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

Abstract

The invention belongs to the technical fields of immunology and pathogenic biology, and particularly discloses an anti-HIV-1 gp120 antibody or an antigen binding fragment thereof and application thereof. The heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No.4 and the light chain variable region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 7. The monoclonal antibody provided by the invention has specific binding capacity with natural HIV-1gp120, can be applied to HIV-1 virus detection, can also be applied to the research of different levels of distribution and action mechanisms of HIV-1 virus in cells, organs and the like, and has good application prospect.

Description

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

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to an anti-HIV-1 gp120 antibody or an antigen binding fragment thereof and application thereof.

Background

Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is the causative agent of acquired immunodeficiency syndrome, which causes AIDs (Acquired Immunodefieiency Syndrome, AIDs). HIV includes both HIV-1 and HIV-2 types, which differ at the gene level by >55%. Of these, HTV-1 is more virulent and infectious, and most cases of AIDS worldwide are caused by HIV-l infection. HIV-2 is less replicative and pathogenic than HIV-1, and cases caused by its infection are mainly present in the western Africa and are fewer in number. Thus, the main research goal worldwide is currently HIV-1. The envelope glycoprotein (env) gene of HIV-1 type codes gp160 protein to be further processed into gp120 and gp41, which has stronger antigenicity, and the receptor binding site on gp120 is a potential target for treating HIV.

The types of HIV epidemic strains in China are increased compared with the third epidemiological investigation, the diversity and the complexity are also in an ascending trend, and a large number of novel Circulating Recombinant Strains (CRFs) are emerged in infected people, and more than 18 HIV Circulating Recombinant Strains (CRFs) are spread in China. The high complexity of HIV-1 epidemic highlights the serious challenges faced by designing measures that are effective in preventing HIV transmission. At present, china is the country with the most infection subtype and the most recombination type, and the main popular HIV strains are CRF_07BC, CRF_08BC, CRF_01AE and B subtype from Thailand, and the infected people of the epidemic strains account for 89.3 percent of the total infected people of the HIV-1 in China. In the face of such complex HIV epidemic situation in China, there is a need to find an effective treatment method aiming at the dominant epidemic strain of HIV in China, and the situation of 'anti-AIDS' is indistinct.

Methods of treatment for HIV mainly include conventional therapies and emerging therapies, with CAR-T therapies and antibody therapies being one of the most advanced methods in the field of HIV treatment. Antibodies function primarily through two aspects. In one aspect, antibodies with neutralizing activity can block viral infection by binding to viral envelope proteins, blocking binding of the virus to cellular receptors. On the other hand, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) recruit immune cells and molecules such as macrophages or complement to eliminate free virus and infected cells.

To date, there are no specific drugs or vaccines for treating or preventing AIDS in humans, but studies have demonstrated that antiviral antibodies can exert antiviral effects by a single or multiple mechanisms, and can include viral infection and any stage of the replicative cycle. The intensive research on neutralizing virus mechanism of anti-virus antibody can clarify the basic mechanism of the antibody for inhibiting virus; on one hand, key steps of the virus life cycle process can be found, so that a hint is provided for the development of antiviral targeting new drugs, cocktail drugs and high-efficiency vaccines, and hope is brought for the treatment of AIDS. Many antibodies are derived from murine sources and other animal sources, and therefore, humanization is required, and there is still an immunogenicity problem in therapy, so that development of antibodies of fully human origin is currently being expected by scientists.

Disclosure of Invention

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

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

It is a further object of the present invention to provide medicaments and means for detecting or treating HIV-1 viral infections.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

In a first aspect the invention provides an anti-HIV-1 gp120 antibody comprising: 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.

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

In a second aspect of the invention, there is provided a recombinant protein comprising: the antibody or antigen-binding fragment thereof of the first aspect of the invention.

Further, the recombinant protein also includes an optional tag sequence that facilitates expression and/or purification.

Further, the tag sequence is selected from at least one of the following group: his tag, GGGS sequence, FLAG tag, etc.

In a third aspect of the invention there is provided a biological material associated with an antibody or antigen-binding fragment thereof of the first aspect of the invention, or a recombinant protein of the second aspect of the invention, the biological material comprising at least one of a 1) to a 16):

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

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).

Further, the transgenic cell line does not comprise propagation material.

Further, the nucleic acid molecule encoding the antibody or antigen binding fragment thereof according to the first aspect of the invention comprises a nucleic acid molecule encoding the heavy chain of the antibody or antigen binding fragment thereof according to the first aspect of the invention and a nucleic acid molecule encoding the light chain of the antibody or antigen binding fragment thereof according to the first aspect of the invention.

