Recombinant antigen for HIV antibody detection

Abstract

This invention relates to the field of immunodiagnostics and provides recombinant HIV antigens for HIV antibody detection. Specifically, it concerns recombinant HIV antigens, nucleic acid molecules, expression vectors, host cells, reagents, kits, and their use. When recombinant HIV antigen proteins are used as the labeling end, the marker activity of the recombinant HIV antigen can be significantly improved, and nonspecific immunobinding activity can be reduced, thereby increasing sensitivity.

Description

Cross-reference to Related Applications

[0001] This application claims priority to a Chinese patent application with application number 2023107487351, titled "Recombinant Antigen for HIV Antibody Detection", filed with the China National Intellectual Property Administration on June 21, 2023, and incorporates the entire content thereof by reference into this application.

Technical Field

[0002] This disclosure belongs to the technical field of immunodiagnosis and relates to a recombinant antigen for HIV antibody detection.

Background Art

[0003] The following description only provides background information related to this disclosure and does not necessarily constitute prior art.

[0004] Currently, there are two types of HIV (human immunodeficiency virus), namely HIV-1 and HIV-2. HIV-1 is the main virus causing ADIS currently. According to the interspecies infection route, HIV-1 can be classified into M type, N type, O type, and P type. Among them, the M type accounts for 90% of HIV-1 virus infections. According to the infection region, the M type can be classified into subtypes A, B, C, D, E, F, G, H, I, J, K, and L. HIV-2 has weaker infectivity and virulence compared to HIV-1. Among the currently known eight types of HIV-2, only two types, group A and group B, cause large-scale infections.

[0005] The HIV virus is a spherical particle with a diameter of 100-120 nm, consisting of two parts: a core and an envelope. The inner region is a conical core surrounded by the structural protein P24 (capsid). The core consists of an ssRNA genome (surrounded by p7 nucleocapsid), reverse transcriptase, integrase and protease, several secondary proteins, and the main core protein. A limited number of HIV envelope glycoproteins (Env) have been found on the surface of the virion, which bind to its main host receptor CD4 and its co-receptors (mainly CCR5 or CXCR4), allowing the virus to enter its target cells. Of these, the HIV-2 env gene encodes the gp160 precursor protein, which is cleaved by the host cell's furin protease in somatic cells to produce gp125 and gp36 proteins.

[0006] HIV antibody detection is one of the main methods for identifying HIV-infected patients, and the gp36 protein is the main antigenic raw material used for HIV-2 antibody detection. However, because HIV gp36 is highly hydrophobic, it is difficult to manufacture products with good detection performance. [Overview of the project] [Problems that the invention aims to solve]

[0007] This disclosure provides the gp36 protein sequence shown in Sequence ID No. 1 for HIV-2 antibody detection. [Means for solving the problem]

[0008] In some embodiments of this disclosure, an HIV-2 antibody is detected using a segment that is part of the gp36 protein sequence shown in SEQ ID NO: 1, for example, a segment that is the amino acids from position x to y of the gp36 protein, where x is an integer from 1 to 60 and y is an integer from 143 to 161.

[0009] Specifically, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 y is 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, and y is 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161.

[0010] In some embodiments of this disclosure, the HIV recombinant antigen includes a segment consisting of amino acids at positions 1 to 143 of gp36 as shown in SEQ ID NO: 2, a segment consisting of amino acids at positions 15 to 143 as shown in SEQ ID NO: 3, or a segment consisting of amino acids at positions 60 to 161 as shown in SEQ ID NO: 4.

[0011] In some embodiments of this disclosure, the cysteine ​​in the recombinant antigen is mutated to another amino acid, and in an optional embodiment, the cysteine ​​is mutated to serine.

[0012] In some embodiments of the present disclosure, the recombinant antigen further comprises a segment of another HIV protein; in an optional embodiment, the recombinant antigen further comprises a segment of HIV-1; and in an optional embodiment, the recombinant antigen further comprises a segment of HIV-1 gp41.

[0013] In some embodiments of this disclosure, the recombinant antigen further comprises a fusion partner.

[0014] Another aspect of this disclosure is the provision of HIV detection reagents / kits containing the recombinant antigen described above.

[0015] Specifically, a tracer marker or solid-phase carrier is coupled to the recombinant antigen in the reagent / kit.

