CN115716872B - anti-HIV antibody, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an anti-HIV antibody, a preparation method and application thereof, wherein the antibody comprises heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of amino acid sequences shown as SEQ ID NO.1, 2 and 3 respectively; and light chain complementarity determining regions CDR1, CDR2, CDR3 of the amino acid sequences shown in SEQ ID NO.5, 6, 7, respectively. The invention also discloses a nucleic acid molecule for encoding the antibody, a vector containing the nucleic acid molecule, a recombinant cell, a pharmaceutical composition and a vaccine. Methods of producing HIV antibodies are also disclosed.

Description

anti-HIV antibody, and preparation method and application thereof

Technical Field

The invention belongs to the fields of cell immunology and genetic engineering, and relates to an anti-HIV antibody, a preparation method and application thereof.

Background

Human immunodeficiency virus (Human immunodeficiency virus, HIV) is a virus responsible for the human immune system deficiency syndrome-AIDS (Acquried immunodeficiency syndrome, AIDS). HIV infection attacks human CD4 ⁺ T lymphocytes, the human immune system is destroyed, the occurrence of organic infection and malignant tumor is easy, the death rate is high, and no thorough cure method exists at present.

HIV belongs to the genus lentivirus of the family retrovirus, and is divided into two subtypes: type 1 and type 2. Of these, HIV-2 is mainly prevalent in western Europe, north America and western Africa, where the strain is less virulent. HIV-1 is widely existed worldwide, and is a main virus strain with strong toxicity for epidemic AIDS in the world. HIV is the most terrible pathogenic microorganism experienced so far in vaccine development and drug development processes, and although a great deal of basic research of biology is carried out by researchers in the field in the process of anti-HIV, the characteristics of high mutation, immune escape, short window period and the like of HIV relative to other pathogens bring great difficulty to the treatment of anti-HIV and the development work of vaccines.

After HIV infection of the body, specific antibody responses can be induced. However, some of these antibodies only have the ability to bind to the virus, which after binding still has the ability to infect host cells, we call the binding antibody. While another part of the antibodies, after binding to the virus, can inactivate the virus, i.e. have the ability to neutralize the virus, we call neutralizing antibodies (Neutralizing Anubody, NAb). Screening and identification of broad-spectrum neutralizing antibodies and recognition epitopes thereof provides an important basis for development of AIDS vaccines by utilizing reverse vaccinology technology, and an infected person capable of generating the broad-spectrum neutralizing antibodies can be used as a model for potentially inducing similar neutralizing antibodies through vaccines, and different natural membrane proteins are used for sequential inoculation by simulating the co-evolution and affinity maturation process of the broad-spectrum neutralizing antibodies with viruses in organisms so as to combine and activate unmutated common progenitor cells of the broad-spectrum neutralizing antibodies, promote differentiation and maturation of specific precursor B cells into plasma cells secreting the broad-spectrum neutralizing antibodies, and realize the aim of generating the broad-spectrum neutralizing antibodies through vaccine inoculation induction.

The prevalence of aids has taken over 3400 tens of thousands of lives since the eighties of the last century. In 2017, 3690 thousands of people worldwide were infected with HIV, with only 59% of HIV-infected people receiving antiretroviral therapy (ART) treatment, as counted by the World Health Organization (WHO). HIV has so far remained one of the greatest public health challenges worldwide, and there is an urgent need to study methods for detecting HIV in depth. Currently, P24 antigen detection is capable of detecting soluble P24 antigen in the blood after the virus has begun to replicate, but is prone to false positives. Therefore, a positive result must be confirmed by a neutralization test, and the result can be used as an auxiliary diagnosis basis for HIV infection. HIV p24 antigen detection was negative, indicating no response in this assay, and HIV infection cannot be excluded. The virus nucleic acid detection method has high sensitivity, however, due to the diversity of HIV genes, no set of primers can cover all HIV sequences, and the detection sensitivity is limited. In addition, the existing virus nucleic acid is expensive in detection instrument and detection reagent, or complex in operation, has high requirements on operators, is difficult to popularize in a common laboratory, is not suitable for rapid detection of a large number of patients, and is also not suitable for wide clinical application.

Therefore, to improve the sensitivity and specificity of detection, shorten the window period, and simply, quickly and reduce the cost has become a requirement and direction of development of HIV detection technology, and many researches are being conducted to find alternative technologies for HIV virus detection.