In a fourth aspect of the invention, there is provided a conjugate comprising: at least one of an antibody or antigen-binding fragment thereof of the first aspect of the invention and a recombinant protein of the second aspect of the invention; 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. Preferably, the detectable label is a fluorescent or luminescent label.

Further, the medicine is other medicines for preventing and/or treating HIV virus infection and/or diseases related to HIV virus infection.

In a fifth aspect of the invention there is provided the use of an antibody or antigen-binding fragment thereof of the first aspect of the invention, a recombinant protein of the second aspect, a biomaterial of the third aspect, and/or a conjugate of the fourth aspect in the manufacture of a product;

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

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

b1 Preventing and/or treating HIV-1 virus infection;

b2 Preventing and/or treating diseases associated with HIV-1 virus.

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

c1 Detecting HIV-1 virus;

c2 Diagnosing a disease associated with HIV-1 virus infection.

In a sixth aspect of the invention, there is provided a product comprising at least one of d 1) -d 4):

d1 An antibody or antigen-binding fragment thereof of the first aspect of the invention;

d2 A recombinant protein according to the second aspect of the invention;

d3 A biomaterial according to the third aspect of the invention;

d4 A conjugate according to the fourth aspect of the invention.

Further, the product comprises at least one of a pharmaceutical composition, a reagent, a test plate, a kit, a test chip, the reagent, the test plate, the test chip, or the kit having at least one of the functions of e 1) -e 2):

e1 Detecting HIV-1 virus;

e2 Diagnosing a disease associated with HIV-1 virus infection.

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

f1 Preventing and/or treating HIV-1 virus infection;

f2 Preventing and/or treating diseases associated with HIV-1 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.

In a seventh aspect of the invention there is provided a method of producing an antibody or antigen-binding fragment thereof according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention by culturing a transgenic cell line according to the third aspect of the invention.

In an eighth aspect of the invention, there is provided a method of detecting the presence or level of HIV virus in a sample comprising: the antibody or antigen-binding fragment thereof of the first aspect of the invention, the recombinant protein of the second aspect, the related biological material of the third aspect and/or the conjugate of the fourth aspect are used.

Further, the method may be used for diagnostic purposes (e.g., the sample is a sample from a patient), or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

In a ninth aspect of the invention, there is provided a method of diagnosing whether a subject is infected with an HIV virus or has a disease associated with an HIV virus, comprising: detecting the presence of HIV virus in a sample from the subject using an antibody or antigen binding fragment thereof of the first aspect, a recombinant protein of the second aspect, a related biological material of the third aspect and/or a conjugate of the fourth aspect of the invention.

Further, the sample includes, but is not limited to, fecal matter from a subject (e.g., mammal, preferably human), oral or nasal secretions, alveolar lavage, and the like.

In a tenth aspect of the invention, there is provided a method for neutralising the virulence of an HIV virus in a sample comprising: contacting a sample comprising HIV virus with a pharmaceutical composition according to the sixth aspect of the invention.

Further, the methods may be used for therapeutic purposes, or for non-therapeutic purposes (e.g., the sample is a cell sample, not a patient or a sample from a patient).

In an eleventh aspect of the invention, there is provided a method for preventing or treating an HIV viral infection or a disease associated with an HIV viral infection in a subject, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of the pharmaceutical composition of the sixth aspect of the invention.

Preferably, the subject is a mammal, e.g., human, mouse.

Preferably, the pharmaceutical composition of the invention may 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.

Drawings

FIG. 1 shows SDS-PAGE results of purified monoclonal antibody 5.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, medical microbiology, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related 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 whole-length immunoglobulin molecules formed by recombinant processing of immunoglobulin gene fragments, as well as antibody fragments. It is typically 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 kappa and lambda 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 linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises 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 (CH 1, CH2 and CH 3). 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 one domain CL. The crystallizable fragment (Fc fragment) corresponds to the CH2 and CH3 functional regions of an antibody, and crystals can be formed, which have no antigen binding activity but have the functions of fixing complement, binding Fc receptor, and the like. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies that highly specifically recognize and bind an epitope or epitope. This is in contrast to polyclonal antibodies, which generally include different antibodies that recognize and bind to different antigenic determinants.

As used herein, "neutralizing antibody" refers to an antibody or antibody fragment that is capable of clearing or significantly reducing the virulence of a pathogen of interest.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the maturation of virus-infected cells and/or virus progeny and/or the release of virus progeny, and an antibody or antibody fragment having neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

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

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby 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, a prokaryotic cell such as e.g. e.coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. 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 in which an antigen or antibody is bound to a solid support surface, and the specific binding of the antigen-antibody and the enzyme labeled on the antibody or antigen are used to catalyze a color reaction of a specific substrate to effect detection of a target.

