An antibody against HIV-P24 and its preparation method and application

Abstract

This invention discloses an anti-HIV-P24 antibody, its preparation method, and its application, relating to the field of antibodies. The anti-HIV-P24 antibody includes a heavy chain variable region and a light chain variable region. This anti-HIV-P24 antibody has good binding activity, providing a new approach and an important source of raw materials for the high-sensitivity detection of HIV-P24.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an anti-HIV-P24 antibody, its preparation method, and its application. Background Technology

[0002] AIDS, also known as Acquired Immunodeficiency Syndrome, is a systemic disease caused by the Human Immunodeficiency Virus (HIV). The main modes of transmission for AIDS include blood transmission, mother-to-child transmission, and sexual transmission. Currently, there are two types of HIV: HIV-1 and HIV-2. HIV-1 is the primary cause of AIDS. Based on different cross-species transmission chains, HIV-1 can be classified into: M, N, O, and P types. M type accounts for 90% of HIV-1 infections. Based on different endemic regions, M type can be further divided into subtypes A, B, C, D, E, F, G, H, I, J, K, and L. HIV-2 has a lower transmissibility and less virulent strains than HIV-1.

[0003] The HIV virus is a spherical particle with a diameter of 100–120 nm, consisting of a core and an envelope. Its inner region consists of a cone-shaped core, which is encapsulated by the structural protein P24 (capsid). The P24 protein is encoded by the gag gene, whose initial translation product is 55 kDa. Under the action of proteases, it is processed into mature proteins of 24 kDa, 17 kDa, and 15 kDa, namely P24, P17, and P15.

[0004] During the period between HIV infection and seroconversion, HIV is usually undetectable in the body; this period is known as the window period. After HIV infection, the HIV P24 antigen can be detected within 2-4 weeks, while HIV antibodies require at least 4-6 weeks. Fourth-generation HIV testing, also known as HIV antigen-antibody testing, can simultaneously detect HIV IgM, IgG, IgA, and the P24 antigen, shortening the window period for HIV immune detection to 2 weeks. Detecting the HIV P24 protein in the blood allows for early diagnosis, enabling more effective control of HIV transmission.

[0005] Currently, there are many types of HIP-P24 antibody raw materials on the market, each with different properties. Therefore, developing a P24 antibody raw material with strong binding activity is of great significance for improving HIV sensitivity. Summary of the Invention

[0006] The purpose of this invention is to provide a novel anti-HIV-P24 antibody, its preparation method, and its application.

[0007] This invention is implemented as follows:

[0008] In a first aspect, embodiments of the present invention provide an anti-HIV-P24 antibody, comprising: a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, 14, 15, 37, 38, 39, 40, 41, 61, 62, 63, 84, 85, 86, 87, 88, 109, 110, 111, 112, 113, 114, 115 or 116; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16, 17, 18, 42, 43, 44, 45, 46, 47, 48, 64, 65, 66, 67, 68, 69, 70, 71, 89, 90, 91, 92, 93, 117, 118, 119 or 120.

[0009] Secondly, embodiments of the present invention provide an anti-HIV-P24 antibody, which includes a CDR region, the CDR region being consistent with the CDR regions in the heavy chain variable region and light chain variable region described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide an anti-HIV-P24 antibody, which includes a CDR region, wherein the aforementioned CDR region includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the aforementioned CDR region is selected from any combination of the following:

[0011]

[0012]

[0013]

[0014] Fourthly, embodiments of the present invention provide a conjugate comprising the antibody described in the foregoing embodiments.

[0015] Fifthly, embodiments of the present invention provide a reagent or kit comprising: the antibody or conjugate described in the foregoing embodiments.

[0016] Sixthly, embodiments of the present invention provide a method for preparing a nucleic acid molecule, a vector, a cell, or an antibody as described in the foregoing embodiments, wherein the nucleic acid molecule encodes the antibody as described in the foregoing embodiments, the vector contains the nucleic acid molecule, the cell contains the vector, and the preparation method includes culturing cells containing the vector.

[0017] Seventhly, embodiments of the present invention provide the use of antibodies as described in the foregoing embodiments or nucleic acid molecules, vectors or cells as described in the foregoing embodiments in the preparation of products for detecting HIV-P24.