[0016] Another aspect of this disclosure provides a nucleic acid molecule encoding the above-mentioned HIV recombinant antigen, a vector containing the nucleic acid molecule, and a host cell containing the vector. [Brief explanation of the drawing]

[0017] [Figure 1] The expression vector map is shown. Since the PE vector itself contains a 6×His tag at its N-terminus (other purification tags can also be used), the resulting HIV protein / recombinant protein both contain a 6-histidine tag, which can facilitate affinity purification using NI-NTA support. [Modes for carrying out the invention]

[0018] When used in the claims and / or specification with the term “including,” the words “a” or “an” can mean “one,” and can also mean “one or more,” “at least one,” and “one or more.”

[0019] As used in the claims and specification, the words “include,” “have,” or “contain” are in a comprehensive or open sense, not excluding additional or undescribed elements or steps of the method.

[0020] Throughout the application documents, the term "approximately" means that the value includes the standard deviation of the error of the apparatus or method used to measure that value.

[0021] While the disclosed terms "or" are defined as substitutes and "and / or," unless explicitly stated that substitutes alone or substitutes together are mutually exclusive, the term "or" in the claims refers to "and / or."

[0022] When detecting antibodies from a sample using an antigen reagent, the antigen fragment that is the reagent usually undergoes several technical processes; therefore, the gp36 antigen described herein may be either a full-length gp36 protein or a truncated gp36 protein. Exemplarily, the full-length gp36 protein may contain the amino acids shown in Sequence ID No. 1, or it may be a truncated gp36 protein based thereon, containing the amino acids from position X to Y of the gp36 protein, where X is an integer selected from 1 to 60 and Y is an integer selected from 143 to 161. For example, the gp36 antigen includes, without limitation, amino acids at positions 1-158, 1-153, 1-148, 1-143, 2-161, 5-158, 8-155, 11-152, 14-148, 15-143, 15-145, 15-147, 25-161, 135-161, 45-161, 50-161, 55-161, and 60-161 of gp36. For example, the gp36 antigen includes a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0023] In this disclosure, the term “antibody” is used in its broadest sense herein and refers to a protein containing an antigen-binding site, covering a variety of natural and artificial antibodies with various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, complete antibodies, and antibody fragments.

[0024] The term "fusion partner" may refer to a tag polypeptide that is fused or linked to a target protein (e.g., the HIV recombinant antigen of the present disclosure), thereby promoting soluble expression, stabilization, and / or purification of the recombinant protein. The tag can be fused or linked to the N-terminus and / or C-terminus of the target protein (optionally, via a linker or protease cleavage site). Such tags are well-known to those skilled in the art and are also described in detail in the prior art literature. For example, such tags include, but are not limited to, histidine (His) tags, glutathione-S-transferase (GST) tags, maltose-binding protein (MBP) tags, thioredoxin (Trx) tags, NusA tags, DsbA tags and DsbC tags of protein disulfide isomerase, SUMO tags, msyB tags, TF tags, initiator tags, ubiquitin tags, Myc tags, Flag tags, fluorescent protein (e.g., GFP) tags, biotin tags, avidin tags, and foldon domains.

[0025] The terms "peptide linker" and "linker peptide" refer to short-chain peptides for linking two molecules (e.g., proteins). A linker peptide can be present in the HIV chimeric antigen of the present disclosure. Usually, a polynucleotide sequence encoding the short-chain peptide is introduced between two DNA fragments encoding two target proteins to be linked (e.g., by PCR amplification or ligase), and the proteins are expressed to obtain a fusion protein, such as target protein 1-linker peptide-target protein 2, more specifically, for example, gp36 antigen-linker peptide-fusion partner, gp36 antigen-linker peptide-other HIV fragments.

[0026] The term "recombinant protein" as used in the present disclosure is a protein obtained using recombinant DNA or recombinant RNA technology and can be obtained in vivo or in vitro.

[0027] As used in this disclosure, the term “polypeptide” refers to any molecule containing three or more amino acid residues linked by peptide bonds. Polypeptides as used in this application include peptides (e.g., tripeptides, oligopeptides, etc.) and peptides that may undergo chemical modifications (e.g., glycation (peptidoglycan), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation). Polypeptides may also refer to proteins.

[0028] As used in this disclosure, the term “signal peptide” refers to a polypeptide that can be linked to a target protein and promote the expression or transport of the target protein. For example, a signal peptide may be linked to the N-terminus of a target protein, and is generally not present in mature proteins secreted by cells because it is typically cleaved and removed.