Disclosure of Invention

It is an object of the present invention to provide a broad-spectrum neutralizing antibody against HIV.

It is a second object of the present invention to provide a method for screening for anti-HIV broad-spectrum neutralizing antibodies.

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

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 broad-spectrum neutralizing antibody comprising heavy chain complementarity determining regions CDR1, CDR2 and CDR3, the sequences of said heavy chain complementarity determining regions CDR1, CDR2 and CDR3 being shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, respectively; and light chain complementarity determining regions CDR1, CDR2 and CDR3, wherein the sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7.

Further, the antibody comprises:

(a) A heavy chain variable region sequence having at least 90%, preferably 95%, sequence identity to the amino acid sequence of SEQ ID NO. 4;

(b) A light chain variable region sequence having at least 90%, preferably 95%, sequence identity to the amino acid sequence of SEQ ID No. 8; or alternatively

(c) A heavy chain variable region sequence as in (a) and a light chain variable region sequence as in (b).

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. 8.

Further, the antibody comprises all or part of an antibody heavy chain variable region and/or an antibody light chain constant region.

Further, the antibody may include a complete antibody molecule having full length heavy and light chains or binding fragments thereof and may be, but is not limited to, fab, modified Fab, fab ', modified F (ab') 2, fv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, bispecific-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above.

In a second aspect the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof according to the first aspect of the invention.

Further, the antibody-encoding nucleic acid molecules may be operably linked to one or more regulatory elements, such as promoters and enhancers, that allow expression of the nucleotide sequence in the intended target cell (e.g., a cell genetically modified to synthesize the encoded antibody).

Further, the nucleic acid molecule comprises: deoxyribonucleic acid (DNA), which includes, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), in particular messenger RNA (mRNA); synthetic forms of DNA or RNA; and hybrid polymers comprising two or more of these molecules.

Further, the nucleic acid molecule may be linear or circular.

Further, the nucleic acid molecules include sense and antisense strands, as well as single and double stranded forms.

Further, the nucleic acid molecule may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases with derivatized sugar, phosphate linked or chemically modified residues.

Further, nucleic acid molecules also include DNA and RNA molecules suitable for direct expression of vectors of the antibodies of the invention in vitro and/or in vivo, e.g., in a host or patient. Vectors for such DNA (e.g., cDNA) or RNA (e.g., mRNA) molecules may be unmodified or modified.

In a third aspect the invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

Further, the vectors include, but are not limited to, linear polynucleotides, plasmids, and viral vectors.

Further, the viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors.

In a fourth aspect, the invention provides a recombinant cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention.

Further, the recombinant cells include prokaryotic cells and eukaryotic cells.

Further, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia.

Further, the bacteria include Escherichia coli, bacillus subtilis, salmonella typhimurium, pseudomonas, streptomyces, and Staphylococcus.

Further, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells.

Further, the mammalian cells include human cells, and may specifically include cells obtained from a human body or commercially available finished cell lines: such as RPE1 cells, SW480 cells, u87MG cells, HOS cells, C8166 cells, MT-4 cells, molt-4 cells, heLa cells, HT1080 cells, 293 cells, TE671 cells, etc.

In a fifth aspect the invention provides a pharmaceutical composition comprising an antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a recombinant cell according to the fourth aspect of the invention.

Further, the antibodies may be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable carriers, and may be formulated as preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

Further, specific examples of some substances which can be pharmaceutically acceptable carriers or components thereof are saccharides such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; phosphate buffer, and the like.

Further, the pharmaceutical compositions of the present invention may be administered orally, by injection, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or by an implanted reservoir.

Further, the pharmaceutical compositions of the present invention may be administered orally in any oral dosage form, including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, common carriers include lactose and corn starch. A lubricant such as magnesium stearate is also typically added. Suitable diluents for oral administration in capsule form include lactose and anhydrous cornstarch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in the oil phase and combined with emulsifying and/or suspending agents. If desired, some sweetener and/or flavoring and/or coloring agents may be added.

Further, the pharmaceutical compositions of the invention may be administered to a host in any convenient manner that is capable of producing the desired therapeutic or diagnostic effect. Thus, the agents may be incorporated into a variety of formulations for therapeutic administration.