[0018] Eighthly, embodiments of the present invention provide a method for detecting HIV-P24, comprising: contacting the sample to be tested with the antibody, conjugate, or reagent or kit described in the foregoing embodiments to form an immune complex.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides a variety of new anti-HIV-P24 antibodies, all of which have good binding activity, providing a new approach for high-sensitivity detection of HIV-P24. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] On one hand, embodiments of the present invention provide an anti-HIV-P24 antibody, including a heavy chain variable region and a light chain variable region;

[0023] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, 14, 15, 37, 38, 39, 40, 41, 61, 62, 63, 84, 85, 86, 87, 88, 109, 110, 111, 112, 113, 114, 115 or 116;

[0024] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 16, 17, 18, 42, 43, 44, 45, 46, 47, 48, 64, 65, 66, 67, 68, 69, 70, 71, 89, 90, 91, 92, 93, 117, 118, 119 or 120.

[0025] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from any combination of the following:

[0026]

[0027]

[0028] On the other hand, embodiments of the present invention provide an anti-HIV-P24 antibody, which includes a CDR region, the CDR region being consistent with the CDR regions in the heavy chain variable region and light chain variable region described in any of the foregoing embodiments.

[0029] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the aforementioned antibody, and LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody described in the first aspect.

[0030] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:13; LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:16.

[0031] In this application, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, provided they exhibit the desired antigen-binding activity. Antigen-binding fragments of antibodies include any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv, and generally have the same binding specificity as the antibody from which they originated. Those skilled in the art will readily understand from the description in this application that antigen-binding fragments of antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this application, those skilled in the art can readily obtain the antigen-binding fragments of antibodies.

[0032] Antigen-binding fragments of antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by automated peptide synthesizers, such as those sold by Applied BioSystems.

[0033] In this application, the terms "CDR region (complementarity-determining region)," "CDR," or "CDRs" refer to the highly variable region of the heavy and light chains of an immunoglobulin, specifically the region containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In a specific embodiment of this application, the CDR region refers to the highly variable region of the heavy and light chains of the antibody.

[0034] In this application, the heavy chain complementarity determination region is denoted by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is denoted by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0035] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of protection of this application.

[0036] Table 1: CDR Definition 1

[0037] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0038] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0039] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0040] 3If neither H35A nor H35B exists, then HCDR1 ends at bit 35; if only H35A exists, then HCDR1 ends at bit 35A; if both H35A and H35B exist, then HCDR1 ends at bit 35B.

[0041] 4 If neither H35A nor H35B exists, then HCDR1 ends at bit 32; if only H35A exists, then HCDR1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0042] 5 If neither H35A nor H35B exists, then HCDR1 ends at bit 33; if only H35A exists, then HCDR1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0043] In some embodiments, the CDR area includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one system or a combination of systems among Kabat, Chothia, AbM, Contact, and IMGT.

[0044] On the other hand, embodiments of the present invention provide an anti-HIV-P24 antibody, which includes a CDR region, wherein the CDR region includes the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3:

[0045] HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, 2, 19, 49, 72 or 94;

[0046] The amino acid sequence is as shown in SEQ ID NO:3, 20, 50, 51, 52, 73, 74, 95, 96, 97, 98, 99, 100 or 101 HCDR2;

[0047] HCDR3 with an amino acid sequence as shown in SEQ ID NO:4, 5, 21, 22, 23, 24, 25, 53, 75, 76, 77 or 103;

[0048] LCDR1 with an amino acid sequence as shown in SEQ ID NO: 6, 26, 27, 28, 29, 54, 78, 79, 80, 105 or 104;

[0049] LCDR2 with an amino acid sequence as shown in SEQ ID NO:7, 8, 30, 31, 32, 33, 55, 56, 57, 58, 81 or 106;

[0050] LCDR3 with an amino acid sequence as shown in SEQ ID NO:9, 10, 34, 59, 60, 82, 83, 107 or 108.

[0051] In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from any combination of the following:

[0052]

[0053]

[0054] In some embodiments, the anti-HIV-P24 antibody further includes a backbone region, the backbone region including at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0055] In this application, the "backbone region," "framework region," or "FR" region includes the heavy chain framework region and the light chain framework region, which refer to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain framework region can be further subdivided into adjacent regions separated by the CDR, including HFR1, HFR2, HFR3, and HFR4; the light chain framework region can be further subdivided into adjacent regions separated by the CDR, including LFR1, LFR2, LFR3, and LFR4.