[0029] As used in this disclosure, the terms “amino acid mutation” or “nucleotide mutation” include “substitution, duplication, deletion or addition of one or more amino acids or nucleotides.” In this disclosure, the term “mutation” means a change in a nucleotide sequence or amino acid sequence. In some embodiments, “mutation” in this disclosure can be selected from “conservative mutation,” “semi-conservative mutation,” or “non-conservative mutation.” In this disclosure, the terms “non-conservative mutation” or “semi-conservative mutation” may refer to a mutation that causes loss or partial loss of protein function. The term “conservative mutation” refers to a mutation in which the normal function of the protein is maintained. A typical example of a conservative mutation is a conservative substitution.

[0030] As used in this disclosure, “conservative substitution” generally refers to the replacement of one amino acid at one or more sites in a protein. Such substitutions may be conservative. Substitutions considered to be conservative include, specifically, substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His or Lys, substitutions of Asn to Glu, Gln, Lys, His or Asp, substitutions of Asp to Asn, Glu or Gln, substitutions of Cys to Ser or Ala, substitutions of Gln to Asn, Glu, Lys, His, Asp or Arg, substitutions of Glu to Gly, Asn, Gln, Lys or Asp, substitutions of Gly to Pro, substitutions of His to Asn, Lys, Gln, Arg or Tyr, and I Examples of substitutions include substitution of le with Leu, Met, Val, or Phe; substitution of Leu with Ile, Met, Val, or Phe; substitution of Lys with Asn, Glu, Gln, His, or Arg; substitution of Met with Ile, Leu, Val, or Phe; substitution of Phe with Trp, Tyr, Met, Ile, or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe, or Trp; and substitution of Val with Met, Ile, or Leu. Conservative mutations also include natural mutations resulting from differences in the individual from which the gene originates, differences in strains, or differences in species.

[0031] In this disclosure, “sequence identity” and “identity percentage” refer to the percentage of the same (identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by: aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides; scoring the number of positions of the same nucleotide or amino acid residues in the aligned polynucleotides or polypeptides; and comparing this to the number of positions of different nucleotide or amino acid residues in the aligned polynucleotides or polypeptides. Polynucleotides differ at one position, for example, due to the inclusion of different nucleotides (i.e., substitution or mutation) or the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides differ at one position, for example, due to the inclusion of different amino acids (i.e., substitution or mutation) or the deletion of amino acids (i.e., the insertion or deletion of one or two amino acids in the polypeptide). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the percentage of identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and multiplying by 100.

[0032] Exemplary, in this disclosure, when a sequence comparison algorithm is used or when comparison and alignment are performed to the greatest extent possible by visual detection and measurement, two or more sequences or subsequences have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of nucleotide or amino acid residues “sequence identity” or “identity percentage.” The determination or calculation of “sequence identity” or “identity percentage” can be performed on any appropriate region of the sequence. For example, a region of at least about 50 residues, at least about 100 residues, at least about 200 residues, at least about 400 residues, or at least about 500 residues. In some embodiments, the sequences are essentially the same in terms of the overall length of any one or two biopolymers (nucleic acids or polypeptides) being compared.

[0033] As used in this disclosure, the term “polynucleotide” refers to a polymer composed of nucleotides. A polynucleotide may be in the form of a single fragment or a portion constituting a larger nucleotide sequence structure, and it is derived from a nucleotide sequence that has undergone at least one quantitative or concentration-wise separation, and the sequence and the nucleotide sequences of its components can be identified, manipulated, and recovered using standard molecular biological methods (e.g., cloning vectors). When a nucleotide sequence is represented as a DNA sequence (A, T, G, C), RNA sequences in which “U” substitutes “T” (A, U, G, C) are also included. In other words, a “polynucleotide” is a nucleotide polymer isolated from other nucleotides (single fragments or whole fragments), or it may be a portion or component constituting a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. “Recombinant polynucleotide” and “recombinant nucleic acid molecule” are types of “polynucleotides.”

[0034] As used in this disclosure, the term “recombinant nucleic acid molecule” refers to a polynucleotide having a sequence that is not naturally linked in nature. Recombinant polynucleotides may be contained in a suitable vector, and the vector can be transformed into a suitable host cell. The polynucleotides are then expressed in the recombinant host cell to produce, for example, “recombinant polypeptides,” “recombinant proteins,” “fusion proteins,” etc.