Further, the pharmaceutical composition further comprises a second therapeutic agent.

Further, the second therapeutic agent is an antiviral agent.

Further, the antiviral agent includes: non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, integrase inhibitors.

Further, the non-nucleoside reverse transcriptase inhibitors include nevirapine, delavirdine, efavirenz, itravirenz and rilpivirine.

Further, the protease in the protease inhibitor can be leupeptin, antalgin, chymotrypsin, elastase aldehyde, pepstatin, phosphoamidin and the like which are separated from actinomycete fermentation liquor, and can respectively inhibit various proteases such as trypsin, papain, chymotrypsin, elastase, pepsin, metalloproteinase and the like.

Further, the protease inhibitor comprises indinavir, saquinavir, ritonavir, nelfinavir, amprenavir.

Further, the fusion inhibitor comprises a polypeptide HIV-1 fusion inhibitor.

Further, the polypeptide HIV-1 fusion inhibitor comprises a C peptide fusion inhibitor, an N peptide fusion inhibitor, a fusion inhibitor taking gp41 Fusion Peptide (FP) as a target and an HIV-1 fusion inhibitor polypeptide modified by unnatural amino acids.

Further, the C peptide fusion inhibitor comprises T-20.

Further, the N-peptide fusion inhibitor comprises DP-107.

Further, the fusion inhibitor targeting gp41 Fusion Peptide (FP) comprises virp.

Further, the unnatural amino acid modified HIV-1 fusion inhibitor polypeptide comprises a D-type loop 18 peptide.

Further, the integrase inhibitors include Raltegravir (RAL), ivermectin Lei Wei (EVG), dortevir (DTG), and Bictegravir (BIC).

In a sixth aspect the invention provides an HIV detection product comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention.

Any test product comprising an antibody of the invention that is capable of detecting the level of HIV expression is included within the scope of the invention.

Further, the detection products include, but are not limited to, detection kits, chips, or high throughput sequencing platforms.

Further, the kit includes a chemiluminescent solution.

Further, the chemiluminescent liquid comprises an enzymatic reaction luminescent agent, a direct chemiluminescent agent and an electrochemical luminescent agent.

Further, enzymatically-reacting luminescences include enzymatically-reacting enzymes and luminescent substrates of enzymatically-reacting enzymes.

Further, enzymatic reaction enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, lysozyme, malate dehydrogenase.

Further, the luminescent substrates of the enzyme reaction enzymes include luminescent substrates of horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, lysozyme and malate dehydrogenase.

Further, the luminescent substrate of horseradish peroxidase comprises luminol or a derivative thereof, and p-hydroxyphenylacetic acid.

Further, the luminescent substrate of the alkaline phosphatase comprises AMPPD, 4-methylumbelliferone phosphate.

Further, the direct chemiluminescent agent includes an agent that does not require catalysis by an enzyme, and that emits light only by changing the pH of the solution.

Further, the electrochemiluminescence agent includes a substance that emits light by performing an electrochemical reaction on the electrode surface.

In a seventh aspect, the invention provides a method for producing an antibody according to the first aspect of the invention, the method comprising: culturing the recombinant cell of the fourth aspect of the invention.

Further, the recombinant cells are cultured in a medium under conditions that allow expression of the polypeptide encoded by the vector and assembly of the antibody or fragment thereof, and the antibody is purified from the recombinant cells or the culture medium of the recombinant cells.

In an eighth aspect the invention provides a vaccine comprising an epitope which specifically binds to an antibody according to the first aspect of the invention.

Further, the vaccine may be a DNA-based vaccine, an RNA-based vaccine, or a virus-transduction-based vaccine.

Further, the vaccine may be prophylactic or therapeutic.

A ninth aspect of the invention provides the use of any one of the following:

(a) Use of an antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a recombinant cell according to the fourth aspect of the invention, a pharmaceutical composition according to the fifth aspect of the invention for the preparation of a medicament for the treatment of HIV infection or an HIV-related disease;

(b) Use of an antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a recombinant cell according to the fourth aspect of the invention, a detection product according to the sixth aspect of the invention for the manufacture of a product for the detection of HIV infection or diagnosis, prognosis or therapy monitoring of an HIV-related disease;

(c) Use of an antibody according to the first aspect of the invention in an HIV immunohistochemical assay.

Further, the detection includes quantitative or qualitative detection.