[0056] In this application, the heavy chain variable region is obtained by connecting the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In some embodiments, the skeletal region is derived from any one of the following: cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, and humans.

[0058] In some embodiments, the skeleton region is identical to or has at least 80% identity with the skeleton regions of the heavy chain variable region and the light chain variable region described in any of the foregoing embodiments.

[0059] In some embodiments, the at least 80% refers to a range of any one or any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%.

[0060] In some embodiments, the anti-HIV-P24 antibody further includes a heavy chain constant region and / or a light chain constant region.

[0061] In this application, the division of the variable and constant region sequences is based on the IMGT partitioning method, see Lefranc, M.-P. IMGT. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire.IMGT ,the international ImMunoGenetics information system http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGLC,IMGT Repertoire.IMGT ,the international ImMunoGenetics information system http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region or the N-terminus of the constant region delineated by IMGT. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this application.

[0062] In some embodiments, the species source of the heavy chain constant region and / or the light chain constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, and humans.

[0063] In some embodiments, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0064] In some embodiments, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0065] In some embodiments, the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0066] In some embodiments, the heavy chain constant region sequence is as shown in SEQ ID NO:11 or 35 or has at least 80% identity with it.

[0067] In some embodiments, the light chain constant region sequence is as shown in SEQ ID NO:112 or 36 or has at least 80% identity with it.

[0068] On the other hand, embodiments of the present invention provide a conjugate comprising: the anti-HIV-P24 antibody described in any of the foregoing embodiments.

[0069] In some embodiments, the conjugate further includes a conjugation portion conjugated to the anti-HIV-P24 antibody. The conjugation portion is selected from one or more of biotin or biotin derivatives, solid-phase carriers, and markers.

[0070] In some embodiments, the solid support is selected from microspheres, plates, and membranes.

[0071] In some embodiments, the aforementioned markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or probed by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0072] In some embodiments, the markers include any one or more of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0073] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this application.

[0074] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0075] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0076] In optional embodiments, the radioactive isotope includes, but is not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0077] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0078] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0079] In optional embodiments, the colloid includes, but is not limited to, colloidal selenium, colloidal metal, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0080] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold and colloidal silver.

[0081] In some embodiments, the marker is colloidal gold.

[0082] It should be noted that when an antibody is labeled with a marker to become a labeled antibody, it does not affect the characteristics of the antibody's immune response, nor does it change the activity of the marker itself.

[0083] On the other hand, embodiments of the present invention provide a reagent or kit comprising: the anti-HIV-P24 antibody or the conjugate described in any of the foregoing embodiments.

[0084] On the other hand, embodiments of the present invention provide a method for preparing a nucleic acid molecule, a vector, a cell, or an anti-HIV-P24 antibody as described in any of the foregoing embodiments, wherein the nucleic acid molecule encodes the anti-HIV-P24 antibody as described in any of the foregoing embodiments, the vector contains the nucleic acid molecule, the cell contains the vector, and the preparation method includes culturing cells containing the vector.

[0085] Based on the amino acid sequence of the anti-HIV-P24 antibody disclosed in this application, those skilled in the art will readily conceive of preparing the anti-HIV-P24 antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the anti-HIV-P24 antibody as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-HIV-P24 antibody of this application, it falls within the protection scope of this application.

[0086] On the other hand, embodiments of the present invention provide the application of anti-HIV-P24 antibodies as described in any of the foregoing embodiments, or nucleic acid molecules, vectors, or cells as described in any of the foregoing embodiments, in the detection of HIV-P24 or the preparation of products for the detection of HIV-P24.

[0087] In some embodiments, the product includes, but is not limited to, reagents, kits, test strips, or test plates.

[0088] On the other hand, embodiments of the present invention provide a method for detecting HIV-P24, comprising:

[0089] The anti-HIV-P24 antibody, conjugate, or reagent or kit described in any of the foregoing embodiments is brought into contact with the sample to be tested to form an immune complex.

[0090] In some embodiments, the presence of HIV-P24 or the amount of HIV-P24 in the sample to be tested is determined based on the signal of the immune complex.

[0091] In some embodiments, the immune complex further includes a second antibody that binds to the anti-HIV-P24 antibody.

[0092] In some embodiments, the immune complex further includes a second antibody that binds to HIV-P24.