[0035] As used in this disclosure, the term “vector” refers to a DNA construct that contains a DNA sequence operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host.

[0036] As used in this disclosure, the term “recombinant expression vector” refers, for example, to a DNA structure that expresses a polynucleotide encoding a given polypeptide. A recombinant expression vector includes, for example, i) a set of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) a structure or coding sequence that is transcribed into mRNA and translated into a protein; and iii) transcriptional subunits of appropriate transcription and translation start and termination sequences. Recombinant expression vectors can be constructed in any suitable manner. The properties of the vector are not particularly important, and any vector can be used, such as plasmids, viruses, phages, and transposons.

[0037] In this disclosure, the term “host cell” refers to any cell type useful for transformation, transfection, transduction, etc., of the gene editing elements, nucleic acid constructs, or recombinant expression vectors of this disclosure. The term “recombinant host cell” covers host cells different from the parent cell after the introduction of the gene editing elements, nucleic acid constructs, or recombinant expression vectors, and recombinant host cells are specifically realized by transformation. The host cells of this disclosure may be prokaryotic or eukaryotic cells, and may be any cell to which the recombinant nucleic acid molecules or recombinant expression vectors of this disclosure can be introduced.

[0038] In this disclosure, the terms “transformation, transfection, and transduction” have the general meaning understood by those skilled in the art, namely, the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include, but are not limited to, any method of introducing nucleic acids into cells, including, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0039] As used in this disclosure, the terms “sample,” “specimen,” “test sample,” or “test sample” include any type of sample for which we want to determine whether or not it contains HIV antibodies. Exemplarily, a test sample may be any product produced by a subject or any product derived from a product produced by a subject. Samples may be taken from any tissue or body fluid, for example, from blood samples (including blood-derived samples), serum samples, lymph samples, saliva samples, or synovial fluid. Blood-derived samples may be a predetermined portion of a patient’s blood or a vaccinated person’s blood, for example, a predetermined cell-containing portion, or a plasma or serum portion. In some embodiments, a sample may be any sample containing antibody products of a humoral immune response.

[0040] As used in this disclosure, “diagnosis” includes the detection or identification of a subject’s disease state or condition, the determination of the likelihood that a subject has a given disease or condition, the determination of the likelihood that a subject with a disease or condition will respond to treatment, the determination of the prognosis (or likelihood of progression or mitigation) of a subject with a disease or condition, and the determination of the effectiveness of treatment for a subject with a disease or condition. For example, a diagnosis can be used to detect the presence or likelihood of infection with HIV in a subject, or the likelihood that such a subject will respond favorably to a compound (e.g., a drug (e.g., a pharmaceutical)) or other treatment.

[0041] As used in the context of this disclosure, the terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, goats, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0042] The methods disclosed herein may be carried out in vitro, ex vivo, or in vivo, or the products may exist in vitro, ex vivo, or in vivo. The term "in vitro" refers to experiments using materials, biological substances, cells and / or tissues under laboratory conditions or in a culture medium, while the term "in vivo" refers to experiments and procedures using complete multicellular organisms. In some embodiments, methods carried out in vivo may be carried out in non-human animals. "Ex vivo" refers to events that exist or occur outside a living organism, for example, outside the body of a human or animal, for example, events that exist or may occur in tissues (e.g., whole organs) or cells taken from a living organism.

[0043] (Technical aspects) In some optional embodiments, the HIV recombinant antigen includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 1, obtained through one or more amino acid substitutions, duplications, deletions, or additions.

[0044] In some optional embodiments, the HIV recombinant antigen includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 2, obtained through one or more amino acid substitutions, duplications, deletions, or additions.

[0045] In some optional embodiments, the HIV recombinant antigen includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 3, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 3, obtained through one or more amino acid substitutions, duplications, deletions, or additions.

[0046] In some optional embodiments, the HIV recombinant antigen includes a polypeptide of the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide having or partially having the activity of the polypeptide of the sequence shown in SEQ ID NO: 4, obtained through one or more amino acid substitutions, duplications, deletions, or additions.

[0047] In some optional embodiments, the HIV recombinant antigen includes a polypeptide of the amino acid sequence shown in SEQ ID NO: 5, or a polypeptide having or partially having the activity of the polypeptide of the sequence shown in SEQ ID NO: 5, obtained through one or more amino acid substitutions, duplications, deletions, or additions.

