CN118063600A - Anti-HIV-1 gp120 antibody or antigen binding fragment thereof and application thereof - Google Patents

Abstract

The present invention belongs to the technical field of immunology and pathogenic biology, and specifically discloses an anti-HIV-1gp120 antibody or an antigen-binding fragment thereof and its application. The heavy chain variable region of the monoclonal antibody or its antigen-binding fragment includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO.4, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO.7. The monoclonal antibody provided by the present invention has specific binding ability with natural HIV-1gp120, which can be used in HIV-1 virus detection, and can also be used in the distribution of HIV-1 virus at different levels such as cells and organs and the study of its mechanism of action, and has good application prospects.

Description

Anti-HIV-1gp120 antibody or antigen-binding fragment thereof and application thereof

Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-HIV-1gp120 antibody or an antigen-binding fragment thereof and an application thereof.

Background technique

Human Immunodeficiency Virus (HIV) is the pathogen that causes AIDS (Acquired Immunodefieiency Syndrome, AIDs). HIV includes two types, HIV-1 and HIV-2, and the difference between the two at the genetic level is >55%. Among them, HIV-1 has stronger virulence and infectivity, and most AIDS cases worldwide are caused by infection with HIV-1. Compared with HIV-1, HIV-2 has weaker replication ability and pathogenicity, and the cases caused by its infection are mainly present in western Africa, and the number is relatively small. Therefore, the main research target in the world is HIV-1. The envelope glycoprotein (env) gene of HIV-1 encodes gp160 protein, which is further processed into gp120 and gp41, and has strong antigenicity. The receptor binding site on gp120 is a potential target for treating HIV.

Compared with the third epidemiological survey, the types of HIV epidemic strains in my country have increased, and the diversity and complexity are also on the rise. In addition, a large number of new circulating recombinant strains (CRF) have emerged in the infected population, and more than 18 HIV circulating recombinant strains (CRF) are spreading in China. The high complexity of HIV-1 epidemic highlights the severe challenges faced in designing effective measures to prevent HIV transmission. my country is currently the country with the most infected subtypes and the most recombinant types. The main prevalent HIV strains are CRF_07BC, CRF_08BC, CRF_01AE and subtype B from Thailand. The infected people of these epidemic strains account for 89.3% of the total HIV-1 infected people in my country. Faced with such a complex HIV epidemic in my country, it is urgent to find effective treatments for the dominant prevalent strains of HIV in my country, and the "anti-AIDS" situation is urgent.

HIV treatment methods mainly include conventional therapies and emerging therapies, among which CAR-T therapy and antibody therapy are one of the most cutting-edge methods in the field of HIV treatment. Antibodies work mainly in two ways. On the one hand, antibodies with neutralizing activity can block the binding of viruses to cell receptors by binding to viral envelope proteins, thereby blocking viral infection. On the other hand, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) can recruit immune cells and immune molecules such as macrophages or complement, thereby clearing free viruses and infected cells.

So far, humans have no specific drugs or vaccines to treat or prevent AIDS, but research has shown that antiviral antibodies can exert antiviral effects through single or multiple mechanisms, which can include any stage of the viral infection and replication cycle. In-depth research on the mechanism of antiviral antibodies neutralizing viruses can, on the one hand, clarify the basic mechanism of such antibodies in inhibiting viruses; on the other hand, it can discover the key steps in the viral life cycle, thereby providing inspiration for the development of new antiviral targeted drugs, cocktail drugs and highly effective vaccines, bringing hope to the treatment of AIDS. At present, many antibodies are derived from mice and other animal sources and need to be humanized. There are still problems with immunogenicity in treatment, so scientists currently have high hopes for the development of fully human antibodies.

Summary of the invention

One of the purposes of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof against HIV-1 gp120.

The second object of the present invention is to provide a method for screening fully human antibodies against HIV-1 gp120.

The third object of the present invention is to provide drugs and methods for detecting or treating HIV-1 virus infection.

In order to achieve the above object, the present invention adopts the following technical solution:

The first aspect of the present invention provides an anti-HIV-1gp120 antibody, which comprises: a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a heavy chain complementary determining region, and the heavy chain complementary determining region includes: CDR-H1 shown in the amino acid sequence of SEQ ID NO.1, CDR-H2 shown in the amino acid sequence of SEQ ID NO.2, and CDR-H3 shown in the amino acid sequence of SEQ ID NO.3; the light chain variable region includes a light chain complementary determining region, and the light chain complementary determining region includes: CDR-L1 shown in the amino acid sequence of SEQ ID NO.5, CDR-L2 having the amino acid sequence AAS, and CDR-L3 shown in the amino acid sequence of SEQ ID NO.6.

Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.7.

The second aspect of the present invention provides a recombinant protein comprising: the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

Furthermore, the recombinant protein also includes an optional tag sequence for facilitating expression and/or purification.

Furthermore, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag, etc.

The third aspect of the present invention provides a biological material related to the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the recombinant protein of the second aspect of the present invention, wherein the biological material comprises at least one of a1) to a16):

a1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect, or the recombinant protein of the second aspect of the present invention;

a2) an expression cassette comprising the nucleic acid molecule described in a1);

a3) a vector comprising the nucleic acid molecule described in a1);

a4) a vector comprising the expression cassette described in a2);

a5) a transgenic cell line comprising the nucleic acid molecule described in a1);

a6) a transgenic cell line comprising the expression cassette described in a2);

a7) a transgenic cell line comprising the vector described in a3);

a8) a transgenic cell line comprising the vector described in a4);

a9) a microorganism comprising the nucleic acid molecule described in a1);

a10) a microorganism comprising the expression cassette described in a2);

a11) a microorganism comprising the vector described in a3);

a12) a microorganism comprising the vector described in a4);

a13) a virus comprising the nucleic acid molecule described in a1);

a14) a virus comprising the expression cassette described in a2);

a15) a virus comprising the vector described in a3);

a16) A virus comprising the vector described in a4).

Furthermore, the transgenic cell line does not contain any reproductive material.

Furthermore, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention and a nucleic acid molecule encoding the light chain of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

The fourth aspect of the present invention provides a conjugate comprising: at least one of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention; and a coupling portion.

Further, the coupling part comprises at least one of a detectable marker, a drug, a toxin, an electron-dense marker, biotin/avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, and a viral coat protein. Preferably, the detectable marker is a fluorescent or luminescent marker.

Furthermore, the drug is other drug for preventing and/or treating HIV virus infection and/or diseases related to HIV virus infection.

The fifth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the invention, the recombinant protein of the second aspect, the biomaterial of the third aspect, and/or the conjugate of the fourth aspect in the preparation of a product;

The product comprises at least one of a pharmaceutical composition, a reagent, a detection plate, a test kit, and a detection chip.

Furthermore, the pharmaceutical composition has at least one of the functions b1)-b2):

b1) Prevention and/or treatment of HIV-1 infection;

b2) Prevention and/or treatment of diseases associated with HIV-1 virus.

Furthermore, the reagent, detection plate, detection chip or kit has at least one function among c1)-c2):

c1) Detection of HIV-1 virus;

c2) Diagnosis of diseases associated with HIV-1 virus infection.

A sixth aspect of the present invention provides a product, comprising at least one of d1) to d4):

d1) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

d2) the recombinant protein of the second aspect of the present invention;

d3) the biomaterial according to the third aspect of the present invention;

d4) The conjugate according to the fourth aspect of the present invention.

Furthermore, the product comprises at least one of a pharmaceutical composition, a reagent, a test plate, a test kit, and a test chip, and the reagent, the test plate, the test chip, or the test kit has at least one of the functions of e1)-e2):

e1) Detection of HIV-1 virus;

e2) Diagnosis of diseases associated with HIV-1 infection.

The pharmaceutical composition has at least one of the functions f1)-f2):

f1) prevention and/or treatment of HIV-1 infection;

f2) prevention and/or treatment of diseases associated with HIV-1 virus;

Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier;

Furthermore, the pharmaceutical composition also comprises other drugs for preventing and/or treating orthopoxvirus infection and/or diseases associated with orthopoxvirus infection.

The seventh aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention, which is obtained by culturing the transgenic cell line of the third aspect of the present invention.

The eighth aspect of the present invention provides a method for detecting the presence or level of HIV virus in a sample, comprising: using the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the recombinant protein of the second aspect, the related biological material described in the third aspect and/or the conjugate of the fourth aspect.

