变区的24～34位、50～56位、89～96位氨基酸序列，或依次如轻链可变区的24～34位、50～56位、89～96位氨基酸序列所示。
且，所述氨基酸位点编号是依据Kabat编号系统。
在可选的实施方式中，所述抗体或其功能性片段包含以下互补决定区：
HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由其组成；
HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3、21任一所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4、22任一所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5、23任一所示的氨基酸序列，或由其组成；
LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由其组成。
在本文中，“骨架区”或“FR”区包括重链骨架区和轻链骨架区，是指抗体重链可变区(可以表示为VH)和轻链可变区(可以表示为VL)中除CDR之外的区域；其中，重链骨架区用“HFR”表示，并且可以被进一步细分成被CDR分隔开的毗邻区域，包含HFR1、HFR2、HFR3和HFR4骨架区；轻链骨架区用“LFR”表示，并且可以被进一步细分成被CDR分隔开的毗邻区域，包含LFR1、LFR2、LFR3和LFR4骨架区。
在可选的实施方式中，所述的抗体或其功能性片段还包含重链可变区的骨架区HFR1、HFR2、HFR3和HFR4，和轻链可变区的骨架区LFR1、LFR2、LFR3和LFR4；
其中，HFR1包括如SEQ ID NO:7所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
HFR2包括如SEQ ID NO:8所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
HFR3包括如SEQ ID NO:9所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
HFR4包括如SEQ ID NO:10所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
LFR1包括如SEQ ID NO:11、24任一所示的氨基酸序列或与其具有至少80％同源性的 氨基酸序列；
LFR2包括如SEQ ID NO:12所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
LFR3包括如SEQ ID NO:13、25任一所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
LFR4包括如SEQ ID NO:14所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列。
在可选的实施方式中，所述抗体还或其功能性片段具有以下的骨架区：
HFR1氨基酸序列如SEQ ID NO:7所示或与其具有至少80％同源性；
HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80％同源性；
HFR3氨基酸序列如SEQ ID NO:9所示或与其具有至少80％同源性；
HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80％同源性；
LFR1氨基酸序列如SEQ ID NO:11、24任一所示，或与其具有至少80％同源性；
LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80％同源性；
LFR3氨基酸序列如SEQ ID NO:13、25任一所示，或与其具有至少80％同源性；
LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80％同源性。
在本文中，重链可变区由以下编号的CDR与FR按如下组合排列连接获得：HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4；轻链可变区由以下编号的CDR与FR按如下组合排列连接获得：LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4。
需要说明的是，在其他的实施例中，本公开提供的抗体或其功能性片段的各骨架区氨基酸序列可以与上述对应骨架区(SEQ ID NO:7、8、9、10、11、12、13或14)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
在可选的实施方式中，所述抗体或其功能性片段以KD≤10 -7M、KD≤10 -8M、KD≤10 -9M、KD≤10 -10M或KD≤10 -11的亲和力结合HIV-1 P24。
在可选的实施方式中，所述抗体或其功能性片段以KD≤3.23×10 -9M或9.42×10 -10M的亲和力结合HIV-1 P24。
KD的检测参考本公开实施例中的方法进行。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15、26任一所示；
所述轻链可变区氨基酸序列如SEQ ID NO:16、27、28、29、30任一所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:16所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:27所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:28所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:29所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:30所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:16所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:27所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:28所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:29所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:30所示。