[0048] In some optional embodiments, the HIV recombinant antigen includes a polypeptide of the amino acid sequence shown in SEQ ID NO: 6, or a polypeptide having or partially having the activity of the polypeptide of the sequence shown in SEQ ID NO: 6, obtained through one or more amino acid substitutions, duplications, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 6.

[0049] In some optional embodiments, the HIV recombinant antigen includes a polypeptide of the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide having or partially having the activity of the polypeptide of the sequence shown in SEQ ID NO: 7, obtained through one or more amino acid substitutions, duplications, deletions, or additions.

[0050] In some optional embodiments, flexible linking peptides can be selected as linking peptides. Linking peptides include, but are not limited to, GS, GGSGG (SEQ ID NO: 8), KESGSVSSEQLAQFRSLD (SEQ ID NO: 9), GSAGSAAGSGEF (SEQ ID NO: 10), GGSGEFGGSGG (SEQ ID NO: 11), etc.

[0051] In some embodiments, the HIV recombinant antigen further comprises a tagged polypeptide at its N-terminus and / or C-terminus. Exemplarily, the tagged polypeptide is a His tag, preferably a 6×His tag, and is linked to the N-terminus of the HIV antigen so that it can be purified using NI-NTA affinity chromatography against the HIV recombinant antigen. In some optional embodiments, the HIV recombinant antigen and the tagged polypeptide can be linked via a linking peptide.

[0052] In some embodiments, a tracer marker or solid-phase carrier is coupled to an HIV recombinant antigen, the tracer marker being selected from at least one of acridinium esters, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, gold colloid, fluorescent microspheres, tris(2,2'-bipyridyl)ruthenium(II) dichloride hexahydrate, quantum dot luminescent materials, and upconversion luminescent materials, and the solid-phase carrier being selected from at least one of magnetic microspheres, plastic microspheres, microplastics, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0053] In some embodiments, the Disclosure provides recombinant nucleic acid molecules comprising nucleotide sequences encoding any of the above-described HIV recombinant antigens, the recombinant nucleic acid molecules may be DNA, RNA, or a combination thereof.

[0054] In some embodiments, the disclosure provides recombinant expression vectors that include the recombinant nucleic acid molecule described above and can introduce the recombinant nucleic acid molecule into cells. In some embodiments, the recombinant expression vector includes one or more regulatory elements, which may be elements such as promoters, enhancers, silencers, and insulators commonly used in the art. The regulatory elements are operably ligated to the recombinant nucleic acid molecule, thereby mediating the transcription and translation of the recombinant nucleic acid molecule.

[0055] The term "operably linked" includes a situation in which a regulatory element sequence (e.g., a promoter and / or enhancer) is covalently linked to a predetermined nucleic acid molecular sequence, thereby placing the expression of the nucleic acid sequence under the influence or control of the regulatory element (forming an expression cassette). Therefore, the regulatory element is operably linked to the nucleic acid molecule when it can act on the transcription of the nucleic acid molecule. The resulting transcription factor can then be translated into a predetermined polypeptide or protein.

[0056] In this disclosure, vectors suitable for constructing recombinant expression vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus (e.g., vectors derived from mouse leukemia virus (MLV)), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0057] In some embodiments, the vector is a prokaryotic vector containing various elements expressed in prokaryotic cells. For example, the vector includes, but is not limited to, pET series vectors, Duet series vectors, pGEX series vectors, pHY300 vectors, pHY300PLK vectors, and PE vectors. The vector is preferably a PE vector.

[0058] In some embodiments, the Disclosure provides recombinant host cells comprising the recombinant nucleic acid molecule or recombinant expression vector described above. Recombinant host cells are obtained by transforming, transfecting, or transducing a host cell with the recombinant nucleic acid molecule or recombinant expression vector. In the Disclosure, the host cell may be a eukaryotic cell or a prokaryotic cell, as long as it is capable of expressing a recombinant receptor-binding protein by introducing the recombinant nucleic acid molecule or recombinant expression vector of the Disclosure.

[0059] In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. The bacteria may be cocci (e.g., Micrococcus, Thermococcus, Streptococcus), bacilli (e.g., Enterobacter, Bacillus, Actinomycetes, Acetobacter, Lactobacillus), or Spirillum (e.g., Brachyspira). In some embodiments, the host cell is E. coli ER2529, E. coli BL21(DE3), and preferably E. coli ER2566.