Further, the method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

The ninth aspect of the present invention provides a method for diagnosing whether a subject is infected with HIV virus or suffers from a disease associated with HIV virus, comprising: using the antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the related biological material of the third aspect and/or the conjugate of the fourth aspect of the present invention to detect the presence of HIV virus in a sample from the subject.

Furthermore, the sample includes but is not limited to excrement, oral or nasal secretions, alveolar lavage fluid, etc. from a subject (eg, a mammal, preferably a human).

The tenth aspect of the present invention provides a method for neutralizing the virulence of HIV virus in a sample, comprising: contacting a sample containing HIV virus with the pharmaceutical composition of the sixth aspect of the present invention.

Further, the methods may be used for therapeutic purposes, or for non-therapeutic purposes (eg, the sample is a cell sample rather than a patient or a sample from a patient).

The eleventh aspect of the present invention provides a method for preventing or treating HIV infection or a disease associated with HIV infection in a subject, comprising: administering a preventively or therapeutically effective amount of the pharmaceutical composition of the sixth aspect of the present invention to a subject in need thereof.

Preferably, the subject is a mammal, such as a human or a mouse.

Preferably, the pharmaceutical composition of the present invention can be administered to a subject by any suitable route of administration. Such routes of administration include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the SDS-PAGE result of the purified monoclonal antibody 5.

Detailed ways

The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, medical microbiology, and immunology laboratory operation procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "antibody" may include whole antibody molecules, full-length immunoglobulin molecules, particularly naturally occurring full-length immunoglobulin molecules, or full-length immunoglobulin molecules formed by recombinant immunoglobulin gene fragments, and antibody fragments. It is usually an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair having a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a domain CL. The crystallizable fragment (Fc segment) is equivalent to the CH2 and CH3 functional regions of the antibody, which can form crystals. It has no antigen binding activity, but has functions such as fixation of complement and binding to Fc receptors. The term "antibody" is not limited to any specific method for producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

As used herein, the term "monoclonal antibody" refers to a substantially homogeneous antibody population that recognizes and binds an antigenic determinant or epitope with high specificity. This is in contrast to polyclonal antibodies, which generally include different antibodies that recognize and bind different antigenic determinants.

As used herein, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence of a target pathogen.

As used herein, the term "neutralizing activity" refers to the functional activity of antibodies or antibody fragments that bind to antigenic proteins on viruses, thereby preventing virus-infected cells and/or the maturation of viral progeny and/or the release of viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

As used herein, the term "ELISA" is an enzyme-linked immunosorbent assay, which is an immunoassay method that combines an antigen or antibody on the surface of a solid carrier and utilizes the specific binding of the antigen and antibody and the color development reaction of a specific substrate catalyzed by an enzyme labeled on the antibody or antigen to detect the target.

As used herein, the term " _IC50 " refers to the half inhibitory concentration of the antagonist being measured. In this context, it refers to the antibody concentration at which the antibody neutralizes the virus to 50% of the maximum effect in an in vitro neutralization assay.

In order to obtain neutralizing antibodies with protective effects, the present invention adopts a single B cell antibody screening technology, which uses a flow cytometry sorting method to fluorescently label specific antigens, and uses the principle that antigens can specifically bind to BCR on the surface of B cells to screen out single target B cells, and obtains the heavy chain and light chain genes of antibodies by PCR amplification. The antibody genes are constructed on an expression vector and transfected into eukaryotic cells to express monoclonal antibodies. The inventors of this application first obtained peripheral blood from HIV-infected patients, separated PBMCs, reacted PBMCs with gp120 antigens and fluorescently labeled them, and then sorted single cells into a 96-well plate pre-added with cell lysate by a flow cytometry sorter, followed by RT-PCR and PCR amplification to obtain the encoding antibody variable region sequence. Further, the antibody variable region is connected to the vector for transformation, and the obtained product is transfected into cells for eukaryotic expression to obtain antibodies, and the antibodies are captured by a prefabricated gravity column of Protein A/G. A series of functional tests are performed on the antibodies. The results show that the antibodies can specifically bind to HIV-1gp120.