在可选的实施方式中，所述抗体或其功能性片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区；如本文所用，“重链恒定区”可以表示为CH；“轻链恒定区”可以表示为CL。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区，所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，所述恒定区的种属来源为人。
在可选的实施方式中，所述重链恒定区序列(CH)如SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；所述轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
需要说明的是，在其他的实施方式中，本公开提供的恒定区序列可以与上述恒定区(SEQ ID NO:17或18)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
在可选的实施方式中，上述功能性片段选自所述抗体的VHH、F(ab’) 2、Fab’、Fab、Fv、Fd、scFv、单链抗体和双价抗体或结构域抗体中的任意一种。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本公开记载的内容容易理解到，上述抗体的功能性片段可以通过比如酶消化的方法(包括胃 蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开中完整抗体的结构基础上，本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如Applied BioSystems等销售的自动肽合成仪合成获得。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19、31任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、32、33、34、35任一所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:32所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:33所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:34所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:35所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:32所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:33所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:34所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:35所示。
本公开的一些实施方式还提供了一种抗HIV-1 P24的抗体或其功能性片段，所述抗体或其功能性包含重链可变区和/或轻链可变区，所述重链可变区包含上述的HCDR1～3和上述的HFR1～4，所述轻链可变区包含上述的LCDR1～3和上述的LFR1～4。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15、26任一所示；
所述轻链可变区氨基酸序列如SEQ ID NO:16、27、28、29、30任一所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:16所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可 变区氨基酸序列如SEQ ID NO:27所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:28所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:29所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:15所示；所述轻链可变区氨基酸序列如SEQ ID NO:30所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:16所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:27所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:28所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:29所示。
在可选的实施方式中，所述重链可变区氨基酸序列如SEQ ID NO:26所示；所述轻链可变区氨基酸序列如SEQ ID NO:30所示。
在可选的实施方式中，所述抗体或其功能性片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区；如本文所用，“重链恒定区”可以表示为CH；“轻链恒定区”可以表示为CL。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区，所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，所述恒定区的种属来源为人。
在可选的实施方式中，所述重链恒定区序列(CH)如SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；所述轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
需要说明的是，在其他的实施方式中，本公开提供的恒定区序列可以与上述恒定区(SEQ ID NO:17或18)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
在可选的实施方式中，上述功能性片段选自所述抗体的VHH、F(ab’) 2、Fab’、Fab、Fv、Fd、scFv、单链抗体和双价抗体或结构域抗体中的任意一种。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据 本公开记载的内容容易理解到，上述抗体的功能性片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开中完整抗体的结构基础上，本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如Applied BioSystems等销售的自动肽合成仪合成获得。