[0060] In some embodiments, HIV recombinant antigens can be produced by expression in recombinant host cells, which may be prokaryotic or eukaryotic. In some embodiments, the recombinant host cells are prokaryotic, for example, obtained by introducing a recombinant expression vector into E. coli.

[0061] Recombinant host cells are cultured under conditions suitable for protein expression. After the culture is complete, the recombinant HIV antigen protein is collected from the cell culture medium or fermentation medium, and then the recombinant HIV antigen protein is purified.

[0062] In some embodiments, the steps for purifying the HIV recombinant antigen protein include Ni-NTA affinity chromatography, ion exchange chromatography, and protein dialysis.

[0063] In some embodiments, the Disclosure provides a method for diagnosing HIV-related diseases, comprising contacting an HIV recombinant antigen, reagent, or kit described above with an HIV antibody in a biological sample to detect the presence of an HIV-1 antibody in the biological sample.

[0064] In some optional embodiments, the above method includes an immunoassay on a biological sample, the immunoassay being said to be ELISA, fluorescence immunochromatography, gold colloid immunochromatography, chemiluminescence measurement, electrochemiluminescence measurement, indirect immunofluorescence (IFA), or radioimmunoassay (RIA).

[0065] Unless otherwise specified, the experimental techniques and methods used in these examples are all conventional techniques or methods. For example, in the following examples, experimental methods for which specific conditions are not explicitly stated generally conform to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples can all be obtained from general suppliers.

[0066] Example 1: Production of HIV recombinant antigen 1.1 Preparation of gp36 recombinant antigen (Plasmid design) Based on the sequences shown in SEQ ID NOs: 2, 3, and 4, a fusion partner GST was introduced to the N-terminus of the recombinant antigen, and expression plasmids were constructed based on the nucleic acid encoding the protein sequence. The gene sequence contained restriction enzyme sites BamHI and EcoRI at both ends. Mutant genes were treated with the restriction enzymes BamHI and EcoRI, respectively, and the treated gene fragments were ligated into a PE vector (see Figure 1) to obtain the corresponding expression plasmids.

[0067] (Induced expression) Using the heat shock method, the expression plasmid constructed as described above was transformed into E. coli expression strain ER2566 (NEB), plated onto LB plates containing 100 μg / mL Amp, and incubated at 37°C for 16 hours. Single colonies were picked, positive strains correctly identified by PCR of the bacterial suspension were selected and sequenced, and the correctly sequenced strains were inoculated into LB medium containing 50 μg / mL Amp and incubated with shaking at 37°C. OD 600 When the value reached 0.6-0.8, 1.0 mM IPTG was added, and the cells were incubated at 37°C for 2-4 hours. The expression status of recombinant proteins was identified by SDS-PAGE, and the purified proteins were named HIV-G-1 (SEQ ID NO: 2), HIV-G-2 (SEQ ID NO: 3), and HIV-G-3 (SEQ ID NO: 4), respectively.

[0068] 1.2 Preparation of gp36-gp41 chimeric antigens Creating a fusion expression antigen of gp36 (SEQ ID NO: 4) and gp41 (SEQ ID NO: 5) involved designing a gp41-gp36 chimeric sequence (SEQ ID NO: 5 + SEQ ID NO: 4, with a restriction enzyme site, amino acid residue RS, between them) and obtaining recombinant protein HIV-G-4 (SEQ ID NO: 6) in a similar manner to Example 1.1.

[0069] 1.3 Creation of cysteine ​​mutant antigens Based on HIV-G-4, a cysteine ​​mutant antigen was designed (cysteine ​​was mutated to serine), and recombinant protein HIV-G-5 (SEQ ID NO: 7) was obtained in a similar manner to Example 1.1.

[0070] Example 2: Evaluation of the sensitivity and specificity of recombinant HIV antigen of this disclosure using a double antigen sandwich method on a gold colloid chromatography platform. 2.1 Detection of HIV-2 type specimens (1) HIV antigen labeling To 5 mL of 4 / 10000 gold colloid, an appropriate amount of 0.2 M K2CO3 was added and stirred for 5 minutes. HIV-labeled antigen was added and stirred for another 5 minutes. An appropriate amount of 10% BSA was then added to block and complete the labeling process. The mixture was centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, the precipitate was redissolved with gold colloid reconstitution solution, and finally, the solution was diluted to 0.5 mL (1 / 10 of the volume of the gold colloid solution) with gold colloid reconstitution solution. Finally, the gold colloid with concentrated labeled antigen was diluted to a certain multiple with gold colloid reconstitution solution to obtain a gold colloid working solution, and the gold colloid was plated. The plated gold colloid was freeze-dried in a freeze-dryer (1-2 hours) or dried overnight in a drying room at 37°C.