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

The following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

Example 1: Preparation of anti-HIV-1 gp120 antibodies

1. Isolation of mature B cells

(1) Collect peripheral blood from patients infected with HIV, separate PBMCs using lymphocyte separation solution, and freeze in a liquid nitrogen tank for later use;

(2) Take out the PBMCs cryopreservation tube from the liquid nitrogen tank, quickly put it into a 37°C water bath, and shake it from time to time to completely thaw it within 1 minute. Then, take out the cells under sterile conditions and wash them by blowing in PBS;

(3) Centrifuge at 1000 r/min for 10 min, discard the supernatant, add 500 μL PBS to blow up the cells, then add 8 mL PBS and centrifuge at 1000 r/min for 10 min to wash the PBMCs. Repeat the washing twice and count the cells using a cell counter. The number of PBMC cells should be more than 10^6. Finally, resuspend the PBMC cells to 100 μL.

(4) Take the resuspended PBMC cells and add His-tagged antigen to a final concentration of 400 nM (1 mg/mL/45 KDa = 20 μM) and incubate on ice for 30 min;

(5) Precool the centrifuge to 4°C, centrifuge at 300 g for 5 min, discard the supernatant, add 1 ml of PBS to wash twice, and resuspend to 100 μL with PBS again;

(6) Add antibodies and incubate on ice for 30 min. The specific antibody names, characteristics and usage are shown in the following table:

Antibody Fluorescence cell Usage amount Anti-human CD235a APC Red cell 1:20 Anti-human CD3 APC T cells 1:20 Anti-human CD16 APC NK cell 1:20 Anti-human CD19 APC CY7 B cells 1:20 Anti-human CD38 APC Plasma B cells 1:20 Anti-human CD27 Pacific Blue Memory B cell 1:50 Anti-human IgG FITC 1:20 Anti-His PE 1:10

(7) Add 1 ml of PBS, wash twice, centrifuge at 4°C, 300 g for 5 min, discard the supernatant and resuspend to 1000 μL with PBS;

(8) Specific B cells were sorted into 96-well plates using a flow cytometer, CD3-CD16-CD235a-CD19+(CD27+ or CD38+)IgG+His+ positive cells were collected and sorted into 96-well PCR Multiplate ^™ 96-Well PCR Plates. One cell was sorted per well, and 8 μL of RNase-free water and 1 μL of RT buffer were pre-added to each well of the 96-well plate.

2. Antibody gene amplification and linear expression vector construction

Antibody gene amplification: The sorted mature B cells were subjected to RT-PCR in a 96-well plate to obtain cDNA, which was used as a template for nested PCR. The PCR products were identified by 1% agarose gel electrophoresis, and those identified as positive were recovered using an agarose gel recovery kit, and the PCR products were sequenced.

Construction of linear expression vector: The amplified heavy and light chain genes were respectively connected to the pCAGGS expression vector with the light and heavy chain constant region sequences by homologous recombination under the catalysis of enzymes, reacted at 65°C for 5 minutes, and then cooled on ice. They were then added to DH5α competent cells for transformation, activated on a shaker at 37°C for 1 hour, centrifuged at 5000 rpm for 5 minutes, and 100 μl of supernatant was retained in the precipitate. The rest of the supernatant was discarded, and 50 μl was taken out of the suspension and spread on an LB agar plate containing ampicillin resistance. After growing for 12-16 hours, a single colony was picked for colony PCR and the product was sequenced and compared with the aforementioned PCR product sequence. If they were consistent, it was successful.

The specific sequences of the antibody light chain and heavy chain are shown in Table 1.

Table 1. Sequence information

3. Antibody expression and purification

One day before the experiment, 293T cells were plated on a 6-well cell plate. When the cells were fully grown on the next day, each well was transfected with a vector plasmid carrying the antibody light and heavy chain genes using PEI. 2 μg of plasmid was transfected into each well of the 6-well plate for each light and heavy chain. The cell supernatant was collected once 3 days after transfection and once after 7 days. The specific binding activity of the cell supernatant to the antigen was first determined by ELISA experiment, and antibodies with antigen-specific binding activity were preliminarily screened.

PEI was used to co-transfect the light chain and heavy chain plasmids into Expi293F cells for large-scale expression. The antibodies were then captured using a Protein A/G prefabricated gravity column. The antibodies with binding activity were purified using a Protein A affinity chromatography column. After a large number of experimental studies, a neutralizing monoclonal antibody targeting HIV-1gp120 was discovered. The SDS-PAGE results in Figure 1 show that a highly pure monoclonal antibody was obtained, with molecular weights of the heavy and light chains being approximately 50 kDa and 25 kDa, respectively.