As used herein, the term "IC ₅₀" refers to the half-inhibitory concentration of the antagonist being measured. In this context, the concentration of antibody at which the antibody neutralises virus to 50% of maximum effect in an in vitro neutralisation assay is shown.

In order to obtain a neutralizing antibody with a protective effect, a single B cell antibody screening technology is adopted, a specific antigen is subjected to fluorescent marking by a flow cell sorting method, a single target B cell is screened out by utilizing the principle that the antigen can be specifically combined with a B cell surface BCR, a heavy chain gene and a light chain gene of the antibody are obtained by a PCR amplification method, the antibody gene is constructed on an expression vector, and the monoclonal antibody is transfected into eukaryotic cells to express the monoclonal antibody. The inventor firstly obtains peripheral blood of a patient infected by HIV virus, separates PBMC, reacts the PBMC with gp120 antigen and carries out fluorescent marking, then sorts single cells to a 96-well plate which is added with cell lysate in advance through a flow cell sorter, and then carries out RT-PCR and PCR amplification to obtain the coded antibody variable region sequence. Further, the antibody variable region is connected and transformed with a carrier, the obtained product is transfected into cells for eukaryotic expression to obtain the antibody, and the antibody is captured through a Protein A/G prefabricated gravity column. A series of functional assays were performed on the antibodies. The results show that the antibodies are capable of specifically binding HIV-1gp120.

The invention will now be described in further detail with reference to the drawings and examples.

The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.

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

1. Sorting of mature B cells

(1) Collecting peripheral blood of a patient infected with HIV virus, and isolating PBMC using lymphocyte separation fluid; freezing in a liquid nitrogen tank for standby;

(2) Taking out the PBMCs cell cryopreservation tube from the liquid nitrogen tank, rapidly putting the PBMCs cell cryopreservation tube into a water bath at 37 ℃ and shaking the PBMCs cell cryopreservation tube from time to time, completely thawing the PBMCs cell cryopreservation tube within 1 minute, taking out the PBMCs cell cryopreservation tube under the sterile condition, putting the PBMCs cell cryopreservation tube into PBS, blowing and washing the PBMCs cell cryopreservation tube;

(3) Centrifuging at 1000r/min for 10min, removing supernatant, adding 500 μ LPBS to blow cells, adding 8mL PBS, centrifuging at 1000r/min for 10min, washing PMBCs cells, repeatedly cleaning twice, counting cells with a cell counter, wherein the number of PBMC cells is more than 10-6, and finally re-suspending PBMC cells to 100 μL;

(4) Taking the re-suspended PBMC cells, adding His-tagged antigen, and incubating on ice for 30min, wherein the final concentration is 400nM (1 mg/mL/45 kDa=20 mu M);

(5) Pre-cooling to 4deg.C, centrifuging at 300g for 5min, discarding supernatant, adding 1ml PBS, washing twice, and re-suspending to 100 μL with PBS;

(6) The antibodies were added and incubated on ice for 30min, the specific antibody names, properties and amounts used are given in the following table:

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

(7) Adding 1ml PBS, washing twice, centrifuging at 4 ℃ for 5min, discarding the supernatant, and re-suspending to 1000 mu L with PBS;

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

2. Antibody gene amplification and linear expression vector construction

Antibody gene amplification: and (3) carrying out RT-PCR on the mature B cells obtained by sorting in a 96-well plate to obtain cDNA, and carrying out nested PCR by taking the cDNA as a template. The PCR products were identified by 1% agarose gel electrophoresis, positively identified were recovered with agarose gel recovery kit, and the PCR products were sequenced.

Construction of a linear expression vector: the amplified heavy and light chain genes are respectively connected with pCAGGS expression vector with light and heavy chain constant region sequence through homologous recombination under the catalysis of enzyme, the mixture is reacted for 5min at 65 ℃, then is placed on ice for cooling, then is added into DH5 alpha competent cells for conversion, shaking table activation is carried out for 1h at 37 ℃ and centrifugation is carried out for 5min at 5000rpm, 100 mu l of supernatant is left in sediment, the rest supernatant is abandoned, 50 mu l of supernatant is taken after the suspension is re-suspended, the supernatant is coated on LB agar plates with ampicillin resistance, after the supernatant grows for 12-16h, single colony is taken for colony PCR and the product is sequenced, and the comparison with the PCR product sequence is carried out, if the supernatant is consistent, the success is indicated.

Specific sequences of the antibody light and heavy chains are shown in table 1.

TABLE 1 information on sequences

3. Expression purification of antibodies

The day before the experiment, the 293T cells are plated with 6-hole cell plates, when the cells grow full the next day, each hole cell is transfected with vector plasmid with antibody light and heavy chain genes by PEI, each hole 6-hole plate of the light and heavy chain is transfected with 2 mug plasmid, cell supernatant is collected once after 3 days of transfection, cell supernatant is collected once after 7 days, the specific binding activity of the cell supernatant to antigen is firstly measured by ELISA experiment, and the antibody with the antigen specific binding activity is screened in a preliminary step.