[0071] (2) Coating of HIV antigen HIV antigen-coated T-lines, diluted to 1.0 mg / mL with a coating diluent (10 mM PB + 150 mM NaCl + 2% sucrose, pH 7.4), were placed in a 37°C incubator and kept for 1-2 hours.

[0072] (3) Preparation of gold colloid test strips Gold colloid test strips were cut to the specified width using a strip cutter to obtain strips, which were then assembled and the sample was added for detection. The gold colloid color card shows a total of 10 levels: C1, C2, C3, C4, C5, C6, C7, C8, C9, and B. From left to right, the color of the corresponding T-line gradually becomes lighter, with C1 having the darkest T-line color, C9 having the lightest T-line color, B being colorless, and B+ being nearly colorless. In Table 2, "C3+" means that the color is darker than C3 but not yet reached C2, and the same applies to the other "+" values.

[0073] Detection results: The shades of color were determined using color cards, and the results are as follows: [Table 1] The results demonstrate that HIV-G-1, HIV-G-2, and HIV-G-3, as described herein, can all be used as antigens to detect HIV-2 type specimens with high sensitivity and specificity.

[0074] 2.2 Detection of HIV specimens (including confirmed HIV-2 positive specimens and Abbott-manufactured HIV-positive specimens) The procedure is the same as in Example 2.1, except that two T lines on the test strip are coated with the P81 antigen for HIV-1 and the P82 antigen for HIV-2, respectively.

[0075] The detection results were as follows: The intensity of the colors was determined using color cards, and the results were as follows: (The value to the left of the symbol " / " indicates the detection result of HIV-2 antibodies, and the value to the right indicates the detection result of HIV-1 antibodies.) [Table 2] The results demonstrate that both HIV-G-4 and HIV-G-5 described herein can effectively detect HIV-positive specimens as antigens.

[0076] Example 3: Investigation of thermal stability The stability assessment of recombinant HIV antigens includes performing accelerated stability tests for 3 days and 7 days under environmental conditions of 37°C, and detecting the formation of aggregates using an SDS-PAGE protein gel without mercaptoethanol treatment.

[0077] 3.1 Formation status of aggregates The table below summarizes the aggregation status of SDS-PAGE protein gels without mercaptoethanol treatment when HIV recombinant antigens were stored at 4°C and tested at 37°C for 3 and 7 days. Fewer aggregates indicate better stability. It was found that the mutation of cysteine ​​to serine in the HIV recombinant antigen significantly improves thermal stability. [Table 3]

[0078] (Related sequences) Sequence ID 1 LATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY Sequence ID 2 LATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVF Sequence ID 3 LSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQQEKNMYELQKLNSWDVF Sequence ID 4 AIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY Sequence ID 5 TLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIEESQNQQEKNEKDLLALDSWKNLWNWFDIT Sequence ID 6 AIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY RS TLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIEESQNQQEKNEKDLLALDSWKNLWNWFDIT Sequence ID 7 AIEKYLKDQAQLNSWGSAFRQVSHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY RS TLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQQLLGIWGSSGKLISTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIEESQNQQEKNEKDLLALDSWKNLWNWFDIT

[0079] In this specification, any description containing the terms “one example,” “several examples,” “example,” “specific example,” or “several examples” means that the specific features, structures, materials, or matters described using such example or example are included in at least one example or example of this disclosure. In this specification, a general description using the above terms does not necessarily refer to the same example or example. Furthermore, the specific features, structures, materials, or matters described may be combined in an appropriate manner in any one or more examples or examples. Furthermore, to the extent that they do not contradict each other, a person skilled in the art may combine or link different examples or examples and features of different examples or examples described herein.

[0080] While embodiments of the present disclosure are shown and described above, it should be understood that these embodiments are illustrative and should not be considered limitations to the present disclosure. Those skilled in the art can modify, change, substitute, and transform the above embodiments within the scope of the present disclosure. [Industrial applicability]

[0081] The inventors analyzed the gp36 protein of different HIV-2 strains and designed the gp36 protein sequence shown in Sequence ID No. 1. Based on this sequence, the recombinant HIV antigen designed can effectively detect clinical HIV-2 positive specimens and possesses high sensitivity and specificity. When this recombinant HIV antigen protein is used as the labeling end, the marker activity of the recombinant HIV antigen can be significantly improved, while nonspecific immunobinding activity can be reduced, thereby increasing sensitivity.