4. Determination of antibody-antigen binding activity

(1) Coating: dilute gp120 antigen with PBS buffer to add 100 ng antigen per well and incubate at 37°C for 2 h;

(2) Blocking: Use 5% skim milk powder diluted with PBS and block at 37°C for 2 h or 4°C overnight. After the block is completed, discard the solution, wash with PBST 5 times, and pat dry.

(3) Primary antibody: Add the antibody to be tested, use the supernatant of cells without plasmid transfection as a negative control, and PBS as a blank control, incubate in a 37°C incubator for 30 min, then wash 5 times with PBST and pat dry;

(4) Secondary antibody: Add HRP-labeled goat anti-human IgG diluted 1:5000 in blocking solution and incubate in a 37°C incubator for 30 min, then wash five times with PBST and pat dry;

(5) Color development: Mix 50 μl of color development solution A and 50 μl of color development solution B and add them to the wells for 15 min in the dark.

(6) Termination: Add 50 μl of stop solution to terminate the reaction and measure the absorbance at 450 nm using an ELISA reader.

The results are shown in Table 2. Antibody No. 5 (shown in bold in the column of antibodies 1-8) can specifically bind to HIV-1 viral protein gp120, while the negative control (shown in bold in the column of antibodies 17-20) has no specific binding, and the positive control (shown in bold in the column of H-23 plasma) has specific binding.

Table 2. ELISA results

5. Evaluation of antibody neutralization ability against HIV

Add culture medium to a 96-well plate in advance, add 180 μL to A1-H1, 110 μL to A3-H9 and C10-F10, and 220 μL to C11-F11. Add 20 μL of sample plasma to each of A1-H1, mix for 30 times, take 165 μL and transfer to A2-H2, and then transfer 55 μL in sequence for 3-fold dilution, for a total of 8 gradients. For pseudovirus dilution, prepare the virus solution with 1 μL HIV-1 pseudovirus stock solution + 109 μL culture medium per well. Add 110 μL of virus solution to the sample well and virus control, mix 3 times; place in an incubator, incubate at 37°C for 1 hour, transfer the incubated sample solution, virus control and cell control to a 96-well white plate, 100 μL per well, and repeat twice;

Digest TZM-bl cells, resuspend to a cell concentration of 2.5×10 ⁵ /mL, and add DEAE-dextran hydrochloride (12.5 mg/mL) to make its concentration 25 μg/mL; add 100 μL of cell suspension (i.e. 2.5×10 ⁴ cells) to a 96-well white plate, and the final concentration of DEAE-dextran hydrochloride is 12.5 μg/mL; culture at 37°C, 5% CO ₂ for 48 hours; detect fluorescence value: remove the 96-well white plate, discard the culture supernatant; add 100 μL of Bright-Lite Luciferase, incubate at room temperature for 2 minutes and then mix; detect the luminescence value of each well on a fluorescence detector, and calculate the inhibition rate: virus inhibition ratio (%) of each plasma dilution = (virus control V-test sample T)/(virus control V-cell control C) × 100%; use GraphPadPrism to take the log10 value of the dilution, and then select the model log (inhibitor) vs. Normalized response--Variable slope to calculate the ID50 value (50% inhibitory dose).

The results are shown in Table 3, which show that the antibody has a neutralizing effect on a total of 9 subtypes of the 6 subtypes of HIV-1 virus.

Table 3. Antibody neutralization results for HIV-1 virus

Claims (11)

1. A monoclonal antibody against HIV-1gp120, or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region comprising: CDR-H1 shown in the amino acid sequence of SEQ ID NO.1, CDR-H2 shown in the amino acid sequence of SEQ ID NO.2 and CDR-H3 shown in the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises a light chain complementarity determining region comprising: CDR-L1 shown in the amino acid sequence of SEQ ID NO.5, CDR-L2 having the amino acid sequence AAS, and CDR-L3 shown in the amino acid sequence of SEQ ID NO. 6.

2. The monoclonal antibody or antigen-binding fragment thereof against HIV-1gp120 according to claim 1, wherein the amino acid sequence of the heavy chain variable region of said antibody is shown in SEQ ID No. 4; the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7.

3. The monoclonal antibody or antigen-binding fragment thereof against HIV-1gp120 according to claim 1 or 2, wherein the nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof is shown in SEQ ID No.11 and/or SEQ ID No. 14.