[0093] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0094] In this application, restriction endonucleases, T4 DNA ligases, and DNA polymerases were purchased from New England Biolabs, Taq DNA polymerase from TaKaRa, the VO1 expression vector was constructed in our laboratory, gel extraction kits and plasmid extraction kits were commercially available, and primer synthesis and gene sequencing were outsourced. The HIV-P24 monoclonal antibody (hereinafter referred to as W1-W5 antibody) sequences were obtained from rabbit (W1) and mouse (W2-W5) hybridoma cell sequencing.

[0095] Example 1: Construction and screening of mutant libraries.

[0096] 1. Construction of WT template plasmid

[0097] (1) Antibody gene synthesis

[0098] The VH and VL sequences of the antibody sequences named W1, W2, W3, W4, and W5 were optimized using E. coli codons, and then the antibody gene sequences were used for gene synthesis. The light and heavy chain variable region sequences of W1, W2, W3, W4, and W5 are shown in the table below.

[0099] Antibody Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence W1 SEQ ID NO:14 SEQ ID NO:16 W2 SEQ ID NO:37 SEQ ID NO:48 W3 SEQ ID NO:61 SEQ ID NO:67 W4 SEQ ID NO:84 SEQ ID NO:92 W5 SEQ ID NO:109 SEQ ID NO:119

[0100] The amino acid sequences of the heavy chain constant region and light chain constant region of the mutant antibody W1 are shown in SEQ ID NO:11-12, respectively; the amino acid sequences of the heavy chain constant region and light chain constant region of the mutant antibodies W2, W3, W4 and W5 are shown in SEQ ID NO:35-36, respectively.

[0101] (2) Amplification of WT antibody gene fragment

[0102] The synthesized antibody sequence was amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. The antibody gene fragment was then purified using a gel recovery kit.

[0103] (3) Enzyme digestion and ligation of WT antibody gene fragment

[0104] The antibody gene fragment and the VO1 vector plasmid were simultaneously digested with restriction endonucleases, and then purified using a gel extraction kit to obtain the antibody gene fragment and VO1 vector with sticky ends. Next, the antibody gene fragment and VO1 vector were ligated with T4 DNA ligase at 22°C for 4 hours. The ligation product was recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells, and the entire bacterial culture was plated on ampicillin-resistant plates and incubated overnight at 37°C.

[0105] (4) Extraction and sequencing verification of WT template plasmid

[0106] The following day, single-clone colonies were selected, and colony PCR and gel electrophoresis were performed using Taq DNA polymerase. Bacteria with the correct inserted antibody gene sequence were selected for culture and amplification. WT template plasmids were obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0107] 2. Construction of single-point mutation libraries

[0108] (1) Primer design and synthesis

[0109] Using degenerate base codons, single-point saturation mutation upstream and downstream primers for the VH and VL full CDR regions were designed and primers were synthesized.

[0110] (2) PCR amplification of single-point saturation mutant plasmid

[0111] The PCR method was used, and the reaction system was prepared according to Table 2. Then, the PCR reaction conditions in Table 3 were used to amplify and prepare the single-point saturated mutant library plasmid. Finally, the WT template plasmid was digested with restriction endonuclease at 37°C for 1 hour to obtain the mutant library plasmid.

[0112] Table 2: PCR System

[0113] WT template plasmid 50ng DNA polymerase 1μl DNA polymerase buffer 10μl dNTP (2.5mM) 4μl Upstream primer (10 μM) 1μl Downstream primer (10 μM) 1μl <![CDATA[ddH2O]]> Adjust the volume to 50 μl

[0114] Table 3: PCR reaction conditions

[0115] Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ time 5min 30s 30s 2min 5min ∞

[0116] Steps 2 through 4: 22 cycles.

[0117] (3) Single-point saturation mutant plasmid transformation

[0118] Take 10 μl of the reaction product and transform it into 100 μl of TG1 Escherichia coli competent cells. Spread the entire bacterial culture onto an ampicillin-resistant plate and incubate overnight at 37°C.