Further, methods of diagnosing HIV-associated diseases typically involve obtaining a biological sample (e.g., blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy) from a patient, contacting the sample with HIV antibodies, and determining whether the antibodies preferentially bind to the sample compared to a control sample or predetermined cutoff value, thereby indicating the presence of HIV virus.

Definition of the definition

The term "Fab" fragment refers to papain digestion of an antibody to produce two identical antigen binding fragments, each having a single antigen binding site, and a residual "Fc" fragment. The F (ab') 2 fragment is pepsin treated, has two antigen binding sites and is still capable of cross-linking the antigen.

The term "Fv" is the smallest antibody fragment that contains a complete antigen recognition site and an antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity compared to the entire binding site.

The term "single chain Fv" or "sFv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that is capable of allowing the sFv to form the structure required for antigen binding.

The term "specific binding" refers to the formation of a complex of two molecules that is relatively stable under physiological conditions, specific binding being characterized by high affinity and low to medium capacity, non-specific binding generally having low affinity and medium to high capacity. In general, when the affinity constant K is higher than 10 ⁶ M ^-1 Or preferably above 10 ⁸ M ^-1 Binding is considered specific when it is. Non-specific binding is reduced, if necessary, by changing the binding conditions without substantially affecting specific binding. The above conditions are well known in the art and suitable conditions can be selected by those skilled in the art using conventional techniques. The conditions are typically concentrated with antibody concentration, solution ionic strength, temperature, time for binding, and non-related molecules (e.g., serum albumin, milk casein)Degree, etc.

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a nucleotide polymer. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. In general, nucleic acid molecules are described by a sequence of bases, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented by 5 'to 3'.

The term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto, and includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acid molecules to which they are operably linked.

The term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. Variability is concentrated in three segments in the light and heavy chain variable regions known as Complementarity Determining Regions (CDRs) or hypervariable regions. The variable regions of the natural heavy and light chains each comprise four FR regions (the more conserved portions of the variable regions) which are generally in a β -sheet configuration, joined by three CDRs which form a connecting loop, to form part of a β -sheet structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. The constant regions are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions.

The term "non-nucleoside reverse transcriptase inhibitors" refers to a class of small drug molecules that act by binding to a hydrophobic binding pocket near the polymerization site of HIV reverse transcriptase.

The term "protease inhibitor" refers broadly to a substance that binds to groups on the center of protease molecule activity, causing the protease to decrease or even disappear, but not denature the enzyme protein.

The term "integrase inhibitor" refers to a drug that inhibits integrase, i.e., inhibits the process of replication of retrovirus, blocking the integration of catalytic viral DNA with the host chromosomal DNA.

The terms "treatment" or "alleviation" are used interchangeably and refer to both therapeutic treatment and prophylactic or preventative measures; wherein the aim is to prevent or slow down (alleviate) the targeted pathological condition or disorder. Subjects in need of treatment include those already with the disorder, those prone to have the disorder, or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the invention, the patient exhibits an observable and/or measurable decrease or absence of one or more of: a reduced number of infected cells or a lack of infected cells; the percentage of total cells infected is reduced; and/or to some extent, alleviate one or more symptoms associated with a particular infection; reduced morbidity and mortality, and improvement in quality of life problems.

Drawings

FIG. 1 is a diagram of pcDNA3.1 (+) -Fc expression vector;

FIG. 2 is a diagram of the sorting of peripheral blood mature B cells of humanized GTL mice;

FIG. 3 is a graph of the detection of the auto-reactivity of BN3 antibodies (Hep-2 Elisa);

FIG. 4 is a graph showing the detection of the binding activity of BN3 antibodies to the HIV surface antigen gp 140-oligomer.

Detailed Description

Humanized GTL mice are obtained by adding CD34 to human fetal umbilical cord blood ⁺ Hematopoietic stem cells, fetal liver and thymus tissue transplantation into NOD/Scid/IL2 Rgamma ^null Human hematopoietic cells and lymphocytes were allowed to develop in vivo in the GTL mice. Since human B cells are in the developmental environment of mice, the proportion of such autoreactive/autoreactive B cell clones that may be cleared for the human body is reduced and thus more retained. Based on the above, we set up a humanized GTL mouse model, study the development condition of human B cells in GTL mice, and find that 75% of mature B cells have self/multi-reactivity and the proportion is quite high. In the case of these self/poly-reactive antibodiesDuring the course of the study, antibodies were found that were characteristic of HIV broad-spectrum neutralizing antibodies.