Claims

1. The amino acid segment comprises the amino acids from position x to y of the HIV-2 gp36 protein, where x is an integer from 1 to 60, and y is an integer from 143 to 161, and the amino acid sequence of the HIV-2 gp36 protein is shown in SEQ ID NO:

1. HIV recombinant antigen.

2. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, The recombinant antigen according to claim 1, wherein y is 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161.

3. The recombinant antigen according to claim 1, wherein the HIV recombinant antigen comprises the HIV-2 gp36 protein sequence shown in SEQ ID NO: 2, the HIV recombinant antigen comprises the HIV-2 gp36 protein sequence shown in SEQ ID NO: 3, or the HIV recombinant antigen comprises the HIV-2 gp36 protein sequence shown in SEQ ID NO:

4.

4. The recombinant antigen according to claim 1, wherein the cysteine ​​in the sequence of the recombinant antigen is mutated to another amino acid, and optionally, the cysteine ​​is mutated to serine.

5. The recombinant antigen according to claim 1, further comprising a segment of another HIV protein, optionally comprising a segment of HIV-1, optionally comprising a segment of HIV-1, optionally comprising a segment of HIV-1 gp41, optionally comprising a gp41 protein sequence of HIV-1 represented by SEQ ID NO: 5, optionally comprising a fusion partner.

6. The recombinant antigen according to any one of claims 1 to 5, wherein the recombinant antigen comprises the sequence shown in SEQ ID NO: 6 or SEQ ID NO:

7.

7. A recombinant HIV antigen containing one of the following polypeptides. a) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 1, after one or more amino acid substitutions, duplications, deletions, or additions. b) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 2, after one or more amino acid substitutions, duplications, deletions, or additions. c) A polypeptide having the amino acid sequence shown in SEQ ID NO: 3, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 3, after one or more amino acid substitutions, duplications, deletions, or additions. d) A polypeptide having the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 4, after one or more amino acid substitutions, duplications, deletions, or additions. e) A polypeptide having the amino acid sequence shown in SEQ ID NO: 5, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 5, after one or more amino acid substitutions, duplications, deletions, or additions. f) A polypeptide having the amino acid sequence shown in SEQ ID NO: 6, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 6, after one or more amino acid substitutions, duplications, deletions, or additions. g) A polypeptide having the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide having or partially having the activity of the polypeptide having the sequence shown in SEQ ID NO: 7, after one or more amino acid substitutions, duplications, deletions, or additions to the amino acid sequence shown in SEQ ID NO:

7.

8. A reagent for detecting HIV, comprising the recombinant antigen described in any one of claims 1 to 7.

9. The reagent according to claim 8, wherein a tracer marker or a solid-phase carrier is coupled to the recombinant antigen, the tracer marker is selected from at least one of acridinium ester, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, gold colloid, fluorescent microspheres, tris(2,2'-bipyridyl)ruthenium(II) dichloride hexahydrate, quantum dot luminescent material and upconversion luminescent material, and the solid-phase carrier is selected from at least one of magnetic microspheres, plastic microspheres, microplastics, microplates, glass, capillaries, nylon and nitrocellulose membranes.

10. A kit for detecting HIV, comprising a recombinant antigen according to any one of claims 1 to 7, or a reagent according to claim 8 or 9.

11. A nucleic acid molecule encoding an HIV recombinant antigen according to any one of claims 1 to 7, a vector containing the nucleic acid molecule, and a host cell containing the vector.

12. Use of a recombinant antigen according to any one of claims 1 to 7 or a reagent according to claim 8 or 9 in the manufacture of an HIV detection kit.

13. A diagnostic method for HIV-related diseases, The method includes contacting an HIV recombinant antigen according to any one of claims 1 to 7, a reagent according to claim 8 or 9, or a kit according to claim 10 with an HIV antibody in a biological sample to detect the presence of an HIV-1 antibody in the biological sample. Optionally, the method includes an immunoassay on a biological sample, the immunoassay being ELISA, fluorescence immunochromatography, gold colloid immunochromatography, chemiluminescence measurement, electrochemiluminescence measurement, indirect immunofluorescence (IFA), or radioimmunoassay (RIA). method.

Images