[0119] 3. Screening of single-point mutation libraries

[0120] (1) Antibody expression in mutant library

[0121] The following day, 500 μl of culture medium was added to a 96-well plate beforehand. For each single-point mutation library, 92 single-clone colonies were selected, and WT, negative, and blank control colonies were set up. After incubation at 37°C for 5-6 hours, the bacterial culture was transferred to a new 96-well plate and incubated at 37°C for 1-2 hours. Finally, induction medium was added, and the plate was incubated overnight at 37°C to express antibodies, obtaining the antibody expression supernatant of the mutant library.

[0122] (2) Mutant library screening and sequencing

[0123] HIV-P24 was added to ELISA plates at a rate of 0.1 μg / ml and 100 μl / well. The plates were incubated overnight at 4°C. The next day, the plates were blocked with 1%–2% skim milk powder. The antibody expression supernatant from the mutant library was added to ELISA plate wells at a rate of 100 μl / well. WT, negative, and blank controls were also included. The plates were incubated at room temperature for 2 hours. The standard ELISA detection method was used for subsequent washing, color development, and reading. Finally, the data were processed and analyzed. Clones with enhanced activity (candidate clones) were sent for sequencing. The sequencing results were analyzed to select the mutation sites of unique candidate mutant clones (see Table 4) for constructing combined mutant libraries. (Ratio value explanation: The ratio value represents the degree of affinity enhancement. When the ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT. When the ratio value is greater than 1, it means that the affinity is enhanced. That is, the larger the ratio value, the greater the degree of affinity enhancement.) The results in Table 4 show that the binding activity of the selected single-point mutant clones is significantly better than that of WT, with a significant improvement in activity.

[0124] Table 4: Screening results and mutation sites of candidate clones

[0125]

[0126]

[0127] 4. Construction of combined mutant libraries

[0128] (1) Library primer design and synthesis

[0129] Based on the mutation sites on VH and VL, amplification primers for the combined mutant library were designed and outsourced to a company for primer synthesis.

[0130] (2) Fragment amplification and ligation

[0131] According to the PCR system in Table 2 and the PCR reaction conditions in Table 3, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel electrophoresis. The antibody mutant fragments were then spliced ​​into complete antibody fragments using the overlap PCR method.

[0132] Finally, the antibody fragment was inserted into the V01 vector using an enzyme digestion and ligation method to form a complete antibody expression plasmid: the method is the same as that for "WT antibody gene fragment digestion and ligation". 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells, and the entire bacterial culture was plated on ampicillin-resistant plates and incubated overnight at 37°C.

[0133] Screening of 5-combination mutant libraries

[0134] The following day, three monoclonal bacterial culture plates were selected for antibody supernatant expression, ELISA screening, and positive clone sequencing analysis. The specific procedures are as follows.

[0135] Add 500 μl of culture medium to each 96-well culture plate beforehand. Select single colonies for each 96-well bacterial culture plate as follows: Select 92 single colonies and set up WT, negative and blank control colonies. The remaining operations are the same as those for single point mutation library screening.

[0136] Multiple clones were obtained through screening of the combined mutant library. Specific screening results and mutation site information are shown in Table 5 (Ratio value explanation: The Ratio value represents the degree of affinity enhancement. When the Ratio value equals 1, it indicates that the affinity of the mutant clone is the same as that of WT; when the Ratio value is greater than 1, it indicates an enhanced affinity, i.e., the larger the Ratio value, the greater the degree of affinity enhancement). The results show that the combined mutant antibodies in Table 5 exhibit significantly better binding activity than WT.

[0137] Table 5: Information on candidate clones with combined mutations

[0138]

[0139]

[0140] Example 2: Validation of antibody eukaryotic recombinant expression.

[0141] 1. Construction of eukaryotic recombinant expression plasmids

[0142] pcDNA TM 3.4 TOPO The vector is a recombinant antibody eukaryotic expression vector constructed. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the variable region gene sequences of multiple candidate clones obtained by screening the above combined mutant library (see Table 5) and multiple clones obtained by screening the single-point mutant library (see Table 4), VL and VH gene-specific amplification primers and constant region overlap primers for the corresponding antibody sequences were designed. The primers at both ends are respectively equipped with HindIII and EcoRI restriction sites and protective bases. The Light Chain gene fragment and Heavy Chain gene fragment were amplified by PCR amplification.

[0143] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery, the Heavy Chain and Light Chain genes were ligated into the 3.4A expression vector and transformed into DH5α competent cells. After bacterial growth, single colonies were picked for PCR identification of positive clones. Positive clones were then sequenced to confirm sequence accuracy. Plasmids were extracted from correctly sequenced clones for later use.