本公开的一些实施方式还提供了一种HIV-1 P24的抗体或其功能性片段，包括重链和/或轻链，所述重链包括上述的重链可变区和上述的重链恒定区；所述轻链包括上述的轻链可变区和上述的轻链恒定区。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19、31任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、32、33、34、35任一所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:32所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:33所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:34所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:35所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:32所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:33所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:34所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:31所示；所述轻链的氨基酸序列如SEQ ID NO:35所示。
本公开的一些实施方式还提供了一种抗体偶联物，所述抗体偶联物包括上述的抗体或其功能性片段以及与其偶联的偶联部分。
在可选的实施方式中，所述偶联部分包括选自纯化标签(如His标签)；可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致 密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记中的一种或多种。
在可选的实施方式中，所述偶联部分选自固相载体。
在可选的实施方式中，所述固相载体选自微球、板或膜。
在可选的实施方式中，所述固相载体包括但不限于磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
在可选的实施方式中，所述固相载体为磁性微球。
本公开的一些实施方式还提供了一种试剂或试剂盒，所述试剂或试剂盒包括上述的抗体或其功能性片段或上述的抗体偶联物。
本公开的一些实施方式还提供了一种编码上述抗体或其功能性片段的核酸分子。
核酸通常是RNA或DNA，核酸分子可以是单链或双链的。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时采用DNA核酸。
本公开的一些实施方式还提供了一种含有上述核酸分子的载体。
本公开的一些实施方式还提供了一种含有上述载体的重组细胞。
本公开的一些实施方式还提供了一种制备抗体或其功能性片段的方法，其包括：培养如上所述的细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
在本公开的抗体或其功能性片段的氨基酸序列的基础上，本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、重组表达)制备得到该抗体或其功能性片段，例如从能够重组表达如上任一项所述的抗体或其功能性片段的重组细胞的培养产物中分离纯化得到该抗体或其功能性片段，这对本领域技术人员来说是容易实现的，基于此，无论采用何种技术制备本公开的抗体或其功能性片段，其均属于本公开的保护范围。
本公开的一些实施方式还提供了一种试剂或试剂盒，所述试剂或试剂盒包含上文任一项所述的抗体或其功能性片段或上述的抗体偶联物。
本公开的一些实施方式还提供了一种上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸或上述所述的细胞在制备试剂盒中的用途。
在可选的实施方式中，所述试剂盒用于检测HIV或HIV-1 P24蛋白、诊断HIV引起的相关疾病、筛选或评估抗HIV药物。
本公开的一些实施方式还提供了一种上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒，用于检测HIV或HIV-1 P24蛋白、诊断HIV引起的相关疾病、筛选或评估抗HIV药物中的用途。
本公开的一些实施方式还提供了一种检测HIV或HIV-1 P24蛋白的方法，包括：A)在足以发生结合反应的条件下，采用上述任一项所述的抗体或其功能性片段、上述所述的抗体 偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒与待测样品接触以进行形成免疫复合物；以及B)检测所述免疫复合物的存在，所述免疫复合物的存在指示所述待测样品中所述HIV或HIV-1 P24蛋白的存在。
本公开的一些实施方式还提供了一种诊断HIV引起的相关疾病的方法，包括：A)在足以发生结合反应的条件下，采用上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒与来自受试者的样品接触以进行结合反应；以及B)检测结合反应产生的免疫复合物。
本公开的一些实施方式还提供了一种筛选或评估抗HIV药物的方法，包括：采用上述任一项所述的抗体或其功能性片段、上述所述的抗体偶联物、上述所述的核酸、上述所述的细胞或上述的试剂或试剂盒对接触所述抗HIV药物前后的待测样品中的HIV进行检测；确定所述待测样品接触所述抗HIV药物前后的HIV含量差值，基于所述差值筛选或评估所述抗HIV药物。
为使本公开中实施方式的目的、技术方案和优点更加清楚，下面将对本公开中实施方式中的技术方案进行清楚、完整地描述。实施例实施方式中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，2011)，所述文献中的每个文献均通过引用明确并入本文中。
以下实施例中，限制性内切酶、rTaq DNA聚合酶购自Takara公司。MagExtractor-RNA 提取试剂盒购自TOYOBO公司。BD SMART TM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
本公开提供了抗HIV-1 P24的抗体、检测HIV-1 P24的试剂和试剂盒，该抗体可以特异性结合HIV-1 P24，对其具有较高的亲和力，用该抗体检测HIV或HIV-1 P24具有较好的灵敏度或特异性。本公开为HIV-1 P24的检测提供了更为丰富的抗体选择。