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 antibodies

1. Sorting of mature B cells

Buffer1: 500 ml 1 XPBS+2 ml 0.5M EDTA+25 ml 10% BSA (2 mM EDTA, 0.5%BSA)

(1) Collecting 200 μl of peripheral blood of humanized GTL mice, and adding 200 μl Buffer1;

(2) 10 volumes of ACK lysing buffer (Fisher/BioWhittaker) were added, incubated at room temperature for 10 min, and centrifuged at 1500 rpm for 10 min;

(3) Removing the supernatant, adding 10 ml Buffer1 to wash cells, and centrifuging at 1500 rpm for 10 min;

(4) The supernatant was discarded and 10. Mu.l Buffer1 was added to resuspend the cells; adding streaming antibody to mark mature B cells:

anti-CD5/FITC (UCHT2, eBioscience)

anti-CD19/PE (SJ25-C1, BD Pharmingen)

anti-CD10/APC (BC96, eBioscience)

anti-CD27/PE-Cy7 (O323, eBioscience)

anti-IgM/PE-Cy5 (G20-127, BD Pharmingen)

(5) Flow cytometry (FACS Sorter) sorted mature B cells (CD 5-CD19+CD10-CD 27-IgM+), single cells were collected into 96-well PCR plates with 4. Mu.l cell lysates placed inside;

(6) The sorted cells were placed on dry ice and stored in a-70 ℃ freezer as soon as possible.

(7) Results: as shown in fig. 2, mature B cells were obtained by sorting.

2. Identification of antibodies

(1) cDNA is synthesized by reverse transcription, and a SuperScript cube III First-Strand Synthesis System synthesis system of Invitrogen company is used;

(2) First round PCR, amplifying antibody heavy chain VH gene and light chain VL gene by using a hotspot kit of Qiagen company;

(3) Second round of nested PCR, amplifying antibody VH gene and VL gene, using a hotspot kit of Qiagen company;

(4) Ligating the VH fragment and the VL fragment of the second round PCR product into T vectors respectively, and transforming JM109 competent bacteria;

(5) Identifying positive clones containing the VH fragments and the VL fragments by a PCR method;

(6) VH and VL products from the same cell, identified as positive by PCR, were selected and assembled into single chain antibody scFv.

(7) JM109 competent bacteria were transformed by ligating scFv into pcDNA3.1 (+) -Fc expression vector containing IgG1-Fc (containing IgG1-Fc tag, FIG. 1) via SfiI and Not I cleavage sites.

(8) Positive clones containing scFv fragments were identified by PCR and sequenced to obtain scFv/pcDNA3.1 (+) -Fc clones with the correct sequence.

(9) The antibody sequences are shown in Table 1

TABLE 1 antibody sequences

3. Eukaryotic expression and affinity purification of antibodies

HEK 293T cells were transiently transfected with the correct scFv/pcDNA3.1 (+) -Fc plasmid sequenced using lipofectamine2000 (Invitrogen). 293T cells were plated in 15 cm plates containing 20 ml medium and transfected with 40. Mu.g plasmid DNA, 100. Mu.l lipofectamine2000 at 90% cells according to the instructions. After 8-10h of transfection, the liquid is changed, cell supernatant is collected 48h after liquid change, protein A affinity chromatography (Protein A sepharose CL-4B, GE Healthcare) is used for purifying antibodies (expressed antibodies contain Fc tags), and scFv-Fc antibody proteins are obtained after ultrafiltration and concentration.

Example 2 detection of antibody Properties

1. Autoreactivity detection

The autoreactivities of the antibodies were detected using a clinically standard antinuclear antibody detection kit (QUANTA Lite ™ ANA ELISA, INOVA Diagnostics, inc.) in which negative, low positive and high positive reaction samples were provided. 4E10 is an HIV broad-spectrum neutralizing antibody, also an auto/poly-reactive antibody, and we have prepared 4E10 scFv-Fc as a positive control antibody in experiments. The detection steps are as follows:

(1) Coating: the Elisa plates in the ANA kit have been antigen coated;

(2) An antibody: 50. Mu.l of scFv-Fc protein (50. Mu.g/ml) was added and reacted at room temperature for 2 hours;

(3) And (2) secondary antibody: removing liquid in the hole, washing with PBST for 3 times, adding an HRP-labeled goat anti-human IgG-Fc antibody, and reacting for 1h at room temperature;

(4) Color development: removing liquid in the hole, washing with PBST for 3 times, adding TMB substrate solution, and developing for 3 minutes at room temperature and in dark place in 100 mu l of each hole;

(5) And (3) detection: add 2N H ₂ SO ₄ 100 μl of the reaction was terminated and the detection was performed with a microplate reader at 450 nm.