The light chain and heavy chain plasmids are co-transfected into an Expi293F cell by PEI for large-scale expression, then the antibody with binding activity is purified by a Protein A affinity chromatography column through a Protein A/G prefabricated gravity column, a neutralizing monoclonal antibody targeting HIV-1gp120 is found after a large amount of experimental researches, and SDS-PAGE results of FIG. 1 show that a high-purity monoclonal antibody is obtained, wherein the molecular weight of the heavy chain and the light chain is respectively about 50kDa and about 25 kDa.

4. Determination of antibody and antigen binding Activity

(1) Coating: diluting gp120 antigen with PBS buffer until 100ng antigen is added to each well, and incubating for 2h at 37 ℃;

(2) Closing: 5% nonfat dry milk diluted with PBS, blocking at 37deg.C for 2h or overnight blocking at 4deg.C; after the completion, the liquid is discarded, PBST is cleaned for 5 times, and the liquid is beaten dry;

(3) An antibody: adding an antibody to be detected, taking cell supernatant without plasmid transfection as negative control, taking PBS as blank control, placing in a 37 ℃ incubator for incubation for 30min, then washing for 5 times by using PBST, and drying by beating;

(4) And (2) secondary antibody: adding a sealing liquid 1:5000 diluted HRP marked goat anti-human IgG is placed in a 37 ℃ incubator for incubation for 30min, then PBST is used for cleaning for 5 times, and the goat anti-human IgG is patted dry;

(5) Color development: 50 μl of each of the color developing solution A and the color developing solution B was mixed and put into the well for 15min to develop color in dark place

(6) And (3) terminating: the reaction was terminated by adding 50. Mu.l of a termination solution, and absorbance measurement was performed at 450nm by an ELISA reader.

As a result, as shown in Table 2, antibody No. 5 (indicated by the bold portions of columns 1 to 8) was able to specifically bind HIV-1 virus protein gp120, while the negative control (indicated by the bold portions of columns 17 to 20) was not specifically bound, and the positive control (indicated by the bold portions of columns H-23 plasma) was specifically bound.

TABLE 2 ELISA results

5. Antibody neutralizing HIV virus ability assessment

Culture medium was previously added to a 96-well plate, 180. Mu.L of A1-H1, 110. Mu.L of A3-H9 and C10-F10, and 220. Mu.L of C11-F11 were added. 20 mu L of sample plasma is added to A1-H1, and after mixing evenly for 30 hours, 165 mu L is transferred to A2-H2, and then 55 mu L is sequentially transferred for 3 times dilution, and total gradient is 8. The pseudovirus is diluted, and virus liquid is prepared according to 1 mu L of HIV-1 pseudovirus stock solution and 109 mu L of culture medium in each hole. Adding 110 mu L of virus liquid into the sample hole and the virus control, and uniformly mixing under 3 conditions; placing the sample liquid, the virus control and the cell control in an incubator, incubating for 1h at 37 ℃ and transferring the incubated sample liquid, the virus control and the cell control into a 96 white board, wherein 100 mu L of the sample liquid, the virus control and the cell control are repeated in each hole;

TZM-bl cells were digested, resuspended at a cell concentration of 2.5X10 ⁵/mL, and DEAE-dextran hydrochloride (12.5 mg/mL) was added to a concentration of 25. Mu.g/mL; to 96 Kong Baiban were added 100. Mu.L of the cell suspension (i.e., 2.5X10 ⁴ cells), and the final DEAE-dextran hydrochloride concentration was 12.5. Mu.g/mL; culturing at 37 ℃ with 5% CO ₂ for 48h; detecting fluorescence value: taking out 96 Kong Baiban, and discarding the culture supernatant; 100 mu L of Bright-Lite Luciferase is added to each well, and the mixture is uniformly mixed after incubation for 2min at room temperature; detecting the luminescence value of each hole on a fluorescence detector, and calculating the inhibition rate: the ratio of viral inhibition (%) = (viral control V-test sample T)/(viral control V-cell control C) ×100% for each dilution of plasma; the ID50 value was calculated by taking the log10 value from GRAPHPAD PRISM of the dilution and then selecting the model log (inhibitor) vs. normalized response- -Variable slope (50%inhibitory dose).

The results are shown in Table 3, where the antibodies have neutralizing effect against 9 total subtype viruses out of the 6 subtypes of HIV-1 virus.

TABLE 3 antibody neutralization of HIV-1 Virus results

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.