实施例
以下结合实施例对本公开中的特征和性能作进一步的详细描述。
实施例1：6F13RMb1抗体的制备
1、表达质粒构建
(1)6F13RMb1抗体基因制备
发明人前期通过杂交瘤制备技术获得分泌抗人HIV-1 P24单克隆抗体(6F13RMb1抗体)的杂交瘤细胞株，从分泌抗HIV-1 P24单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain(重链)及Light Chain(轻链)基因克隆各4个克隆送基因测序公司进行测序。
(2)6F13RMb1抗体可变区基因的序列分析
将上述步骤1-(1)测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，轻链可变区(VL)基因序列为333bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，重连可变区(VH)基因序列为381bp，属于VH1基因家族，其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2022132981-appb-000005
vector为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述步骤1-(2)获得的pMD-18T载体中抗体可变区基因测序结果，设计6F13RMb1抗体的VL和VH基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，通过PCR扩增方法扩出0.74KB的Light Chain基因片段和1.46kb的Heavy Chain基因片段。Heavy Chain和Light Chain基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，得到Heavy Chain和Light Chain的重组表达质粒。
2、稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
将步骤1-(3)步骤制备得到的质粒用超纯水稀释至40μg/100μL，于离心管中将CHO细胞的细胞浓度调节至1.43×10 7cells/mL，取100μL上述质粒与700μL细胞混合，转入电转杯进行电转，电转后的第3、5、7天取样计数，第7天收样检测。
采用包被液(主要成分NaHCO3)稀释HIV-1 P24抗原(购自菲鹏)至3μg/mL，每孔100μL，4℃过夜；次日，使用洗涤液(主要成份Na 2HPO 4+NaCl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，于37℃条件下培养1h，拍干；加入稀释后的CHO细胞上清，100μL/孔，于37℃条件下培养30min(部分上清1h)；洗涤液清洗5次，拍干；加入羊抗人IgG-HRP，每孔100μL，于37℃条件下培养30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔，主要成份柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)，加入显色液B液(50μL/孔，主要成份柠檬酸+EDTA·2Na+TMB+浓HCl)，培养10min；加入终止液(50μL/孔，EDTA·2Na+浓H 2SO 4)；在酶标仪上450nm(参考630nm)处读OD值。
结果显示加入CHO细胞上清稀释1000倍后反应OD仍大于1.0，未加CHO细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的6F13RMb1抗体对HIV-1 P24抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂：Buffer 50μL、步骤1-(3)制备得到的质粒100μg/管、PvuⅠ酶10μL、无菌水补至500μL，37℃水浴酶切过夜；先用等体积的酚/氯仿/异戊醇(下层)(酚：氯仿：异戊醇的体积比为25:24:1)，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染，加压筛选稳定细胞株
步骤2-(2)制备得到的质粒用超纯水稀释至40μg/100μL，于离心管中将CHO细胞的细胞浓度调至1.43×10 7cells/mL，取100μL上述质粒与700μLCHO细胞混合，转入电转杯进行电转，次日计数；25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔，并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批量培养(简称批培)，调整CHO细胞的细胞密度至0.5×10 6cells/mL，取2.2mL进行批培，将CHO细胞的细胞密度间接至0.3×10 6cells/mL，取2mL进行保种；7天6孔批培后，将上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3、重组抗体生产
(1)细胞扩培
将步骤2-(3)获得的细胞复苏之后先在125mL规格的摇瓶中培养，接种体积为30mL，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h后，以50万cells/mL接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控 制接种密度为50万cells/mL左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳的浓度为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3％，Feed 7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA亲和层析柱进行亲和纯化，纯化步骤采用本领域常规方法进行。取6.6μg纯化的抗体进行还原性SDS-PAGE。电泳图如图1所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。结果表明，纯化后的抗体为6F13RMb1。
经上述步骤获得的6F13RMb1，经测序及Kabat分析，6F13RMb1的重链CDR1、CDR2、CDR3分别如SEQ ID NO:1～3的氨基酸序列所示，轻链CDR1、CDR2、CDR3分别如SEQ ID NO:4～6的氨基酸序列所示，重链可变区具有如SEQ ID NO:15所示的氨基酸序列、轻链可变区具有如SEQ ID NO:16所示的氨基酸序列，重链具有如SEQ ID NO:19所示的氨基酸序列、轻链具有如SEQ ID NO:20所示的氨基酸序列。