4. The anti-HIV-1 gp120 monoclonal antibody, or antigen-binding fragment thereof, according to claim 1, wherein the monoclonal antibody is selected from one or more of lgG, lgA, lgM, lgE and lgD.

5. The monoclonal antibody or antigen-binding fragment thereof against HIV-1gp120 according to claim 1, wherein the antigen-binding fragment is selected from one or more of Fab fragments, fab ' fragments, F (ab ') ₂ fragments, fd ' fragments, fv fragments, scFv fragments, ds-scFv fragments, dAb fragments, single chain fragments, bivalent antibodies and linear antibodies.

6. A recombinant protein comprising: the anti-HIV-1 gp120 monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-5; preferably, the recombinant protein further comprises an optional tag sequence that facilitates expression and/or purification.

7. A biological material associated with the monoclonal antibody against HIV-1gp120 or antigen binding fragment thereof according to any one of claims 1-5 or the recombinant protein according to claim 6, wherein the biological material comprises at least one of a 1) -a 16):

a1 A nucleic acid molecule encoding the anti-HIV-1 gp120 monoclonal antibody of any one of claims 1-5, or an antigen-binding fragment thereof, or the recombinant protein of claim 6;

a2 An expression cassette comprising a 1) the nucleic acid molecule;

a3 A vector comprising a 1) the nucleic acid molecule;

a4 A vector comprising a 2) the expression cassette;

a5 A transgenic cell line comprising a 1) said nucleic acid molecule;

a6 A transgenic cell line comprising a 2) said expression cassette;

a7 A transgenic cell line comprising a 3) the vector;

a8 A transgenic cell line comprising a 4) the vector;

a9 A) a microorganism comprising a 1) said nucleic acid molecule;

a10 A microorganism comprising the expression cassette of a 2);

a11 A) a microorganism comprising a 3) said vector;

a12 A) a microorganism comprising a 4) said vector;

a13 A) a virus comprising a nucleic acid molecule as described in a 1);

a14 A virus comprising a 2) said expression cassette;

a15 A virus comprising a 3) the vector;

a16 A virus comprising the vector of a 4).

8. An immunoconjugate comprising the monoclonal antibody or antigen-binding fragment thereof, directed against HIV-1gp120 of any one of claims 1-5 and/or the recombinant protein of claim 6; a coupling moiety, and a coupling moiety,

Further, the coupling moiety comprises at least one of a detectable label, a drug, a toxin, an electron dense label, biotin/avidin, a spin label, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, and a viral coat protein.

9. Use of the monoclonal antibody or antigen binding fragment thereof against HIV-1gp120 of any one of claims 1-5, the recombinant protein of claim 6, the biomaterial of claim 7 and/or the immunoconjugate of claim 8 for the preparation of a product.

10. The use according to claim 9, wherein the product comprises at least one of a pharmaceutical composition, a reagent, a test plate, a kit, a test chip;

further, the pharmaceutical composition has at least one of the functions b 1) -b 2):

b1 Preventing and/or treating HIV viral infection;

b2 Preventing and/or treating diseases associated with HIV virus;

Further, the reagent, assay plate, assay chip or kit has at least one of the functions c 1) -c 2): c1 Detecting HIV virus;

c2 Diagnosing a disease associated with HIV viral infection.

11. A product comprising at least one of d 1) -d 4):

d1 A monoclonal antibody or antigen-binding fragment thereof against HIV-1gp120 according to any one of claims 1-5 of the invention;

d2 A recombinant protein according to claim 6 of the present invention;

d3 A biomaterial according to claim 7 of the present invention;

d4 The immunoconjugate of claim 8; the product is at least one of a pharmaceutical composition, a reagent, a detection plate, a kit and a detection chip,

Further, the reagent, assay plate, assay chip or kit has at least one of the functions e 1) -e 2): e1 Detecting HIV virus;

e2 Diagnosing a disease associated with HIV viral infection;

further, the pharmaceutical composition has at least one of the functions f 1) -f 2):

f1 Preventing and/or treating HIV viral infection;

f2 Preventing and/or treating diseases associated with HIV virus;

further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier;

Further, the pharmaceutical composition may further comprise other medicaments for preventing and/or treating an orthopoxvirus infection and/or a disease associated with an orthopoxvirus infection.