[0144] 2. Sample preparation of recombinant antibodies

[0145] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cells / ml, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / ml. 6 Cells / ml were used as cell dilution buffers. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. Antibodies were obtained by affinity purification using a protein A affinity chromatography column.

[0146] 3 Affinity Analysis

[0147] The recombinant antibody obtained above was subjected to affinity testing and analysis: the binding and dissociation curves of the antigen and antibody were tested on a Biacore 8K+ instrument. The instrument automatically fitted and obtained the affinity constant, binding rate, and dissociation rate (KD represents the equilibrium dissociation constant, i.e., the affinity constant; the smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate). The results showed that the affinity of the obtained mutant antibody with HIV-P24 was 1.0 × 10⁻⁶. -12 M < KD < 1.0 × 10 -8 M has a higher affinity.

[0148] The sequence information involved in this application is shown in Table 6.

[0149] Table 6: Amino Acid Sequence List

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-HIV-P24 antibody, characterized in that, It includes: the variable region of the heavy chain and the variable region of the light chain; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, 14, 15, 37, 38, 39, 40, 41, 61, 62, 63, 84, 85, 86, 87, 88, 109, 110, 111, 112, 113, 114, 115 or 116; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 16, 17, 18, 42, 43, 44, 45, 46, 47, 48, 64, 65, 66, 67, 68, 69, 70, 71, 89, 90, 91, 92, 93, 117, 118, 119 or 120.

2. The anti-HIV-P24 antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region are selected from any combination of the following:

3. An anti-HIV-P24 antibody, characterized in that, It includes a CDR region, which is consistent with the CDR region in the heavy chain variable region and the light chain variable region as described in claim 1 or 2; Optionally, the CDR area includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, AbM, Contact, and IMGT, or by a combination of multiple systems.

4. An anti-HIV-P24 antibody, characterized in that, It includes a CDR region comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the CDR region is selected from any combination of the following:

5. The antibody according to any one of claims 1 to 4, characterized in that, The antibody further includes a backbone region, which includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4. Optionally, the skeletal region is derived from any one of the following: cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, and humans; Optionally, the skeleton region is consistent with or has at least 80% identity with the skeleton regions of the heavy chain variable region and the light chain variable region as described in claim 1 or 2.

6. The antibody according to any one of claims 1 to 5, characterized in that, The antibody also includes a heavy chain constant region and / or a light chain constant region; Optionally, the species source of the heavy chain constant region and / or the light chain constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, and humans; Optionally, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the light chain constant region includes a light chain constant region selected from κ-type or λ-type; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO:11 or 35 or has at least 80% identity with it; Optionally, the light chain constant region sequence is as shown in SEQ ID NO:12 or 36 or has at least 80% identity with it.

7. A coupling, characterized in that, It includes: the antibody according to any one of claims 1 to 6; Optionally, the conjugate further includes a conjugation portion conjugated to the antibody, the conjugation portion being selected from any one or more of: biotin or biotin derivatives, solid-phase carriers, and markers; Optionally, the solid support is selected from any one of microspheres, plates, and membranes; Optionally, the label includes any one or more of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labelers; Optionally, the marker is colloidal gold.

8. A reagent or kit, characterized in that, It includes: The antibody according to any one of claims 1 to 6 or the conjugate according to claim 7.

9. A method for preparing a nucleic acid molecule, vector, cell, or antibody as described in any one of claims 1 to 6, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1 to 6, the vector contains the nucleic acid molecule, the cell contains the vector, and the preparation method includes culturing cells containing the vector.

10. The use of the antibody as described in any one of claims 1 to 6 or the nucleic acid molecule, vector, or cell as described in claim 9 in the preparation of a product for detecting HIV-P24.

11. A method for detecting HIV-P24, characterized in that, It includes: The antibody according to any one of claims 1 to 6, the conjugate according to claim 7, or the reagent or kit according to claim 8 is brought into contact with the sample to be tested to form an immune complex; Optionally, based on the signal of the immune complex, it is determined whether the sample to be tested contains HIV-P24 or the amount of HIV-P24. Optionally, the immune complex further includes a second antibody, which binds to the antibody; Optionally, the immune complex further includes a second antibody that binds to HIV-P24.

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