4、发明人对6F13RMb1的结构进行分析，并进行突变引物设计，重复步骤1-(3)至步骤3-(2)，经活性鉴定，筛选得到5个突变抗体，命名为6F13MRb2、6F13MRb3、6F13MRb4、6F13MRb5和6F13MRb 6。6F13MRb1至6(简称6F13MRb1～6)六个抗体的重链和轻链的氨基酸序列如表2所示。
表2：6F13MRb1～6的重链和轻链的氨基酸序列
Figure PCTCN2022132981-appb-000006
实施例2：不同抗体的亲和力分析、活性鉴定以及性能评估
1、亲和力分析
利用AMC传感器，将实施例1纯化获得的6F13MRb1～6抗体和对照抗体用PBST稀释到40μg/mL，HIV-1 P24抗原(购自菲鹏)用PBST进行梯度稀释。
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69 GLY溶液及缓冲液3(PBST)进行传感器再生，输出数据，具体数据如表3所示。
表3：不同抗体的亲和力结果
Figure PCTCN2022132981-appb-000007
Figure PCTCN2022132981-appb-000008
注：KD表示平衡解离常数即亲和力；Kon表示结合速率常数；Kdis表示解离速率常数。
2、活性鉴定
使用包被液(主要成分NaHCO 3)稀释HIV-1 P24抗原(购自菲鹏)至1μg/mL，每孔100μL，4℃过夜；次日，采用洗涤液(主要成份Na 2HPO 4+NaCl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，于37℃条件下培养1h，拍干；加入稀释后的实施例1纯化获得的6F13MRb1～6抗体和对照抗体，100μL/孔，于37℃条件下培养30min；洗涤液清洗5次，拍干；加入羊抗人IgG-HRP，每孔100μL，于37℃条件下培养30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔，主要成份柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)，加入显色液B液(50μL/孔，主要成份柠檬酸+EDTA·2Na+TMB+浓HCl)，培养10min；加入终止液(EDTA·2Na+浓H 2SO 4)，50uL/孔；在酶标仪上450nm(参考630nm)处读OD值，检测结果如表4所示，6F13MRb1～6抗体的结合活性高于对照抗体的结合活性。
表4：不同抗体的结合活性结果
Figure PCTCN2022132981-appb-000009
3、性能评价
将实施例1纯化获得的6F13MRb1～6抗体作为包被抗体，分别与另一株HIV p24抗体(购自菲鹏)配对使用，在酶联免疫平台上检测性能差异，测试500份已定值标本，具体性能见表5。
表5：不同抗体的最低检出线和与标本定值相关性结果
/ 最低检出线 与标本定值相关性
对照抗体 5pg/ml 0.9899
6F13RMb1 2.5pg/ml 0.9921
6F13RMb2 2.5pg/ml 0.9936
6F13RMb3 1pg/ml 0.9997
6F13MRb4 2.5pg/ml 0.9942
6F13MAb5 1pg/ml 0.9984
6F13MRb6 1pg/ml 0.9991
实施例3：抗体稳定性检测
将实施例1获得的6F13MRb1～6抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天抗体样品进行状态观察，并对21天抗体样品进行活性检测，利用酶免法检测OD结果，具体的操作步骤参考实施例2的步骤2，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降越势(表6)。因此，进一步说明实施例1获得的6F13RMb1～6抗体稳定。
表6：抗体的稳定性结果
抗体浓度(ng/ml) 125 15.625 0
4℃，21天样品 1.385 0.375 0.012
-80℃，21天样品 1.342 0.367 0.021
37℃，21天样品 1.327 0.333 0.016
上述实施例为本公开较佳的实施方式，但本公开的实施方式并不受上述实施例的限制，其他的任何未背离本公开的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本公开的保护范围之内。
工业实用性
本公开提供的抗HIV-1 P24的抗体，其对HIV-1 P24的亲和力较好，使用该抗体检测HIV具有较好的灵敏度或特异性。本公开为HIV的检测提供了更为优秀的抗体选择，因此，本公开提供的抗HIV-1 P24的抗体、检测HIV试剂和试剂盒均具备优异的实用性能和广阔的市场应用前景。

Claims (18)

  1. 一种抗HIV-1 P24的抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，其特征在于，所述HCDR1～HCDR3包括与SEQ ID NO:15、SEQ ID NO:26任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括与SEQ ID NO:16、SEQ ID NO:27～30任一项所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
  2. 根据权利要求1所述的抗体或其功能性片段，其特征在于，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由Kabat、Chothia、IMGT、AbM或Contact系统定义。
  3. 根据权利要求1所述的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段包含以下互补决定区：
    HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由其组成；
    HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由其组成；
    HCDR3，其包含SEQ ID NO:3、21任一所示的氨基酸序列，或由其组成；
    LCDR1，其包含SEQ ID NO:4、22任一所示的氨基酸序列，或由其组成；
    LCDR2，其包含SEQ ID NO:5、23任一所示的氨基酸序列，或由其组成；
    LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由其组成。