(6) Results: as shown in FIG. 3, the scFv-Fc antibody (designated BN 3) showed strong positivity against nuclear reaction, almost equivalent to 4E10 scFv-Fc. Indicating that BN3 reacts with human nuclear antigen and is an autoreactive antibody.

2. Multiple reactivity detection

The multi-reactivity of antibodies is a reaction of detecting antibodies and a variety of commonly recognized antigens in nature, including human single-stranded DNA (ssDNA), human double-stranded DNA (dsDNA), recombinant human insulin (insulin), human cardiolipin (cardiolipin), bovine Serum Albumin (BSA), bacterial Lipopolysaccharide (LPS), and the like. Antibody multi-reactivity derived from humanized GTL mice was evaluated by an electrochemiluminescence-based MSD (Gaithersburg, MD, USA) platform. In the experiment, 4E10 scFv-Fc was used as positive control antibody.

(1) Coating: 384 well MSD plates were coated with ssDNA, dsDNA, insulin, cardiolipin, BSA, LPS 10 μg/ml each as a multi-reactive antigen and incubated overnight at 4 ℃;

(2) Closing: the wells were discarded, washed 3 times with PBST, 100 μl of 10% FBS was added to block the Elisa plate, and incubated for 1h at room temperature;

(3) An antibody: removing liquid in the hole, washing with PBST for 3 times, adding 5 mug/ml scFv-Fc protein 15 mug, and incubating at room temperature for 2h;

(4) And (2) secondary antibody: removing the liquid in the hole, washing with PBST for 3 times, adding 15 mu l of goat anti-human IgG-Fc antibody with a SULFO label in the amount of 1 mu g/ml, and reacting for 2 hours at room temperature;

(5) The wells were discarded and the PBST washed 3 times and the plates read at MSD Sector Imager 2400 according to the instructions.

(6) Results: BN3 reacted with ssDNA, dsDNA, insulin, cardiolipin, BSA, LPS antigen showed strong positivity, almost equivalent to 4E10 scFv-Fc, indicating that BN3 reacted with both of these antigens, a multi-reactive antibody.

3. HIV antigen binding reaction assay

Based on MSD platform technology, BN3 binding experiments with HIV surface antigen gp140-trimer were set up, and 384 well MSD plates were coated with 5. Mu.g/ml gp140-trimer (from HIV-YU2 strain) as described above. In the experiment, the 4E10 scFv-Fc was used as a positive control antibody, and antibodies showing negative and low positive reactions in the autoreactivity detection result were used as a negative control and a low positive reaction control in the experiment.

As shown in FIG. 4, BN3 and gp 140-oligomer show strong binding activity, about 15% of the binding activity of 4E10 scFv-Fc, 40 times of that of the negative control and 6.6 times of that of the low positive control, which shows that BN3 has HIV antigen binding activity and has further development value.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (9)

1. An anti-HIV broad spectrum neutralizing antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3, wherein the sequences of heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, respectively; and light chain complementarity determining regions CDR1, CDR2 and CDR3, wherein the sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7.

2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 4; the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 8.

3. A nucleic acid molecule encoding the antibody of claim 1 or 2.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A recombinant cell comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A pharmaceutical composition comprising the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, or the recombinant cell of claim 5.

7. An HIV detection product comprising the antibody of claim 1 or 2.

8. A method for producing the antibody of claim 1 or 2, comprising: culturing the recombinant cell of claim 5.

9. Use for non-diagnostic purposes of any one of the following:

(a) Use of the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, the recombinant cell of claim 5, the detection product of claim 7 for the preparation of a product for detecting HIV infection or diagnosis of an HIV-related disease;

(b) Use of the antibody of claim 1 or 2 in an HIV immunohistochemical assay.

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