  4. 根据权利要求1～3任一项所述的抗体或其功能性片段，其特征在于，所述的抗体或其功能性片段还包含重链可变区的骨架区HFR1、HFR2、HFR3和HFR4，和轻链可变区的骨架区
    LFR1、LFR2、LFR3和LFR4；
    其中，HFR1包括如SEQ ID NO:7所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    HFR2包括如SEQ ID NO:8所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    HFR3包括如SEQ ID NO:9所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    HFR4包括如SEQ ID NO:10所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    LFR1包括如SEQ ID NO:11、24任一所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    LFR2包括如SEQ ID NO:12所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    LFR3包括如SEQ ID NO:13、25任一所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列；
    LFR4包括如SEQ ID NO:14所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列。
  5. 一种抗HIV-1 P24的抗体或其功能性片段，其特征在于，所述抗体或其功能性包含重链可变区和/或轻链可变区，所述重链可变区包含权利要求1～3任一项所述的HCDR1～3和权利要求4所述的HFR1～4，所述轻链可变区包含权利要求1～3任一项所述的LCDR1～3和权利要求4所述的LFR1～4；
    可选地，所述重链可变区氨基酸序列如SEQ ID NO:15、26任一所示；
    所述轻链可变区氨基酸序列如SEQ ID NO:16、27、28、29、30任一所示。
  6. 根据权利要求1～5任一项所述的抗体或其功能性片段，还包含恒定区；
    可选地，所述恒定区包括重链恒定区和/或轻链恒定区；
    可选地，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区；
    可选地，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；可选地，所述恒定区的种属来源为人；
    可选地，所述重链恒定区为SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；所述轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
  7. 根据权利要求1～6任一所述的抗体或其功能性片段，其特征在于，所述功能性片段选自所述抗体的F(ab’) 2、Fab’、Fab、Fv和scFv中的任意一种。
  8. 一种抗HIV-1 P24的抗体或其功能性片段，包括重链和/或轻链，其特征在于，所述重链包括权利要求5所述的重链可变区和权利要求6所述的重链恒定区；
    所述轻链包括权利要求5所述的轻链可变区和权利要求6所述的轻链恒定区；
    可选地，所述重链的氨基酸序列如SEQ ID NO:19、31任一所示；
    所述轻链的氨基酸序列如SEQ ID NO:20、32、33、34、35任一所示。
  9. 一种抗体偶联物，其特征在于，所述抗体偶联物包含权利要求1～8任一项所述的抗体或其功能性片段以及与其偶联的偶联部分；
    可选地，所述偶联部分选自纯化标签或可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记、药物中的一种或多种；
    可选地，所述偶联部分选自固相载体，例如磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
  10. 一种核酸，其特征在于，所述核酸编码权利要求1～8任一项所述的抗体或其功能性片段。
  11. 一种细胞，其特征在于，所述细胞包含权利要求10所述的核酸。
  12. 一种制备权利要求1～8任一项所述的抗体或其功能性片段的方法，其特征在于，所述方法包括培养权利要求11所述的细胞。
  13. 一种试剂或试剂盒，其特征在于，所述试剂或试剂盒包括权利要求1～8任一项所述的抗体或其功能性片段或权利要求9所述的抗体偶联物。
  14. 权利要求1～8任一项所述的抗体或其功能性片段、权利要求9所述的抗体偶联物、权利要求10所述的核酸或权利要求11所述的细胞在制备试剂或试剂盒中的用途；
    可选地，所述试剂或试剂盒用于检测HIV或HIV-1 P24蛋白、诊断HIV引起的相关疾病、筛选或评估抗HIV药物。
  15. 权利要求1～8任一项所述的抗体或其功能性片段、权利要求9所述的抗体偶联物、权利要求10所述的核酸、权利要求11所述的细胞或权利要求13所述的试剂或试剂盒，用于检测HIV或HIV-1 P24蛋白、诊断HIV引起的相关疾病、筛选或评估抗HIV药物中的用途。
  16. 一种检测HIV的方法，其特征在于，包括：
    A)在足以发生结合反应的条件下，采用权利要求1～8任一项所述的抗体或其功能性片段、权利要求9所述的抗体偶联物、权利要求10所述的核酸、权利要求11所述的细胞或权利要求13所述的试剂或试剂盒与待测样品接触以进行形成免疫复合物；以及
    B)检测所述免疫复合物的存在，所述免疫复合物的存在指示所述待测样品中所述HIV或HIV-1 P24蛋白的存在。
  17. 一种诊断HIV引起的相关疾病的方法，其特征在于，包括：
    A)在足以发生结合反应的条件下，采用权利要求1～8任一项所述的抗体或其功能性片段、权利要求9所述的抗体偶联物、权利要求10所述的核酸、权利要求11所述的细胞或权利要求13所述的试剂或试剂盒与来自受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  18. 一种筛选或评估抗HIV药物的方法，其特征在于，包括：
    采用权利要求1～8任一项所述的抗体或其功能性片段、权利要求9所述的抗体偶联物、权利要求10所述的核酸、权利要求11所述的细胞或权利要求13所述的试剂或试剂盒对接触所述抗HIV药物前后的待测样品中的HIV进行检测；
    确定所述待测样品接触所述抗HIV药物前后的HIV含量差值，基于所述差值筛选或评估所述抗HIV药物。
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